Supplement to: ISCT 2014 ANNUAL MEETING ABSTRACTS

Transcription

CYTOTHERAPY
Volume 16
Number 4S April Supplement 2014
www.celltherapyjournal.org
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April Supplement 2014
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Number 4S
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Volume 16
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Supplement to:
ISCT 2014 ANNUAL MEETING ABSTRACTS
Pages S1-S130
ELSEVIER
JCYT_v16_i4_sS_COVER.indd 1
3/8/2014 7:58:04 PM
Senior Editor
Associate Editors
John Barrett, MD
Catherine Bollard, MD
Bethesda, MD, USA
Children's National Health System
Washington, DC, USA
Massimo Dominici, MD
University of Modena and Reggio Emilia
Modena, Italy
Edwin M. Horwitz, MD, PhD
The Children’s Hospital of Philadelphia
Philadelphia, PA, USA
John Rasko, MBBS, PhD
RPA Hospital
Sydney, Australia
Katayoun Rezvani, MD, PhD, FRCP, FRCPath
MD Anderson Cancer Center
Houston, TX, USA
Aims and Scope
Cytotherapy publishes cutting edge findings, clinical trials of cell-based therapies, and news and opinion on all aspects of the
rapidly expanding field of cell-based treatments for cancer, degenerative disorders, immunotherapy and stem cell transplantation.
The journal focuses especially on the practical translation of scientific developments in the laboratory into clinical practice.
Cytotherapy is an essential global resource for clinical researchers, oncologists, hematologists, physicians, and regulatory experts
involved in cell processing and therapy.
The journal’s scope covers:
Stem cell processing and transplantation
Cell-based therapies of malignant and non-malignant blood diseases
Cancer
Stem cell plasticity
Autoimmune diseases
Immunotherapy
Congenital disorders
Novel molecular therapies
Gene therapy
Microvesicle biology and therapeutic application
Published by Elsevier
Editorial Board
Reza Abdi, MD
Brigham and Women’s Hospital
Boston, MA, USA
Ellen Areman, MS, SBB
Ellen Areman Consulting
Glen Burnie, MD, USA
Malcolm Brenner, MD, PhD, FRCP
Baylor College of Medicine
Houston, TX, USA
Richard Champlin, MD
Houston, TX, USA
Nancy Collins, PhD
University of Toledo
Toledo, OH, USA
Patrizia Comoli, PhD
University of Pavia
Pavia, Italy
Robert Deans, PhD
Athersys
Cleveland, OH, USA
Colleen Delaney, MD, MSC
Seattle Cancer Care Alliance
Seattle, WA, USA
Ekaterina Doubrovina, MD, PhD
Memorial Sloan-Kettering Cancer Center
New York, NY, USA
Allen Eaves, MD, PhD
STEMCELL Technologies, Inc.
Vancouver, BC, Canada
JH Frederik Falkenburg, MD, PhD
Leiden University Medical Center
Leiden, The Netherlands
Peiman Hematti, PhD
University of Wisconsin-Madison
School of Medicine & Public Health
Madison, WI, USA
Helen Heslop, MD
Houston, TX, USA
Lawrence Lamb, PhD
University of Alabama at Birmingham
Birmingham, AL, USA
Ping Law, PhD
NUH Singapore
Singapore
Ann Leen, PhD
Texas Children’s Cancer Center
Houston, TX, USA
Bruce Levine, MD, PhD
University of Pennsylvania School
of Medicine
Philadelphia, PA, USA
Douglas Losordo, MD
Northwestern University, Feinberg School
of Medicine
Chicago, IL, USA
Alejandro Madrigal, MD, PhD,
MRCPath, FRCP, DSc
The Anthony Nolan Research Institute
London, UK
Richard Maziarz, MD
Oregon Health & Science University
Portland, OR, USA
David McKenna, MD
Masonic Cancer Center
Minneapolis, MN, USA
Gregor Reid, PhD
University of Western Ontario
London, ON, Canada
Clio Rooney, PhD
Houston, TX, USA
Claudia Rossig, MD
University of Münster
Münster, Germany
Scott Rowley, MD, FACP
Hackensack University Medical Center
Hackensack, NJ, USA
Khalid Shah, MS, PhD
Massachusetts General Hospital
Harvard Medical School
Boston, MA, USA
Warren Sherman, MD, FACC, FSCAI
Mount Sinai Hospital
New York, NY, USA
Akihiro Shimosaka, PhD
Research Foundation for
Community Medicine
Tokyo, Japan
Donna Skerrett, MD
New York Presbyterian Hospital
New York, NY, USA
Ineke Slaper-Cortenbach, PhD
Universitair Medisch Centrum Utrecht
Utrecht, The Netherlands
Paul Szabolcs, MD
Duke University Medical Center
Durham, NC, USA
Satoshi Takahashi, MD, PhD
The Institute of Medicine Science
Tokyo, Japan
Adrian Gee, PhD
Houston, TX, USA
Ian McNiece, PhD
Houston, TX, USA
David Gottlieb, MB, BS, MD
Westmead Hospital
Westmead, NSW, Australia
Jos Melenhorst, PhD
National Institutes of Health, NHLBI
Bethesda, MD, USA
Rupert Handgretinger, MD
Children’s University Hospital
Tuebingen, Germany
Jeff Molldrem, MD
Houston, TX, USA
Frits Van Rhee, MD, PhD, MRCP,
FRCPath
University of Arkansas
Little Rock, AR, USA
Patrick Hanley
Children’s National Health System
Washington, DC, USA
Tuna Mutis, MD, PhD
Universitair Medisch Centrum Utrecht
Utrecht, The Netherlands
Dominic Wall, BSc, PhD
Peter McCallum Cancer Center
East Melbourne, VIC, Australia
Shelly Heimfeld, PhD
Fred Hutchinson Cancer Research Center
Seattle, WA, USA
Robert S. Negrin, MD
Stanford University Hospital
Stanford, CA, USA
Joseph Wu, MD, PhD
Stanford University Hospital
Stanford, CA, USA
Terry E. Thomas, PhD
STEMCELL Technologies, Inc.
Vancouver, BC, Canada
Table of Contents: Volume 16 Number 4S April Supplement 2014
S1
Welcome from the ISCT 2014 Annual Meeting Co-Chairs
S3
Program at a Glance
S5
Author Index
S7
Oral Abstracts
S18
Poster Abstracts
S112 Twenty years of the International Society for Cellular Therapies: the past, present and future of cellular therapy
clinical development
S Abbott, G Mackay, M Durdy, S Solomon, C Zylberberg
S120 Immunotherapy: opportunities, risks and future perspectives
M Hildebrandt, K Peggs, L Uharek, CM Bollard, HE Heslop
The opinions or views expressed in this professional education supplement are those of the authors and do not necessarily reflect the opinions or
recommendations of the International Society for Cell Therapy.
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or may be derived from the professional literature or other clinical sources. Because of the differences between in vitro and in vivo systems and
between laboratory animal models and clinical data in humans, in vitro and animal data may not necessarily correlate with clinical results.
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Ó 2014 International Society for Cellular Therapy. All rights reserved.
Cytotherapy, 2014; 16: S1eS2
Welcome, Willkommen, Bienvenue à Paris la Ville Lumière!
On behalf of the entire 2014 Organizing Committee, we would like to extend a warm and friendly
welcome to you to Paris for the 20th Anniversary Meeting of the International Society for Cell
Therapy (ISCT). This year, we have a packed program of world-class science brought to
France’s vibrant capital for 4 days. What better way to celebrate 20 years of the ISCT!
The perfect city in which to celebrate this significant accomplishment, Paris is a joy to visit, boasting
numerous impressive architectural monuments,
world-class museums and art galleries. It is among
the four fashion capital cities around the world, is
regularly quoted as the world’s food capital,
hosting reams of Michelin-starred
restaurants, and its endless contributions to the
arts make the city a highly desirable places to live.
When it comes to our field of science, Paris boasts
a similarly high level of competence,
containing more than 17 universities, more than
10 hospitals, numerous research institutes and a
thriving stem cell and cell therapy community. It
is an honor for the ISCT to be back in Europe for
this important meeting, the 20th time that we have held our annual meeting in the region. We hope that
you will enjoy the distinct European feel of the program.
All this excellently complements our exciting 2014 scientific program, designed to drive the
translation of all cellular therapies for the benefit of patients worldwide. We have six Plenary Sessions
covering a wide range of cell therapy applications, including
within the settings of neurological disorders, solid organ
transplantation and tissue engineering, cardiovascular diseases
and immunotherapy, not to mention our Presidential Plenary
session on gene-modified cell therapy. We are delighted that
so many leading individuals from around the world are
speaking across the program, with every region represented, as
well as numerous clinicians presenting work in complement
with scientists and industrial leaders. Our technical sessions
and workshops promise to address cutting-edge matters facing
our community, not just looking at manufacturing and
processing issues but reaching across to policy decisions, legal
and regulatory challenges and ethical perspectives.
As more and more of us witness the successful translation of cell therapy practices to patients, our
Commercialization Committee also gains momentum and has come up with an extensive track of sessions
that promise to demonstrate the very latest product and clinical developments at industry level. Likewise,
ISSN 1465-3249 Copyright Ó 2014, published by Elsevier Inc. on behalf of International Society for Cellular Therapy.
http://dx.doi.org/10.1016/j.jcyt.2014.01.011
S2
the Quality and Operations track is overflowing with
technical know-how, with 13 sessions covering the state
of the art in practical aspects related to production and
uses. With a record number of abstracts submitted to
this conference, there is a stellar selection available
within the Oral Abstract Presentation Sessions for you to
fit into your schedule. We encourage you also to join us
for a full slate of pre-conference workshops on April 23,
including the annual Global Regulatory Perspectives
Workshop and Flow Cytometry and Cell Analysis
Workshop.
All of this would not be possible without the help and assistance from a huge number of individuals who
have strived to put together the program over the past 12 months, not forgetting to thank our
worldwide speakers for coming to Paris for this week to join us in our meeting. We are also very grateful to our
sponsors and exhibitors, both loyal supports and new connections, without which this meeting would not
be possible.
We hope that you have a highly enjoyable 4 days with us here in Paris, and thank you for joining us for this
special event.
Merci et à bientôt à Paris!
Luc Senseb
e, MD, PhD
Etablissement Français
Du Sang
Toulouse, France
Emily Culme-Seymour, PhD
London Regenerative
Medicine Network
London, United Kingdom
Martin Hildebrandt, MD
Technische
Universität München
Munich, Germany
20th Annual ISCT Meeting
April 23-26, 2014 Paris, France
PROGRAM AT A GLANCE
Immunotherapy and Dendritic Cells
Oral Presentation Session 1
Thursday April 24, 11:00 e 12:15
Abstracts 1-5
Poster Session 1
Abstracts 36-86
Cell and Gene Therapy or Cellular Gene Transfer
Abstracts 6-8
Poster Session 1
Abstracts 87-114
Cardiovascular Repair and Regeneration
Abstracts 9-10
Poster Session 1
Abstracts 115-134
Legal and Ethical
Abstracts 11-12
Poster Session 1
Abstracts 164-178
Quality and Operations
Abstracts 13-15
Poster Session 1
Abstracts 135-163
Mesenchymal Stem Cells
Friday April 25, 10:45 e 12:15
Abstracts 16-21
Poster Session 2
Abstracts 219-294
Regenerative Medicine and Tissue Engineering
Abstracts 22-27
Poster Session 2
Abstracts 295-359
Translational Process Development
Saturday April 26, 10:45 e 11:45
Abstracts 28-31
Poster Session 2
Abstracts 360-394
Nervous System Repair
Abstracts 32-33
Poster Session 1
Abstracts 204-218
Hematopoietic Stem Cells
Abstracts 34-35
Poster Session 1
Abstracts 179-203
Author Index
A
Adorable-Wagan, Perie P282
Aghdami, Nasser O23
Almåsbak, Hilde P109
Anderson, Kristina P63
Andrade, Ana P309
Andre, Emilie P217
Andreassen, Grete P150
Aro, Andrea P335
Arturo, Jhann P218, P356
B
Badenes, Sara P272
Baharvand, Hossein P120
Barile, Lucio P125
Barry, Jacqueline P143
Bassi, Giulio P229,
P274, P318
Bastien, Jean-Philippe P84
Bell, Rosemarie O14
Belmonte, Nathalie P65
Beloki, Lorea P49
Beltzer, Jim P391
Bemark, Mats P83
Bernardi, Martina
P366, P367
Bernardo, Maria Ester P236,
P237, P247, P273
Bieback, Karen P145,
P243, P244
Bienek, Carol P378
Bigalke, Iris P53
Blasetti, Nahuel P214
Bleau, Sharon P387
Bootcha, Ratikorn P295
Bose, Bipasha P246
Brix, Liselotte P81
Brunet de la Grange,
Philippe P192
Bull, David P239
C
Cai, Jinge P94
Calmels, Boris P194
Caminal, Marta P363
Carneiro, Giane P128
Ceron, Willy P132
Cervio, Elisabetta, O10
Chabannon, Christian O15
Chai Lai, Ruenn P134
Chambers, Daniel O17
Chang, Li-Ching P89,
P90, P115
Chapel, Alain P106, P332,
P339, P340
Chelluri, Lakshmi P136
Chen, Allen O30
Chen, Guanghua P100, P324
Chieregato, Katia P52
Chin, Sze-Piaw O21, P265,
P267, P268
Chu, Yaya P51
Chua, Alvin P297
Chung, Francisco P86
Claude Roy, Denis P72
Clerget-Chossat,
Nathalie P62
Codinach, Margarita P147
Coll, Ruth P344, P345
Corselli, Mirko P275
Courtman, David O06
Creane, Michael P127
Cui, Jiuwei P39
Cushing, Melissa P183, P184,
P185
Cuthbert, Richard P270
D
da Silva, Claudia P203, P358
Das, Ruud P360, P375
de la Kethulle de Ryhove,
Laurence P222
de Paula, Tatiana P348
Del Fattore, Andrea P66,
P67, P249
Deng, Xuewen P44
Dennett, Richard P377
Deschaseaux, Frederic P232
Di Trapani, Mariano
P271, P276
Dietz, Allan O28
Dold, Catherine P287
Dolnikov, Alla O03, P59, P191
Doronin, Sergey P227
Drake, Rosemary P383
Dupraz Poiseau, Anne P174
Dyson, Pamela P189
E
Egloff, Matthieu P371, P372
Ehrhardt, Rolf P78, P79
Erceg, Slaven P306
Espagnolle, Nicolas P230
F
Feng Choong, Pei P221
Fernandez-Moure,
Joseph P258
Figueiredo, Francisco O25
Follin, Bjarke P124
Francois, Sabine P313
Frank, Joseph P307
G
Gabr, Hala O26, P325, P347
Gadelorge, Melanie P373
Gazzola, Maria-Vittoria
P153, P154
Genser-Nir, Mira P263
Ghersi, Giulio P305
Gibbons, Amanda P380
Ginty, Patrick P142
Godoy, Juliana P118
Grigoriadis, Ioannis
P199, P319
Griley, Bambi P171
Grisendi, Giulia P91
Guan Soh, Teck P186
Guilloton, Fabien P235
Gupta, Pawan P278
H
Haack-Sørensen,
Mandana P292
Hackett, Martha P178
Hagbard, Louise P346
Han, Zhibo P242, P337
Hanley, Patrick O04
Harris, David P314
Hashimoto, Hisayoshi P101
Hegde, Meenakshi P77
Herías, Veronica P116
Ho, Jennifer P296
Hollyman, Daniel P138
Holt, Dolly P238
Hoogduijn, Martin
P226, P266
Hulspas, Ruud P393
Huss, Ralf P107
I
Im, Keon-Il P110
Ismail, Rahman P73
Iudicone, Paola P57
Ivanovic, Zoran O35,
P196, P234
J
Jang, Jae-deog P228, P261
Jankowski, Ron P320
Janssen, William P163
Jenhani, Faouzi P187
Jeong, Hyunsuk P126
Jochheim-Richter,
Andrea P365
Jong, Liesbeth P60
Joo Rhyu, Jung P281
K
Kaigler, Darnell P330
Kaiser, Andrew P42, P108
Kallur, Therese P303
Kamal, Mohamed P225
Kang, Kyung-Sun O19
Kassem, Dina P224
Keever-Taylor, Carolyn
P43, P162
Khan, Aleem P312
Kim, Miyeon P283
Kim, Nayoun P104
Kishi, Naoko O12
Klingemann, Hans P82
Kloess, Stephan P148
Koliakos, George P212
Koon-Teoh, Hoon P97
Krampera, Mauro P279
Krebs, Karin O08
Kreissig, Carla P310
Kumar, Pardeep P204
Kumar, Vijay P342
Kurtzberg, Joanne O32, P188
Kuwahara, Kenrick P38
Kyung Jang, Yun P285
L
Lako, Majlinda P329
Lamana, María O16
Lamers, Cor O31
LeBlon, Courtney P197
Lee, Oscar O27, O33
Lehtinen, Miia O09
Li, Linhong P111
Lim, Okjae P47
Linard, Christine P334
Liu, Kelvin P294
Louis, Chrystal P46
Lourenco, Sofia O18
Lujan, Mike P159
Lukomska, Barbara P299
M
Macpherson, Janet P172
Magnani, Chiara P93
Majumdar, Anish P370
Marit Inderberg-Suso,
Else P74
Mathieu, Noëlle P308
Matko, Sarah P71
Matosevic, Sandro P350
McNiece, Ian O34
Mehta, Sunil P354
Mei, Shirley P98, P390
Meij, Pauline P173
Michalopoulos,
Efstathios P119
Mitra, Arindam P54
Montemurro, Tiziana P317
Moraes, Daniela P219
Mouchotte, Rosa P175
Moviglia, Gustavo P155
Moviglia-Brandolino,
Teresita P211
Müller, Sylvia P251
Murgia, Alba P341
Murrell, Julie P252, P253
N
Nagamura-Inoue,
Tokiko P379
Nakajima, Ryota P298
Nakazawa, Yozo P99
Ng, Angela P322
O
O’Brien, Vincent P290, P385
Oh, Steve P374
Oja, Sofia P269
Ooi, Ghee-Chien P161
S6
Author Index
Osadolor, Isaac P164, P165,
P166, P167, P170
Otero, Gabriela P333
Otsuru, Satoru P257, P259
P
Panterne, Beatrice P193
Papadimitrious, Michael P61
Papadopoulou, Anastasia O02
Patel, Amit O24, O121,
O122, O277, O321, O368
Patel, Sachit P208
Pattanapol, Nakrob P300
Pavelcova, Katerina P64
Perez Lopez, Silvia P130
Perisic, Tatjana P102
Perruchoud Fluri,
Stephanie P123
Peshwa, Madhusudan P112
Petchdee, Soontaree P301
Peyrafitte, Julie-Anne P144
Phinney, Donald P256
Pimpaneau, Valerie P149
Pinzur, Lena P357
Pontikoglou,
Charalampos P255
Popova, Anna P323
Pototschnig, Hanno P353
Prel, Anne P233
Preti, Milena P231
Pujals-Fonts, Noelia P364
Putnam, Amy O01
R
Ramachandran, Niraj P157,
P158, P201
Ramos, Thomas P361
Refaie, Ayman P280
Reza Mirlashari,
Mohammad P328
Ricciardi, Mario P50
Riccobono, Diane P92
Ricordi, Camillo O22
Riis, Simone P326
Ritchie, Rae Record P349
Roelofs, Helene P250
Rosell, Anna P216
Ruella, Marco O05
S
Sadr, Nasser P386
Sage, Beth P87
Saha, Arjun P206, P210
Salmons, Brian P114
Samuel, Edward P381
Sanz-Nogues, Clara P331
Saulnier, Nathalie P315
Sauvage, Vincent P103
Schjetne, Karoline P70
Schmiedeknecht, Gerno P40
Schwartz, Joseph P137
Sengenes, Coralie P254
Seon, Mira P245
Sharma, Manoj P179, P182
Shelley, Chris P88
Shenoy, Sudheer P248
Shoulars, Kevin P198
Slaper-Cortenbach,
Ineke O20
Snykers, Sarah P152
Sohn, Hyun-Jung P48
Spitalieri, Paola O07
*O indicates an oral abstract number; P, poster abstract number.
St. Martin, Katherine P139
Startz, Thomas P80
Stasko, Karl P151
Storms, Robert P215
Su Jeon, Eun P286
Sudhakar Magapu,
Solomon P68
Sutton, Caroline P129
Svalgaard, Jesper P195
T
Takahara, Masashi P58
Takeuchi, Yasuo P55
Talmadge, James P75
Tano, Keiko O13
Tassy, Jennifer P200
Tettamanti, Sarah P105
Theys, Nicolas P146,
P316, P327
Thiagarajah, Kalaivani P160
Thijssen-Timmer,
Daphne P288
Thirumala, Sreedhar P394
Thomas, Natalie P376
Thurman-Newell, Jamie
P384
Ting, Anthony P289
Tonn, Torsten P76
Tra, Wendy P382
Trigo, Guillermo P338
Tsang, Kam P240
Tseng, Pei-Chi P302
Tuma, Jorge P131
Turchetto, Lucia P117
Turner, Marc O29, O85
U
Uhlendorf, Toni P205
Uppal, Sabrina P207
V
Van Campenhout,
Ann P284
van der Aar, Pim P362
Velthuis, Jurjen P45
von Tigerstrom, Barbara
O11, P168, P169
W
Wagey, Ravenska P220
Wagner, Beate P190
Wang, Youwei P262
Weiss, Kirsten P96
Wernersson, Karin P69
Willemsen, Philippe P343
Woo Yim, Hyeon P213
Woods, Erik P291, P351,
P352, P336, P388, P389
Wright, Craig P180
Wrobel, Sandra P359
Y
Yao, Chao-Ling P181, P264
Yong Tan, Kah P304
Yoo, Minjoo P247
Z
Zarkos, Kon P223
Zogas, Nikolaos P56
ABSTRACTS
Oral Abstracts
1
RESULTS FOLLOWING COMPLETION OF PHASE I CLINICAL
TRIAL USING EX VIVO EXPANDED CD4+CD127LO/-CD25+
POLYCLONAL TREGS FOR THE TREATMENT OF RECENTONSET TYPE 1 DIABETES
AL Putnam1, A Lares1, W Liu1, M Lee1, S Gitelman1, K Herold2,
N Warner3, J Bluestone1
1
Diabetes Center, University of California, San Francisco, San Francisco,
California, United States, 2Yale University, New Haven, Connecticut, United
States, 3BD Biosciences, San Jose, California, United States
Study participants with recent-onset type 1 diabetes, between the ages of 18-45,
have been successfully treated with ex vivo expanded polyclonal Tregs in a JDRFsponsored phase I clinical trial. In this clinical trial, we were able to generate
therapeutically relevant numbers of sufficiently pure polyclonal CD4+CD127lo/CD25+ Tregs for clinical use using a flow based isolation procedure. Following
the completion of the trial, 14 study participants were treated with either 5x10e6,
40x10e6, 320 x10e6 or 2.6 x10e9 Tregs following an ex vivo expansion using antiCD3/anti-CD28-coated beads plus IL-2. Cells expanded on average of 650.2 fold
(range 29.8-1366.8) and expressed 76-96.9% FOXP3+ (mean 92.2%) following
the 14 day expansion period. In addition, these CD4+CD127lo/-CD25+ Tregs
exhibit potent suppressor activity, and maintain high CD4 (mean 97.4%; range
95-98.7%) and CD25 expression and low CD127 and CD8 (mean 0.6%; range
0.1-2.4%) expression following the clinical expansions. All anti-CD3/antiCD28-coated beads were removed from the expansion cultures prior to
infusion based on specifications approved by the FDA. Additionally, all sterility release criteria with regard to anaerobic/aerobic bacterial testing, mycoplasma, endotoxin, gram stain, KOH and fungal cultures were negative. All
Tregs were manufactured at UCSF and freshly infused at either Yale University or UCSF illustrating feasibility of transport and stability overnight.
This is the first study where Tregs have been cultured in the presence of
deuterated glucose [D-GLUCOSE (6,6-D2, 99%)] and are able to be tracked
in vivo following infusion into study participants. The tracking results, as well
as mechanistic data (flow cytometry, subset analysis, pSTAT5 and cytokine
analysis), from the entire phase I clinical trial will be presented.
2
SAFETY AND CLINICAL EFFICACY OF RAPIDLY-GENERATED
VIRUS-SPECIFIC T CELLS WITH ACTIVITY AGAINST ADV,
EBV, CMV, HHV6 AND BK VIRUS ADMINISTERED AFTER
ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANT
A Papadopoulou, UL Katari, U Gerdemann, I Tzannou, C Martinez,
K Leung, G Carrum, A Gee, J Vera, RA Krance, MK Brenner, C Rooney,
H Heslop, AM Leen
Center for Cell and Gene Therapy, Baylor College of Medicine, The
Methodist Hospital, Texas Children’s Hospital, Houston, Texas, United States
Viral infections remain a major cause of morbidity and mortality post-transplant.
To address this issue, and with NHLBI-PACT support, we have made virusspecific T-cell lines (VSTs) with activity against 5 common post-transplant
viruses (EBV, CMV, Adv, BK, HHV6), using a simplified 10-day. To date 48
clinical-grade multivirus (m)VSTs have been generated. By exposing 30x10 6
PBMCs to overlapping peptide libraries spanning Adv (Hexon, Penton), CMV
(pp65, IE1), EBV (LMP2, EBNA1, BZLF1), BK (Large T, VP1) and HHV6
(U11, U14, U90) antigens we expanded a median of 35.7x10 7 polyclonal cells
over 9-11 days. pVST specificity was dependent on the donor’s prior viral
exposure; 45/48 lines had Adv activity (Hexon: 47071; Penton: 36686 SFC/
2x10 5), 26/48 against CMV (IE1: 356157; pp65: 1048446), 37/48 against
EBV (LMP2: 13776; EBNA1: 12352; BZLF1: 997), 28/48 against BK
(Large T: 12361; VP1: 20889) and 29/48 against HHV6 (U90: 10978;
U11: 3717; U14: 8426). None of the lines reacted against recipient cells. To
date 11 allogeneic HSCT recipients have received 0.5-2x10 7 pVSTs/m2
without adverse events. Three patients were infused prophylactically while 8
were treated for one or more active infections. A single infusion successfully
controlled active infections associated with all our targeted viruses: CMV
(2 CR, 1 PR); EBV (5 CR); Adv (1 CR); HHV6 (2 CR) and BK (5 CR, 1 PR,
1 NR). Of note, all 3 patients with BK hemorrhagic cystitis had marked
S7
improvement/disappearance of hematuria post-mVSTs. Our only “mixed”
responder cleared EBV and HHV6 but not BK following the infusion of a line
that lacked specificity for this virus, likely reflecting the seronegative status of
the donor. Thus, infusion of mVSTs has been safe and clinically effective
against up to four simultaneous/sequential infections in a single HSCT
recipient. We are planning to assess the activity of “off the shelf” 3rd party
pVSTs for broader implementation.
3
IDENTIFYING THE FACTORS MODULATING THE EFFICACY
OF CAR-T CELL THERAPY
A Dolnikov1,2,3, G Klamer2,3, S Shen1,3, A Chitranjan3, H Carol3,2, R Lock2,3,
T O’Brien1,2,3
1
Cord and marrow transplant laboratory, Sydney Children, Randwick, New
South Wales, Australia, 2Faculty of medicine, UNSW, Randwick, New South
Wales, Australia, 3Children’s Cancer Institute Australia for Medical Research,
Randwick, New South Wales, Australia
T cells modified to express tumour-directed chimeric antigen receptors (CAR)
have shown clinical efficacy in early phase clinical trials. The major hurdle in
CAR-T cell therapy of cancer is inefficient expansion and rapid exhaustion of
infused CAR-T cells. Here we identified the factors that modify CAR-T cell
function using CARs targeting CD19+ B-cell leukaemia. CAR-T cell proliferation correlated with CD19 expression on target cells suggesting limited CAR-T
cell expansion in “low” antigen-expressing tumours. Increasing CAR expression
using epigenetic modification of CAR-T cells promoted anti-tumour activity of
CAR-T cells. We speculate that up-regulation of CAR expression may promote
antigen-specific activation of CAR-T cells and improve their activity in “low”
antigen-expressing tumours. Effector to target ratio appears to be the strongest
factor affecting CAR-T cell function. Low density of target cells promoted
effector/target conjugation, cytokine secretion and target cell killing but
reduced CAR-T expansion. This effect was also demonstrated in vivo using a
‘humanised’ hematochimeric mouse model where treatment of stem cellderived CD19+ B cells with autologous CAR-T cells mimics B cell depletion
observed in human patients. Single infusion of CAR-T cells used at high target
cell ratio resulted in rapid and selective elimination of human B cells. B cell
depletion, however, was not sustained and resulted in complete loss of CAR-T
cells demonstrating that a large number of host target cells triggers CAR-T
cell inactivation justifying the use of lymphodepleting conditioning prior to
CAR-T cell infusion. CAR-T cells used at low target cell ratio resulted in
sustained B cell depletion that did not required lymphodepletion prior to
CAR-T cell infusion. Our data provide the information that may define novel
strategies to enhance therapeutic efficacy of CAR-T cells.
4
EVALUATING MULTIVIRUS-SPECIFIC T CELLS FROM BOTH
CORD BLOOD AND BONE MARROW TRANSPLANT DONORS:
A PHASE 1 PERSPECTIVE
P Hanley1,2, R Krance2, MK Brenner2, AM Leen2, C Rooney2, E Shpall3,
H Heslop2, C Bollard1,2
1
Program for Cell Enhancement and Technologies for Immunotherapy,
Children’s National Medical Center, Washington, District of Columbia,
United States, 2Center for Cell and Gene Therapy, Baylor College of
Medicine, Houston, Texas, United States, 3Stem Cell Transplantation, MD
Anderson Cancer Center, Houston, Texas, United States
CMV, EBV and adenovirus are problematic in patients after stem cell (SCT)
and cord blood transplantation (CBT) and are associated with morbidity and
mortality. Deficiencies in conventional therapeutics have increased interest in
an immunotherapeutic approach to viral disorders. We have developed 2
strategies to grow multivirus-specific donor-derived T-cells (mCTL) from
peripheral blood (PB) and naive cord blood (CB). Using an adenoviral-vector
expressing CMVpp65 to modify monocytes, DC and EBV-LCL we generated
a single culture of mCTL (n¼34). PB mCTL(Mean SFC:adeno:86, EBV:183,
CMV:648) had more spot forming cells (SFC) than CB(adeno:83, EBV:117,
CMV:36) but both contained cells specific for at least 1 virus. We infused 25
patients with PB mCTL and 9 patients with CB mCTL. Patients received
CTL infusions from 35-164days(median 84) post transplant at a median of
5x10e7 cells/m2 with no toxicity or GvHD >grade II. We observed up to a
5-fold increase in CMV- and EBV-specific T-cells by 4weeks post-CTL as
measured by IFN-g ELISPOT assay. 26 viral reactivations were observed in
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S8
Oral Abstracts
patients before or immediately after mCTL infusion. In the absence of conventional therapy, 8 of the 11 patients with CMV infection became negative for
CMV in the blood within 7d of mCTL infusion, with a coinciding rise in
CMV-specific CTL in PB. Each of 8 patients with high EBV loads cleared
their virus, as did 7 of 7 patients with adenoviral infections/disease. Overall the
response rate in both groups was 88%. This study demonstrates that mCTL
derived from the PB of seropositive donors as well as the CB of virus naive
donors expand in vivo and are active against multiple viruses. Furthermore, by
restoring immunity to multiple viruses simultaneously, the need for continued
prophylaxis with pharmacotherapy is eliminated, thus, improving the efficiency
and cost effectiveness of protecting SCT and CBT recipients from these
potentially lethal viruses.
5
ANTI-CD123 CHIMERIC ANTIGEN RECEPTOR REDIRECTED
T CELLS FOR RELAPSED B-CELL ACUTE LYMPHOBLASTIC
LEUKEMIA
M Ruella1, O Shestova1, S Kenderian1, D Barrett2, S Grupp2, J Scholler1,
S Lacey1, M Kalos1, CH June1, S Gill1
1
Pathology and Laboratory Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania, United States, 2Division of Oncology, The
Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States
Figure 2A. Comparison of in vitro anti-tumor activity of CART123
and CART19.
Relapsing/refractory (R/R) B-cell Acute Lymphoblastic Leukemia (ALL) is
associated with a poor prognosis. We have previously shown that anti CD19
Chimeric Antigen Receptor T cells (CART19) induce significant responses in
this population. However, occasional CD19-ve relapses have occurred, likely
due to selective pressure from CART19 cells. Hence, CAR-based therapies
against additional antigens may be useful in the treatment of B-ALL. Twenty
R/R ALL samples, including two CD19-ve relapses, were screened for 30
potential secondary targets using a custom Quantigene RNA panel (Affymetrix)
and results were validated by flow cytometry. CD123 was amongst the most
highly and homogeneously expressed, present in >60% of blasts in 16/20 R/RALL including 2/2 CD19-ve relapses (Figure 1). We utilized the anti-CD123
CAR T cells co-stimulated via 4-1BB (CART123) that we had previously
Figure 2B. In vitro anti-tumor activity of CART19 and CART123
against ALL.
generated for the treatment of acute myeloid leukemia (AML) (Gill et al, ISCT
2013) and performed in vitro comparisons between CART123 and CART19.
These investigations revealed similar proliferation, degranulation, cytokine
production and cytotoxicity (Figure 2 A) and suggested that CART123 could
be used to treat B-ALL. We therefore injected NSG mice with 0.5-2x106 cells
of the CD19+ve CD123dim +ve B-ALL cell line Nalm6, and treated them with
CART19, CART123 or control T cells (1x106 each). As expected, mice treated
with control T cells succumbed quickly to disease. Mice treated with CART19
experienced enhanced survival (*).Mice treated with CART123 had improved
survival (*) compared with control T cell treatment, and intermediate between
control and CART19 (Figure 2 B). Hence, CART123 may represent an
additional approach to treating CD19+ve malignancy.
6
PRODUCTION OF ENDOTHELIAL NO-SYNTHASE GENEENHANCED AUTOLOGOUS PROGENITOR CELL THERAPIES
FOR CARDIO-PULMONARY DISEASES
DW Courtman, L Comanita, DJ Stewart
Regenerative Medicine, Ottawa Hospital Research Institute, Ottawa, Ontario,
Canada
Figure 1. CD123 expression on ALL blasts at baseline and at
CD19-ve relapse.
Endothelial progenitor cells (EPCs) derived from the circulation hold promise
for the treatment of cardiovascular diseases. Yet preclinical studies suggest that
patient derived cells are less effective stimulators of vascular repair. We have
therefore pioneered the development of gene enhanced autologous cell based
treatments for pulmonary hypertension and myocardial ischemia. Our first-inhuman clinical trial (PHACeT, Pulmonary Hypertension: Assessment of Cell
Therapy, NCT00469027) was a dose escalation safety trial using peripheral
blood derived early outgrowth EPCs transiently transfected via electroporation
with a human eNOS plasmid and delivered directly to the pulmonary
circulation. We demonstrated that administration of eNOS-transfected EPCs
to patients with stable, severe PAH was well tolerated, and resulted in a trend
towards short-term hemodynamic improvement. Although, there was no long
term sustained hemodynamic improvement in this small (7 patient), uncontrolled trial; there were significant increases in 6 minute walk time seen at
both 1 month, and persisting to 3and 6 months post cell based gene therapy.
To apply our therapy to cardiac patients we developed a non-mobilized
automated apheresis procedure combined with GMP manufacturing in
environmentally controlled isolator units and in addition adopted a cationic
polymer (manose modified linear polyethylenimine) for eNOS transfection to
limit cellular manipulations. Enhanced eNOS expression was verified by
Western Blotting and cell identity by flow cytometry. With these
manufacturing modifications in place a placebo controlled randomized trial
(Enhanced Angiogenic Cell Therapy-Acute Myocardial Infraction trial,
NCT00936819) testing the role of eNOS transfection in autologous EPCs
was initiated with 4 patients treated to date. Our early results suggest that
eNOS transfected autologous EPCs are safe and potentially effective treatments for cardiovascular disorders.
7
GENERATION OF DISEASE-SPECIFIC INDUCED PLURIPOTENT
STEM CELLS FROM HUMAN FETAL EXTRA-EMBRYONIC
TISSUES
P Spitalieri, V Talarico, A Luchetti, F Brancati, A Botta, G Novelli,
F Sangiuolo
Biomedicine and Prevention, University of Rome “Tor Vergata,” Rome, Italy
The generation of induced pluripotent stem cells (iPSCs) is a innovative
personalized-regenerative technology, which can transform own-self cells into
embryonic stem elike cells, which have regarded as a promising candidate for
cell-based therapy, as well as an ideal target for disease modeling and drug
testing and drug discovery, thus enabling researchers to undertake studies for
treating diseases. The objective of the present study was to reprogramming
patient-specific fetal cells deriving from prenatal diagnosis for several genetic
disorder as Cystic Fibrosis (FC), Myotonic Dystrophy (DM1), b-Thalassemia
(b-Thal), Spinal Muscolar Atrophy (SMA1), Lymphema-Distichiasis Syndrome (FOXC2) and healthy cells. The cell type used for create iPSCs can
significantly influence the reprogramming efficiency and kinetics. Here, we
show that amniotic fluid (LA) and chorionic villus sampling (CVS) represent
an ideal cell resource for rapid and efficient generation of human iPSCs.
The reprogramming were done using a polycistronic lentiviral vector
(hSTEMCCA-loxP) encoding Oct4, Sox2, Klf4 and c-Myc genes necessary to
cell reprogramming. Moreover loxP sites can be excised with Cre recombinase.
Stem cells specific morphological, molecular and immunocytochemical
markers (ALP; OCT4; SSEA4; TRA1-60;TRA1-81) confirmed the successful
reprogramming. Additionally, we evaluated their ability to differentiate into
the three embryonic germ layers (ecto, endo and mesoderm) by immunocytochemical characterization and their ability to in vivo form teratomas. To
date, this represents the first example of iPS cells derived from a very early
extra-embryonic fetal tissues like chorionic villi (hIPS-CVS). These data
suggest that hIPS-CVS/LA can be considered a valid cell model to accomplish pathogenesis studies and possibly represent a valid tool for future
therapeutic applications.
treat HBV-associated liver cancer. We designed a chimeric antigen receptor
(CAR) that is composed of a single chain antibody fragment binding to HBsAg
and CD28 / CD3z signaling domains. This study aimed to proof feasibility of
this approach in vivo addressing the following challenges: (i) T cell-depletion to
generate space for cell engraftment in chronic virus carriers is too perilous,
(ii) viral antigens circulating in high amounts may inactivate transferred T cells
or (iii) trigger uncontrolled immune damage.
Methods: Primary murine CD8+ T cells were isolated, stimulated using an
optimized protocol and grafted with CARs by retroviral transduction. A CAR
that binds HBV envelope proteins and transfers activation signals to the T cell
was compared to a control CAR without a proper signaling domain and a CAR
not binding HBV proteins.
Results: CD8+ T cells engineered to express an HBV-specific CAR, which
recognizes HBV envelope proteins of various subtypes on infected hepatocytes,
were able to engraft and expand in immune competent HBV transgenic mice.
Following adoptive transfer CAR-grafted T cells targeted the liver, remained
functional in vivo, rapidly and efficiently controlled HBV replication while
causing only transient liver damage. The large amount of circulating viral
antigens neither impaired nor over-activated the transferred T cells. Conclusion: HBV-specific cell therapy with CAR-engineered T cells bears the
potential to treat chronic hepatitis B and HBV-associated hepatocellular carcinoma irrespective of the patient’s individual HLA-type.
9
BONE MARROW MONONUCLEAR CELLS FOR ISCHEMIC
CARDIAC FAILURE - A PROSPECTIVE, CONTROLLED,
RANDOMIZED,
DOUBLE-BLINDED
STUDY
OF
CELL
TRANSPLANTATION COMBINED WITH CORONARY BYPASS
SURGERY
T Pätilä1, M Lehtinen1, A Vento1, J Schildt2, J Sinisalo3, M Laine3,
P Hämmäinen1, A Nihtinen4, R Alitalo5, P Nikkinen2, A Ahonen2,
M Holmström6, K Lauerma6, R Pöyhiä7, M Kupari3, E Kankuri8, A Harjula1,8
1
Department of Cardiothoracic Surgery, Helsinki University Central Hospital,
Helsinki, Finland, 2Department of Clinical Physiology, Helsinki University
Central Hospital, Helsinki, Finland, 3Department of Medicine, Helsinki
University Central Hospital, Helsinki, Finland, 4Department of Hematology,
Helsinki University Central Hospital, Helsinki, Finland, 5Stem Cell
Laboratory, HUSLAB, Helsinki, Finland, 6Department of Radiology, Helsinki
University Central Hospital, Helsinki, Finland, 7Department of
Anesthesiology and Intensive Care, Helsinki University Central Hospital,
Helsinki, Finland, 8Institute of Biomedicine, University of Helsinki, Helsinki,
Finland
Objectives: Worldwide, millions of people are killed every year by ischemic
heart failure. Bone marrow mononuclear cell (BMMC) transplantation is a
8
T CELLS REDIRECTED BY A CHIMERIC ANTIGEN RECEPTOR
RECOGNIZING HBSAG EFFICIENTLY CONTROL HBV IN
VIVO IN TRANSGENIC MICE
K Krebs1, N Böttinger1, L Huang2, M Chmielewski3, W Uckert4, H Abken3,
M Heikenwälder1, P Knolle2, U Protzer1
1
Institute of Virology, Technische Universität München / Helmholtz Center
München, München, Germany, 2Institute of Molecular Medicine and
Experimental Immunology, University of Bonn, Bonn, Germany, 3Department of
Internal Medicine I, University Hospital Cologne, Cologne, Germany, 4Molecular
Cell Biology and Gene Therapy, Max Delbrück Center for Molecular Medicine,
Berlin, Germany
Background & aims: Current antivirals suppress HBV but do not clear the
infection. For virus clearance strong effector T cell responses are needed,
which are sparse in chronically infected individuals. Cell therapy using T cells
redirected by HBV-specific receptors may clear HBV and help to prevent and
S9
Scar size at injection sites preoperatively and after follow-up.
S10
Oral Abstracts
promising new method to treat heart failure but results from clinical trials have
been mixed. Here, we present results from our study combining BMMC
therapy with coronary bypass surgery (CABG).
Materials and methods: First, we enrolled 107 ischemic heart failure
patients scheduled for CABG. These patients went through a 4- to 12week period with optimized drug therapy. If left ventricular ejection
fraction (LVEF) remained 45%, a patient was eligible for the actual
study. In a randomized, double-blind manner, the still eligible 39 patients
received intramyocardial injections of BMMCs or vehicle intraoperatively
into the infarction and border area during CABG. We measured global and
segmental LV function and scar size by magnetic resonance imaging
(MRI), and viability by positron emission tomography (PET) and singlephoton emission tomography (SPECT), preoperatively and after 1-year
follow-up.
Results: LVEF, the primary end point measure, improved by a median of
5.6% among controls (IQR 0.2 to 10.1) and by 4.8% in the BMMC patients
(IQR -0.5 to 8.2) (P¼0.59). Wall thickening in injected segments rose by a
median of 4.5% in the control group (IQR -18.1 to 23.9) and by 5.5% in
the BMMC patients (IQR -6.6 to 26.5) (P¼0.68). Viability by PET and
SPECT did not differ between the groups. Myocardial scar volume by MRI
in injected segments rose by a median of 5.1% in the control group (IQR
-3.3 to 10.8) but fell by 13.1% in the BMMC group (IQR -21.4 to -6.5)
(P¼0.0002).
Conclusions: As an adjunct to CABG, BMMC therapy failed to affect
global or local LV systolic function or viability by PET and SPECT
during 1-year follow-up. Interestingly, however, it affected one essential
prognostic marker: myocardial scar size was significantly reduced by
BMMC therapy. Long-term studies are necessary to elucidate this finding’s permanence.
10
EXOSOMES SECRETED BY HUMAN CARDIAC PROGENITORS
CONTAIN MICRO-RNA WITH CARDIOPROTECTIVE AND
PRO-ANGIOGENIC ACTIVITIES
E Cervio1, L Barile1, V Lionetti2, M Matteucci2, M Gherghiceanu3,
L Popescu3, T Torre1, F Siclari1, T Moccetti1, G Vassalli1
1
Cardiocentro Ticino, Lugano, Switzerland, 2Istituto Superiore Sant’Anna,
Pisa, Italy, 3“Victor Babes” National Institute of Pathology, Bucharest,
Romania
Background: Secreted factors account, in part, for beneficial effects of
transplanted cells into infarcted hearts. Injected cells activate endogenous
regenerative processes through paracrine mechanisms including microRNAs
(miRNAs). Exosomes (Exo) act as intercellular carriers of proteins and
miRNAs. We analyzed the miRNA transcriptional profile of Exo from human cardiac progenitor cells (Exo-CPC) in comparison with Exo from
normal human dermal fibroblasts (Exo-F). We studied the effect of hypoxia
on miRNAs in Exo-CPC, as well as the cardioprotective effects of selected
miRNAs.
Methods and Results: CPCs were derived from atrial explants of patients
who underwent heart valve surgery. Exo was characterized ultrastructurally
by electron microscopy. They were 46.2+/-16.9 nm in size and expressed
Exo markers such as CD9, CD63 and CD81. Exo-CPC, but not Exo-F,
inhibited cardiomyocyte apoptosis while also stimulating angiogenesis by
human endothelial cells in vitro. When injected into rat infarcted hearts,
Exo-CPC, but not Exo-F, reduced infarct scar and improved cardiac function in vivo. miRNA transcriptional profiling identified miR-146a-3p, miR181a, miR-132, miR-210-3p, miR- 181b and miR-323-5p among the most
highly upregulated miRNAs in Exo-CPC compared to Exo-F. Of these
miRNAs, miR-323-5p was further upregulated in Exo isolated from CPCs
exposed to hypoxia in vitro. In mouse HL-1 cardiomoycytes subjected to
hypoxia and reoxygenation injury, cell viability was significantly increased
after transfection with pre-miR-323-5p compared with controls. On the
other hand, miR-132 has pro-angiogenic effects.
Conclusion: Compared with Exo-F, Exo-CPC is markedly enriched for
cardioprotective and pro-angiogenic miRNAs. Hypoxia further increases the
miR-323-5p content in Exo isolated from CPCs. In gain-of-function experiments, forced overexpression of this miRNA mediated cardioprotection against
hypoxia/reoxygenation injury.
11
PATIENT PARTICIPATION IN REGULATORY DECISIONS
REGARDING REGENERATIVE MEDICINE
B von Tigerstrom
College of Law, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
Regulatory agencies responsible for ensuring the safety, efficacy, and
quality of medical products for human use are faced with many challenges
in appropriately regulating novel medical technologies, including emerging
regenerative medicine treatments such as stem cell-based therapies. These
agencies have the difficult task of assessing whether the balance of risks
and benefits associated with a new treatment justifies approval, in the
context of substantial uncertainty. In making these decisions, they are
increasingly asked to take into account the perspectives of patients and
patient advocacy organizations. These groups may have important information about the risks and benefits of a new treatment and perspectives on
how they should be balanced, but their participation in regulatory decision
making raises many questions. What type of input should be sought or
received from patients and at what stages in the regulatory process? Who
should speak for patients and how should the diversity of interests and
perspectives among patients be addressed in processes for consultation or
input? In what ways should information and views from patients be used
in regulatory decisions and how much weight should they be given? Are
there special considerations regarding patient participation in the context
of novel forms of treatment, such as stem cell-based therapies, or particular types of conditions, such as rare diseases? This paper examines
existing and proposed models for patient input into regulatory decision
making, seeking to determine how different systems have answered the
questions above. It will identify the various approaches used and their
advantages and disadvantages, with a view to formulating a set of recommendations or best practices for regulatory agencies and patient organizations to consider.
12
TECHNOLOGY POLICY AND INDUSTRY GROWTH: THE
POWER OF LOCAL CLUSTER
N Kishi
Yokohama National University, Yokohama city, Japan
This research focuses on technology policy for the regenerative medicine industry in Germany and Japan, and it suggests the effectiveness of Germany
policy, which transfers authorization to the local government. Germany and
Japan have common points. Both have restricted dealing with ES cell from
ethical viewpoint and have firms with high capability of manufacturing, which
provide a strong base for them to build competitive advantages in cell
culturing equipment. The focus of technology policy in the regenerative
medicine industry is categorized into two types: the first focuses on supporting firms which provide final products such as iPS cell. The second focuses on firms which provide the other production process products, such as
cell culturing equipment. Common situation shows that German and Japanese firms should emphasize on supplying the latter products. However, while
there are only two product marketed in Japan, Germany has already marketed
29 products. This research explains the cause by the different technology
policy. The difference in their technology policy is that the power to
implement the policy in Germany is mostly authorized to the local government more than in Japan. Local government has easier accesses to the small
and medium size firms in the region and has built a network with the local
research institutes. That is why they are superior to finding the appropriate
support for the local firms needs and for making a tie between firms and
research institutions. The result is that there are some local clusters with
definite characteristics in Germany. On the other hand, in Japan, the most
authorization to implement them is still in the central government. In the
dawn of the industry, interdisciplinary knowledge is needed to overcome high
uncertainty. In Germany, diversity of the authorized local clusters realizes
that.
13
A HIGHLY EFFICIENT CULTURE METHOD FOR GROWTH
AND
DETECTION
OF
UNDIFFERENTIATED
HUMAN
PLURIPOTENT STEM CELLS PRESENT AS IMPURITIES IN
CELL-PROCESSED THERAPEUTIC PRODUCTS
K Tano1,2, S Yasuda2, A Umezawa1, Y Sato2
1
Reproductive Biology, National Research Institute for Child Health and
Development, Tokyo, Japan, 2Division of Cellular & Gene Therapy Products,
National Institute of Health Sciences, Tokyo, Japan
Human pluripotent stem cells (hPSCs), i.e. induced pluripotent stem cells
(hiPSCs) and embryonic stem cells, have properties of indefinite proliferation
and pluripotency, offering the possibility of renewable sources of various types of
cells for production of cell-processed therapeutic products (CTPs). For the
clinical application of CTPs derived from hPSCs, quality assessment is critical to
ensure their safety and efficacy. The presence of residual undifferentiated hPSCs
is one of concerned quality issues associated with tumorigencity of hPSCsderived CTPs. Therefore, simple, quantitative and highly sensitive methods
are necessary to detect a small amount of undifferentiated hPSCs present as
impurities in hPSC-derived CTPs. In the present study, we developed a novel
method for detection of undifferentiated hiPSCs by amplification using a
combination of an extracellular matrix component and a defined xeno-free
media. The new culture system allowed robust proliferation of hiPSCs
dissociated into single cells without apoptosis, whereas the dissociated hiPSCs
easily underwent apoptosis under the conventional culture condition. We next
spiked dissociated hiPSCs into primary human somatic cells and examined
whether our culture system is applicable to detection of undifferentiated
hPSCs in CTPs. As an example of the somatic cells, we employed human
mesenchymal stem cells (hMSCs), because “off-the-shelf” hMSCs derived
from hPSCs are one of promising CTPs. Our new culture system detected as
low as 0.001% hiPSCs, which were spiked into hMSCs, as hiPSC colonies
within a week. These results suggest that our culture system is useful for
sensitive and quantitative detection of residual undifferentiated hPSCs in
CTPs and contributes to the quality control of hPSC-derived products during
manufacturing processes.
14
GLOBAL GMP e A COMPARABILITY STUDY TO LINK GOOD
MANUFACTURING PRACTICE STANDARDS FOR WORLD
WIDE COMPLIANCE WITHIN THE CELLULAR THERAPY
INDUSTRY
RH Bell
Qgen, QIMR Berghofer Medical Research Institute, Brisbane, Queensland,
Australia
GMP Standards ensure product safety within cellular therapy fields, a fully
harmonised global standard remains elusive. There is an emerging need for
standardisation with respect to GMP. The difference between individual
country’s regulatory GMP requirements, prohibits manufacturers from
complying with GMP Standards globally. Manufacturers cannot actively promote cellular therapy treatments in other countries with higher GMP Standards. The cost to implement higher GMP Standards is prohibitive and
lengthy. Reducing the number of cellular therapy treatments utilized overseas
restricts the potential for global advancement in cellular therapies. A global
GMP Standard capable of meeting equivalent governmental regulatory requirements worldwide is proposed allowing manufacturers to utilize their
cellular therapy treatments universally. Improving potential for global GMP
Standard harmonisation a comparison between the GMP Standards enforced
by Governmental Regulatory Bodies is proposed. The process of determining
major differences between the PIC/s Guide to Good Manufacturing Practice
for Medicinal Products Annex 13; Australian Code of GMP for Human Blood
and Blood Components, Human Tissues and Human Cellular Therapies Australian Therapeutic Goods Administration (TGA); Clinical Trial Handbook and Clinical Practice Guidelines e TGA; Australian Regulatory
Guidelines for Biologicals (TGA); Unapproved Therapeutic Goods Guideline
TGA; In Vitro Diagnostic Medical Devices TGA; Australian International
Conference on Harmonisation Guidelines (ICH); Committee for Proprietary
Medicinal Products (CPMP); European Medicines Agency (EMEA) and
Federal Drug Administration Guidelines (FDA), is the focus of this study.
These observations conclude that there are differences between the Standards,
GMP Standards are similar and a robust global Standard harmonisation is
achievable if have one common goal e product safely produced in accordance
with standardised good manufacturing practice.
S11
15
JACIE ACCREDITATION STATUS AND OUTCOME AFTER
HEMATOPOIETIC STEM CELL TRANSPLANTATION
C Chabannon1, A Gratwohl2, R Brand3, E McGrath4, A van Biezen3,
A Sureda5, H Baldomero6, P Ljungman7, J Apperley8, A Rambaldi9
1
Centre de Therapie Cellulaire / Cell Therapy Facility, Institut PaoliCalmettes & Inserm CBT-510, Marseille cedex 9, France, 2Hematology,
Medical Faculty, University of Basel, Basel, Switzerland, 3Department of
Medical Statistics and Bioinformatics, Leiden University Medical Centre,
Leiden, Netherlands, 4JACIE Accreditation Office, EBMT, Barcelona,
Spain, 5Department of Haematology, Addenbrookes Hospital, Cambridge,
United Kingdom, 6EBMT Activity Survey Office, EBMT. University of
Basel, Basel, Switzerland, 7Department of Hematology, Karolinska
University Hospital and Dept. of Medicine Huddinge, Karolinska
Institutet, Stockholm, Sweden, 8Haematology, Hammersmith Hospital,
London, United Kingdom, 9Hematology, University Hospital, Bergamo,
Italy
Increasing resources are devoted to quality management systems (QMS) in
healthcare. Studies searching for an impact on clinical outcome remain scarce.
Earlier data indicated a stepwise improvement in outcome after allogeneic
HSCT with each phase of the JACIE accreditation process. We tested whether
working towards achieving JACIE acreditation accelerates improvement in
outcome over calendar time. In a retrospective cohort analysis of 41,623 and
66,281 recipients of allogeneic or autologous HSCT from 1999 to 2006, we
looked for an association of outcome with the transplant team accreditation
status at time of transplant. Primary outcome was overall survival at 72 months,
analyzed by an extended COX proportional hazards model. Confounders,
cluster or stratification variables included disease type, EBMT risk score, age,
donor type, conditioning, calendar year, centre, centre size, and Gross National
Income per capita (GNI/cap) of the center’s country. Overall survival, nonrelapse mortality and relapse incidence improved stepwise from baseline (N¼
33,753; HR¼1) over the preparatory (N¼ 4,890; HR 0.90; 0.85 to 0.96) and
application period (N¼ 1,922; HR 0.87; 0.80 to 0.95) to the accreditation
period (N¼ 1,058; HR 0.87; 0.77 to 0.98) after an allogeneic HSCT, and were
better at 72 months for the 49, 459 patients transplanted in the 162 JACIEaccredited centers compared to the 58,445 patients in 423 unaccredited centers
in November 2012. Overall mortality declined over the 14 years observation
period by a factor of 0.67 per 10 years (HR: 0.67; 0.62-0.73). Improvement was
significantly faster in accredited than in non-accredited centers. No effect of
JACIE accreditation was seen for autologous HSCT. Patients in larger centers
had a significantly better overall survival, as did patients from countries with
higher GNI/cap. Data show the complex association of macroeconomic factors
with outcome after HSCT. They support the use of a QMS for other complex
medical procedures.
16
MESENCHYMAL STROMAL CELLS ENHANCE HEMATOPOIETIC
ENGRAFTMENT IN A MOUSE MODEL OF AUTOLOGOUS
TRANSPLANTATION WITH HIGH RISK OF ENGRAFTMENT
FAILURE
M Fernández, RM Yañez, R Sánchez, J Segovia, JA Bueren, M Lamana
Hematopoietic Innovative Therapies Division, CIEMAT /CIBER-ER /
IIS-FJimenez Díaz, Madrid, Spain
Human mesenchymal stromal cells (MSC) co-transplanted with hematopoietic
stem cells (HSC) reduce the risk of graft failure in patients subjected to haploidentical or unrelated donor HSC allogeneic transplants. However, there are
not clear data regarding whether the engraftment facilitating role of MSCs is
also maintained in an autologous transplantation setting. This is of particular
importance for many HSC gene therapy clinical trials in which few numbers of
HSC are available. Using a congenic HSC mouse transplantation model
(Ly5.1/Ly5.2) in sublethally irradiated recipients (5 Gy), we have observed that
the co-infusion of low numbers of congenic HSCs (1,500 LSK cells/mouse)
with 106 adipose tissue-derived MSC/recipient (mAd-MSCs) significantly
improved engraftment of transplanted recipients with donor cells. With the
aim of approaching to a more clinically relevant model, we have conducted
similar experiments using Fanconi anemia A (Fanca-/-) recipients. While
transplants of >1,500 WT LSK donor cells resulted in donor engraftments
(considered as 10% of donor cells in PB) in all WT recipients, these numbers
S12
Oral Abstracts
resulted in a graft failure in 25-35% of FA recipients that received the same
conditioning regimen of 5 Gy. To guarantee the engraftment of all FA recipients the infusion of at least 5,000 LSK cells per FA recipient was required,
reinforcing the idea of a defective supportive hematopoietic stroma as a result
of the FA mutation. Significantly, when 6.105 mAd-MSCs were co-infused
with 1,500 WT LSK cells in FA recipients, all the transplanted animals showed
significant hematopoietic engraftments, reaching 50% and 100% of donor cells
in PB at 4 and 8 weeks after HSC transplant, respectively. Taken together, our
results demonstrate the hematopoietic facilitating engraftment potential of AdMSCs, not only in an allogeneic context, but also in a clinically relevant model
of autologous transplantation.
Results: 8 patients (4 female, aged 63.5 (57-75) years) with median (IQR)
FVC 60(52.5-74.5)%, DLCO 34.5 (29.5-40)%, 6MWD 460(375.5-540)m and
CT fibrosis score 14.8(12.9-17.05)% were treated. Both dose schedules were
well tolerated with only minor and transient acute adverse effects. MSC
infusion was associated with a transient (1%) fall in SaO2 after 15 minutes, but
no changes in haemodynamics. There was a transient improvement in 6WMD
at 3 months. At 6 months FVC, DLCO, 6MWD and CT fibrosis score were all
unchanged compared to baseline (p>0.05 for all measures). There was no
evidence of worsening fibrosis. Conclusion: Intravenous MSC therapy is
feasible and is associated with a satisfactory short-term safety profile in patients
with moderately severe IPF.
17
A PHASE 1B STUDY OF MESENCHYMAL STROMAL CELL
THERAPY FOR IDIOPATHIC PULMONARY FIBROSIS
DC Chambers1,2, D Enever1, N Ilic3, L Sparks1, J Ayres1, ST Yerkovich1,2,
D Khalil4, K Atkinson5,6, PM Hopkins1,2
1
Queensland Lung Transplant Service, The Prince Charles Hospital, Brisbane,
Queensland, Australia, 2School of Medicine, The University of Queensland,
Brisbane, Queensland, Australia, 3Mater Health Services, Mater Hospitals,
Brisbane, Queensland, Australia, 4Mater Research Insitute, Translational
Research Insitute, Brisbane, Queensland, Australia, 5Stem Cell Therapies
Laboratory, Translational Research Insitute, Brisbane, Queensland,
Australia, 6UQ Centre for Clinical Research, The University of Queensland,
Brisbane, Queensland, Australia
18
MIF-CXCR4 AS A NOVEL AXIS FOR MESENCHYMAL STEM CELL
RECRUITMENT TO TUMORS IN VIVO
S Lourenco1, VH Teixeira1, T Kalber1, A Floto2, S Janes1
1
Division of Medicine - Lungs for Living Research Center, University
College London, London, United Kingdom, 2Respiratory
Medicine, Cambridge Institute for Medical Research, Cambridge,
United Kingdom
Introduction: Idiopathic pulmonary fibrosis (IPF) is a lethal degenerative
disease characterised by fibrosis following failed epithelial repair. Mesenchymal
stromal cells (MSC), a key component of the stem cell niche in bone marrow
and possibly other organs including lung, have been shown to enhance
epithelial repair and are effective in preclinical models of pulmonary fibrosis,
but may be pro-fibrotic in some circumstances. The aim of this study was to
confirm the feasibility and safety, particularly with respect to adverse acute
haemodynamic effects and pro-fibrosis, of intravenous MSC therapy in humans
with IPF.
Methods: In this single centre, non-randomised, dose escalation phase 1b
trial (NCT01385644), patients with moderately severe IPF (DLCO>25%
and FVC>50%) received either 1 (n¼4) or 2*106 (n¼4) placenta-derived
MSC/kg via a peripheral vein from an unrelated donor and were followed
for 6 months with lung function, 6 minute walk distance (6WMD) and CT
chest.
Lung function, 6MWD and lung fibrosis score at baseline, 1, 3 and
6 months after intravenous infusion of MSC. While there was a
marginal fall in FVC at 1 and 3 months, lung function, 6MWD and
fibrosis score were no different from baseline at 6 months (p>0.05
for all measures, median (IQR)). *p<0.05 vs pre-infusion. y1 patient was unable to complete the 6 month 6WMD.
Mesenchymal stromal cells (MSCs) are inherently tumor-homing and can be
isolated, expanded and transduced, making them viable candidates for cell
therapy. This tumor-tropism has been used to deliver anti-cancer therapies to
various tumor models. However, one of the limitations is the high number of
cells required for engraftment and therapeutic benefit. Several studies identified different cytokines, growth factors as playing a role in this mechanism.
However the early players attracting MSCs to tumor sites and the exact
mechanism remain disputed and unclarified. In this study we sought to
discover which molecules are the key effectors of MSC tumor homing in vivo.
Using microarray and cytokine array data, in vitro 2D/3D cell culture assays
as well as in vivo model, we discover MIF as a novel key director of MSC
migration and invasion towards tumor cells (mainly via CXCR4). We
demonstrate physical interaction between MIF-CXCR4, and show downstream activation of the MAPK pathway (ERK and JNK branches) which
appears necessary for tumor homing (shown by blocking experiments).
MIF or CXCR4 knockdown impair MSC motility in a 3D spheroid
model. MDAMB231 transfected with luciferase strawberry marker
(LS) (top and bottom panels) or with MIFshRNAGFP (middle
panel) were grown as spheroids. MSCs were transfected either with
LS (middle panel) or transduced with CXCR4shRNA GFP (bottom panel) or non silencing (NS) GFP (top panel). The pictures are
representative of the final scan from videos. Scale bars: 200mM.
Additionally, we show that MIF is able to upregulate the expression of cytokines described as chemoattractants for MSC (IL8, IL6 and CCL2), reinforcing the role of this molecule as the first trigger for several downstream
signalling pathways. Importantly we show that knock down (using lentiviral
shRNAs) of either CXCR4 (in MSCs) or MIF (in cancer cells) decreases
significantly MSC homing to tumors in an in vivo pulmonary metastasis
model. Interestingly, MIF has been shown by others to be over-expressed in
a large variety of human cancers and closely correlates with tumor aggressiveness and metastatic potential. Therefore, MSC homing to tumors triggered by MIF could be a general mechanism for a variety of cancers. This
improved understanding of MSC tumor tropism will further enable development of novel cellular therapies for cancers by increasing the homing
potential of these cells.
19
NOD2 ACTIVATION PROMOTES ANTI-INFLAMMATORY
ACTIVITY OF HUMAN MESENCHYMAL STEM CELLS
AGAINST INFLAMMATORY BOWEL DISEASE
H Kim, T Shin, B Lee, K Kang
College of veterinary medicine, Seoul National University, Seoul, Korea,
Republic of
Decreased levels or function of nucleotide-binding oligomerization domain
2 (NOD2) are associated with Crohn’s disease. NOD2 regulates intestinal
inflammation, and is also expressed by human umbilical cord blood-derived
mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation.
We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. Colitis was induced in mice by
administration of dextran sulfate sodium or trinitrobenzene sulfonic acid.
Mice were then given intraperitoneal injections of NOD2-activated hUCBMSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. Administration of hUCB-MSCs reduced the severity of colitis
in mice. The anti-inflammatory effects of hUCB-MSCs were greatly
increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP).
Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)10
and infiltration by T regulatory (Treg) cells, and reduced production of
inflammatory cytokines. Proliferation of mononuclear cells was significantly
inhibited by co-culture with hUCB-MSCs that had been stimulated with
MDP. MDP induced prolonged production of prostaglandin (PG)E2 in
hUCB-MSCs via the NOD2eRIP2 pathway, which suppressed proliferation
of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs
in response to MDP increased production of IL10 and Treg cells. In mice,
production of PGE2 by MSCs and subsequent production of IL10 were
required to reduce the severity of colitis. Taken together, these results
indicate that activation of NOD2 is required for the ability of hUCB-MSCs
to reduce the severity of colitis in mice and that NOD2 signaling increases
the ability of these cells to suppress mononuclear cell proliferation by
inducing production of PGE2.
S13
20
TREATMENT OF STEROID RESISTANT GRADE II TO IV ACUTE
GVHD BY INFUSION OF MESENCHYMAL STROMAL CELLS
EXPANDED WITH PLATELET LYSATE - A PHASE I/II STUDY
L van der Wagen1,2, L te Boome1, C Mansilla2, C Lindemans3, M Cuijpers1,
K Westinga4, E Petersen1, E Spierings2, M Bierings3, J Boelens3,
N Wulffraat3, I Slaper-Cortenbach4, J Kuball1
1
Hematology, UMC Utrecht, Utrecht, Netherlands, 2Immunology, UMC
Utrecht, Utrecht, Netherlands, 3Pediatrics, BMT-unit, UMC Utrecht,
Utrecht, Netherlands, 4Clinical Pharmacy,Cell Therapy Facility, UMC
Utrecht, Utrecht, Netherlands
Despite major improvements in the last decade in the field of HSCT, steroidresistent acute graft versus host disease (aGVHD) remains a life-threatening
complication. In an open-label, non-randomized prospective phase I/II study
50 patients with steroid-refractory GVHD grade II-IV were treated with MSC.
Response rates, TRM and other adverse events were assessed for up to 12
months. Immunological changes after infusion of MSC were characterized in
vitro. Anti-viral and antileukemia responses of reactive T-cells were tested and
phenotypical changes in immune cells were followed up as were cytokines
implicated in GVHD. MSC production takes 22 days to expand from bone
marrow to P3, resulting in 59x10e6 MSC per 2-layer CellStack. Between
January 2009 and July 2012, 48 out of 50 patients included were eligible for
analysis (7 children,41 adults). Mean age was 44.9 years (1.3-68.9). Organs
involved were skin (52%), GI tract (88%) and liver (35%). Overall GVHD
grade was II for 12 (25%), III for 33 (69%), and IV for 3 (6%) patients. Mean
number of infusions were 3 (1e4). No severe side effects were observed upon
infusions. Median follow up was 5,0 months (0.3-46.5). Complete overall
response of aGVHD was observed in 24 patients (50%) after a median of 53
days (3e116 days). Overall survival was significantly improved in responders
when compared to non-responders (p <0.001). Patients who relapsed with
GVHD of the gut were again sensitive to steroids, or a second cycle of MSC
(one patient). Immunological monitoring shows that anti-viral and anti-leukemia
reactive T-cells are well preserved in all patients who responded to MSC
treatment. In addition we identified a combination of biomarkers that 2 weeks
after initiation of treatment predicts a complete resolution of GVHD, whilst this
usually becomes clinically apparent after months. Identified biomarkers predict
early an usually late clinical resolution of GVHD and thus might be useful to
early guide clinical decision making.
21
UMBILICAL CORD-DERIVED MESENCHYMAL STROMAL CELL
INFUSION IMPROVES BLOOD SUGAR CONTROL IN
PATIENTS WITH TYPE II DIABETES
S Chin1, Z Cheng2, K Then3, S Cheong4
1
Beverly Wilshire Medical Centre, Kuala Lumpur, Malaysia, 2Cytopeutics,
Cyberjaya, Selangor, Malaysia, 3Cryocord, Cyberjaya, Selangor,
Malaysia, 4Tunku Abdul Rahman University, Kajang, Selangor, Malaysia
Background: Diabetes is a systemic disease with end-organ complications
secondary to hyperglycaemia and tissue ischaemia. In Type II Diabetes Mellitus (T2DM), there is initially insulin resistance followed by insulin depletion
due to beta-islet cell dysfunction. We have previously demonstrated that
mesenchymal stromal cell (MSC) may be induced to become insulin-producing cells in-vitro. MSC may also alleviate beta-islet cell dysfunction and
improve skeletal muscle responsiveness to insulin. Therefore we postulate that
MSC could further improve glycaemic control when added to conventional
medical therapy.
Methods: We recruited 10 patients (mean age 63 years; 8 males) with
T2DM who have been taking three or more oral hypoglycaemic medications at
maximal dosage for at least 6 months. Co-morbidities include serious coronary
artery disease (n¼5), chronic renal disease (n¼4) and previous stroke (n¼2).
Patients with proliferative retinopathy were excluded. Blood samples were
collected at baseline, 3 months and 6 months after treatment. Patients received
umbilical cord-derived MSC intravenously.
Results: All patients showed improvement in blood sugar control as evidenced by fall of HbA1c at 3 months and 6 months compared to baseline
(Mean HbA1c 8.2% vs. 7.5% vs. 7.0%; ANOVA p<0.05). Five patients had at
least one diabetic medicine reduced or stopped at three months. Two patients
with renal disease showed significant improvement in serum creatinine level.
There were no adverse events.
S14
Oral Abstracts
Conclusions: MSC could potentially further improve glycaemic control in
patients on maximum oral hypoglycaemic medications. Several mechanisms are
possible including closer patient surveillance.
22
COMPLETION OF THE FIRST FDA PHASE 3 MULTICENTER
TRIAL OF ISLET TRANSPLANTATION IN TYPE 1 DIABETES
BY THE NIH CIT CONSORTIUM
C Ricordi2,1, B Hering6,1, N Bridges3,1, T Eggerman4,1, A Naji10,1,
A Posselt14,1, P Stock14,1, D Kaufman9,1, CP Larsen8,1, N Turgeon8,1,
J Oberholzer13,1, B Barbaro13,1, O Korsgren12,1, J Markmann11,1,
R Alejandro2,1, MR Rickels10,1, PA Senior7,1, X Luo15,1, X Zhang15,1,
M Bellin6,1, J Lei11,1, W Clarke5,1, L Hunsicker5,1, J Goldstein3,1,
C Czarniecki3,1, A Priore3,1, N Green4,1, A Shapiro7,1
1
NIH Clinical Islet Transplantation Consortium, National Institutes of
Health, Bethesda, Maryland, United States, 2Diabetes Research Institute and
Cell Transplant Center, University of Miami, Miami, Florida, United
States, 3NIAID, NIH, Bethesda, Maryland, United States, 4NIDDK, NIH,
Bethesda, Maryland, United States, 5Clinical Trials Statistical and Data
Management Center, University of Iowa, Iowa City, Iowa, United
States, 6University of Minnesota, Minneapolis, Minnesota, United
States, 7University of Alberta, Edmonton, Alberta, Canada, 8Emory University,
Atlanta, Georgia, United States, 9University of Wisconsin, Madison,
Wisconsin, United States, 10University of Pennsylvania, Philadelphia,
Pennsylvania, United States, 11MGH, Harvard University, Boston,
Massachusetts, United States, 12Uppsala University, Uppsala,
Sweden, 13University of Illinois, Chicago, Illinois, United States, 14University
of California at San Francisco, San Francisco, California, United
States, 15Northwestern University , Chicago, Illinois, United States
The Clinical Islet Transplantation (CIT) Consortium was established in 2005
by the NIH with the goals of advancing CIT through clinical trials, developing
standard procedures for islet manufacturing and patient care, and generating
the information needed for FDA licensure for islet products. In this FDA Phase
3 single-arm study (NCT #00434811), 8 centers and the CIT Data Coordinating Center evaluated the efficacy/safety of CIT for treatment of T1D in
adults with hypoglycemia unawareness and a history of severe hypoglycemic
episodes. The clinical study and islet manufacturing protocols were developed
by the CIT consortium and are available online (http://www.isletstudy.org).
Clinical protocols were approved by the NIH DSMB and local IRBs. Informed
consent was obtained from all participants. All serious adverse events were
reported to and reviewed regularly by the DSMB, the IRBs, and the FDA. 48
subjects received a total of 75 islet infusions, with 22 subjects (45.8%) receiving
1 infusion, 25 (52.1%) receiving 2 infusions, and 1 (2.0%) receiving 3 infusions.Total # IEQ transplanted per subject was 806,587290,340 (meanSD),
corresponding to 11,476.4 4,023.0 IEQ/kg. Induction immunosuppression
(IS) was with rabbit antithymocyte globulin (rATG) and peri-transplant etanercept, anticoagulation and antimicrobial prophylaxis. In those who received a
second islet infusion (>4000 IEQ/kg), basiliximab replaced rATG in the
induction IS. Maintenance IS included sirolimus and reduced-dose tacrolimus.
The primary endpoint was the proportion of subjects with an HbA1c <7.0% at
day 365 and free of severe hypoglycemic events from day 28 to day 365 inclusive following the first islet transplant. Key secondary endpoints included
also the proportion of insulin-independent subjects. Insulin usage dropped
dramatically after transplantation (Figure). Islet graft function and insulin independence was achieved by 94% and 52.1% of subjects at day 365 after the
first islet transplant.
23
REPEATED INTRA ARTICULAR INJECTION OF BONE
MARROW DERIVED MESENCHYMAL STEM CELL IN KNEE
OSTEOARTHRITIS: DOUBLE BLIND RANDOMIZED CLINICAL
TRIAL
N Aghdami1, M Ghorbani Liastani1, M Emadedin1, F Mohseni1, R Fazeli1,
R Moghadasali1, S Mardpour1, E Hosseini1, M Niknejadi2, V Azimian1,
N Jaroughi1, N Labibzadeh1, A Mirazimi Bafghi1
1
Regenerative Biomedicine at Cell Science Research Center, Royan Institute
for Stem Cell Biology and Technology, ACECR, Tehran, Tehran, Iran,
Islamic Republic of, 2Reproductive Imaging at Reproductive Biomedicine
Research Center, Royan Institute for Reproductive Biomedicine, ACECR,
Tehran, Tehran, Iran, Islamic Republic of
Recent non-randomized studies including our three previous phase I/II clinical
trials have shown the safety of mesenchymal stem cells injection in the treatment of osteoarthritis. To investigate the effects of intra articular injection of
autologous bone marrow derived mesenchymal stem cells (BM-MSC) on the
symptoms of moderate to sever knee osteoarthritis we performed a double
blind, placebo-controlled study in 46 patients.
Methods: Patients fulfill criteria for knee OA were randomly assigned into two
groups: group one received BM-MSC (20 million cells, twice at day 0 and week
12th) and group two received carrier media as placebo. Primary end points
were the Western Ontario and McMaster Universities Osteoarthritis Index
(WOMAC) and a visual analogue score (VAS) for pain at baseline and at the
end of weeks 12, 24 and 36. Secondary end points were pain free walking
distance, cartilage thickness, subchondral edema and tumor formation at 24 and
36 weeks.
Results: The results indicated that the intra articular injection of autologous
BM-MSC is very well tolerated. Moreover BM-MSC treated group had
significantly clinical improvement as compare to placebo group in all clinical
end points. In particular, the WOMAC-Total score, WOMAC-Physical
Function sub score, WOMAC-pain sub score and pain free walking distance in
BM-MSC, were superior compared to the placebo group (P ¼0.03, p¼0.02,
p¼0.03 and P ¼0.01, respectively). Primary radiologic data indicated that
subchondral edema decreased in some patients also thickness of cartilage
increased in MSC group.
Conclusion: Our short term follow up (9 months) have shown that repeated
intra articular injection of BM-MSC is safe and effective in reducing functional
impairment and relieving pain in patients with moderate to severe osteoarthritis of the knee. Clinical trial registration: NCT01504464.
24
TARGETED MESENCHYMAL STEM CELL THERAPY FOR
CHRONIC OBSTRUCTIVE PULMONARY DISEASE
J Tuma2, F Silva1, N Cottle1, AN Patel1
1
Regenerative Medicine - Surgery, University of Utah, Salt Lake City, Utah,
United States, 2Regenerative Medicine, Maison de Sante, Lima, Peru
Background: Chronic obstructive pulmonary disease is both an obstructive
and inflammatory disease. Mesenchymal stem cell (MSC) therapy has shown
early promise to modulate inflammation and improve lung function. The
therapy has limitations resulting from cell loss and sub-optimal delivery. Our
goal was to develop a MSC therapy that can use the inflammation in the lungs
which results in SDF-1 expression as a target.
Methods: Patients with severe COPD were randomized to receive either
saline, MSCs, or MSCs primed with CXCR4 protein (the natural ligand for
SDF-1). All clinical events along with pulmonary functions tests (FEV!/FVC)
and oxygen requirements were evaluated.
Results: Thirty patients successfully were enrolled in the study. All patients
were prior or recent smokers. There were no acute adverse events. The MSC
and MSC+CXCR4 group demonstrated improvement in pulmonary functions
tests and oxygen requirement compared to the control group. (see table).
Conclusion: Targeted MSC therapy with CXCR4 demonstrates safety
along with promising results in patients with chronic pulmonary obstruction
disease. Larger trials and follow up will be required to evaluate benefit and
sustainability.
FEV!/FVC pre
FEV!/FVC post
O2L/min pre
O2L/min post
MACE
Saline
MSC
MSC+
CXCR4
0.56
0.54
2.4
2.5
3
0.50
0.56
2.7
2.1*
1
0.49
0.62*
3.1
1.6*
0
*p<0.05.
25
LONG-TERM OUTCOMES OF EX VIVO EXPANDED LIMBAL
STEM CELL TRANSPLANTATION IN HUMANS
FC Figueiredo1, S Kolli4, S Ahmad3, M Lako2
1
Ophthalmology, Newcastle University, Newcastle upon Tyne, United
Kingdom, 2Institute of Genetic Medicine, Newcastle University, Newcastle
upon Tyne, United Kingdom, 3Ophthalmology, Liverpool University,
Liverpool, United Kingdom, 4Ophthalmology, Birmigham University,
Birmingham, United Kingdom
Purpose: To study the clinical outcome of transplanting animal-free cultivated
limbal epithelial cells on human amniotic membrane (AM) to treat patients
with limbal stem cell deficiency (LSCD).
Methods: Prospective, single-centre, noncomparative, interventional case
series.
Participants: Eight eyes of 8 consecutive patients with unilateral LSCD
(chemical burns in 6 eyes, thermal burn and radiation keratopathy in 1 eye
each) were treated at the Dept of Ophthalmology, Newcastle University,
Newcastle upon Tyne, UK between Apr 2006 and Nov 2008.
Intervention: Autologous limbal epithelium from the healthy other eye was
cultivated on AM without animal cells/products, and transplanted onto the eye
with severe LSCD.
Outcome measures: Ocular surface reconstruction, change in visual
acuity, absence of goblet cells on corneal impression cytology (IC) and
complications.
Patient-reported outcomes: Vision impairment and pain/discomfort
scores.
Results: 7 of the patients were male and the mean age was 43 (range 16 73). Mean follow up was 72 months (range 60 - 84). Postoperatively, satisfactory ocular surface reconstruction was obtained in all eyes (100%), as
confirmed by IC. Three eyes developed localised conjunctivalisation, requiring
subsequent sectoral epitheliectomy. Penetrating keratoplasty was performed in
4 eyes. At last examination, visual acuity improved in all 8 eyes. Vision
impairment and pain scores improved in all patients (p<0.05).
Complications: Corneal graft rejection in 1 eye and limbal stem cell failure
in 1 eye.
Conclusions: This study demonstrates that transplantation of autologous
limbal epithelial stem cells cultured on AM without animal cells/products is an
effective method of reconstructing the corneal surface and restoring useful
vision in patients with unilateral LSCD. This procedure has the potential to
become a viable management option for patients with severe LSCD. Currently
conducting Phase II study with more patients and longer follow-up.
26
NEURO-REGENERATIVE
EFFECT
OF
AUTOLOGOUS
MESENCHYMAL STEM CELL THERAPY IN CEREBRAL PALSY
PATIENTS: PILOT CLINICAL TRIAL
W Abo Elkheir2, H Gabr1
1
Immunology, Medical academy, cairo, Egypt, 2Clinical pathology, Faculty of
medicine-Cairo university, CAIRO, Egypt
Background: Stem cell-based therapies provide hope for various CNS diseases
including perinatal hypoxic ischemic insults of the brain. Stem cells have the
S15
capacity to proliferate in culture, migrate and disseminate following implantation within the adult CNS. The neuroregenerative potential of bone marrowderived mesenchymal stem cells (MSCs) is proposed to be through paracrine
effect, stimulation of endogenous stem cells, angiogenesis, in addition to the
disputed possibility of direct transdifferentiation.
Objectives: To study the impact of MSC transplantation on psychomotor
functions in patients with cerebral palsy.
Methods: Fifty two Egyptian patients with cerebral palsy were divided
into: group I (26 patients who underwent autologous intrathecal bone
marrow-derived mesenchymal stem cell injection) and group II (26 patients
who served as control). Both groups were assessed, initially and after one year,
by a group of clinical scales to assess motor, communication and independence skills.
Results: In group I, using Boyd’s developmental progress scale
revealed a statistically highly significant improvement in motor, independence and communication skills after SCT (P value < 0.01). Also,
100 points scale revealed a statistically significant improvement after
SCT (P value < 0.05).
Conclusion: Autologous stem cell transplantation could be a useful tool for
the management of patients with cerebral palsy as it may help in improvement
of motor, independence and communication skills.
27
FABRICATION OF ACELLULAR SCAFFOLDS BY CRYOCHEMICAL DECELLULAURIZATION OF THE WHOLE LIVER
FOR MESENCHYMAL STEM CELL-BASED FUNCTIONAL
HEPATIC ENGINEERING
O Lee1,2, W Jiang1,3
1
Stem Cell Research Center, National Yang-Ming University, Taipei,
Taiwan, 2Department of Medical Research, Taipei Veterans General Hospital,
Taipei, Taiwan, 3Institute of Biomedical Engineering, National Yang-Ming
University, Taipei, Taiwan
Liver transplantation is the ultimate treatment for severe hepatic failure to
date. However, the limited supply of donor organs has severely hampered
this treatment. So far, great potentials of using mesenchymal stem cells
(MSCs) to replenish the hepatic cell population have been shown; nevertheless, there still is a lack of an optimal three-dimensional scaffold for
generation of well-transplantable hepatic tissues. In this study, we utilized a
novel cryo-chemical decellularization method which combines physical and
chemical approach to generate acellular liver scaffolds (ALS) from the whole
liver. The produced ALS provides a biomimetic three-dimensional environment to support hepatic differentiation of MSCs, evidenced by expression of hepatic-associated genes and marker protein, glycogen storage,
albumin secretion, and urea production. It is also found that hepatic differentiation of MSCs within the ALS is much more efficient than twodimensional culture in vitro. Importantly, the hepatic-like tissues (HLT)
generated by repopulating ALS with MSCs are able to act as functional
grafts and rescue lethal hepatic failure after transplantation in vivo. In
summary, the cryo-chemical method used in this study is suitable for
decellularization of liver and creates acellular scaffolds that can support
hepatic differentiation of MSCs and be used to fabricate functional tissueengineered liver constructs.
28
CLASSIFYING PATIENTS AND MONITORING THE OUTCOMES
OF CELL BASED THERAPIES USING IMMUNOMICS
M Gustafson1, Y Lin2, M Maas1, P Bulur1, D Gastineau1,2, A Dietz1,3
1
Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester,
Minnesota, United States, 2Department of Medicine, Mayo Clinic, Rochester,
Minnesota, United States, 3Department of Immunology, Mayo Clinic,
Rochester, Minnesota, United States
We have developed a novel approach to categorize immunity in patients that
uses a combination of whole blood flow cytometry and hierarchical clustering. Our approach is based on flow cytometry capable of enumerating
each of the major leukocyte subsets in unfractionated, whole blood. We have
previously demonstrated this approach using 40 healthy volunteers and
120 patients with glioblastoma, renal cell carcinoma, non-Hodgkin lymphoma, ovarian cancer or acute lung injury. After normalization, we used
unsupervised hierarchical clustering to sort individuals by similarity into
S16
Oral Abstracts
discrete groups we call immune profiles. We identified specific immune
profiles with improved survival (p<0.01). What is particularly noteworthy
is that these immune profiles identified patients with similar immune
status independent of the underlying diagnosis. This analysis also allowed
determination of unique relationships between immune markers. For
example, two critical myeloid phenotypes (the immunosuppressive
CD14+HLA-DRlo/neg monocytes and Lin-CD33+HLA-DR- myeloid
derived suppressor cells) independently segregated among patients. We
have now developed our third generation immune profiling panel. This
panel consists of a 10 color, 8-tube protocol set capable of identifying
more than 100 distinct phenotypes. We are using this panel to identify
unique immune profiles in patients undergoing cell-based therapy
including dendritic cell vaccines and mesenchymal stem cell therapies. To
date, we have evaluated approximately 200 patients and healthy volunteers
using this approach. We will present data on the power of this approach in
cancer and demonstrate its potential to select and monitor patients undergoing cell based therapies.
29
PROGRESS TOWARDS THE CGMP PRODUCTION OF
PLURIPOTENT STEM CELL DERIVED RED BLOOD CELLS
M Turner1,3, J Mountford2,1, L Forrester3, C Ghevaert4,6, R Thomas5,
D Anstee6, W Murphy7, A Courtney8, K Thompson9
1
Scottish National Blood Transfusion Service, Edinburgh, United
Kingdom, 2University of Glasgow, Glasgow, United Kingdom, 3University of
Edinburgh, Edinburgh, United Kingdom, 4University of Cambridge,
Cambridge, United Kingdom, 5University of Loughborough, Loughborough,
United Kingdom, 6NHS Blood and Transplant, London, United
Kingdom, 7Irish Blood Transfusion Service, Dublin, Ireland, 8Roslin Cells Ltd,
Edinburgh, United Kingdom, 9Cell Therapy Catapult, London, United
Kingdom
Blood transfusion is a wide spread and important clinical intervention,
however problems persist both nationally and internationally in maintaining
adequacy of supply, managing the risk of transmission of infectious agents
and immune incompatibility between donor and recipient. Human embryonic and induced pluripotent stem cells (hESCs & hiPSC) have unique
properties in that they can be maintained indefinitely in culture in an undifferentiated state and yet retain the ability to form all the cells and tissues
within the body. They therefore offer a potentially scalable source from
which to generate red cells (RBCs) for use in clinical transfusion. We have
evaluated hESC lines derived under Good Manufacturing Practice (GMP)
conditions in compliance with UK regulatory requirements for clinical
products and are preparing master cell bank stocks of the lead line for
clinical use, RC9. We are able to differentiate these RC9-hESCs to form
haematopoietic progenitor cells (HPC) which subsequently result in 95%
conversion to erythroid cells (GlyA+, CD45-, haemoglobinised) with up to
350,000 fold expansion in cell numbers, in a stroma-free, animal productfree suspension based culture system. The culture process starts with a short
embryoid body stage followed by sequential changes in inductive cytokines
and growth factors taking up to 30 days. We have also demonstrated that
this methodology is similarly effective for hiPSC. The erythroid cells express foetal (alpha/gamma) rather than embryonic (epsilon/zeta) haemoglobin and achieve reasonable enucleation. Many challenges exist in taking
this product through to clinical trial including optimisation of the differentiation an maturation protocol, scale-up and optimisation of process
control in manufacturing, cGMP-translation and cost-control, and the
regulatory and commercial challenges of moving through to first in man
clinical studies.
30
EXPANSION
AND
DIFFERENTIATION
OF
HUMAN
PLURIPOTENT STEM CELLS TO NEURAL PROGENITORS: A
SIMPLIFIED BIOREACTOR PROCESS REPLACING NOGGIN
WITH 2 SMALL MOLECULES
A Chen, YM Lim, S Reuveny, S Oh
Bioprocessing Technology Institute, Singapore, Singapore
Scaling up the production of human pluripotent stem cells (hPSCs) holds the
key to the future realization of cell therapy. Conventionally, the cultivation and
expansion of hPSCs is still based on the static tissue culture plate with limited
surface area and requires repetitive passaging for expansion. We have developed a microcarrier based process for the expansion and differentiation of
hPSCs to neural lineage. Using this platform, hPSCs in serum free medium
can be expanded to 3106 cells/ml with 15 fold expansion in a 100ml spinner
flask. These expanded hPSCs were differentiated to neural progenitors
(NPCs) using two small molecules (Dorsomorphin and SB 431542). The
process was simple as medium exchange without manual manipulation
required for embryoid body formation. By replacing recombinant protein
Noggin with these two small molecules, the neural differentiation was
shortened by 4 days to generate above 90% PSA-NCAM+ NPCs, achieving a
higher cell concentration of 15106 NPCs/ml (398 NPCs/hPSCs seeded) as
compared to 6.1106 NPCs/ml (163 NPCs/hPSCs seeded) achieved using
Noggin based protocol. These NPCs can be further differentiated to dopaminergic neurons with positive expression of tyrosine hydroxylase. In
conclusion, the microcarrier platform is robust and scalable. hPSCs and NPCs
generated are significant higher than the conventional 2D platform. It is our
long term goal to further develop this microcarrier based hPSC expansion and
differentiation to specific cell types for cell therapy, disease studies, drug
screening, and tissue engineering.
31
GMP PROTOCOL TO GENE-ENGINEER AND PROCESS HUMAN
T-CELLS REVISITED: MEDIUM CRITICALLY DETERMINES
YIELD, FUNCTION AND DIFFERENTIATION STATE OF CD8+
T-CELLS
C Lamers, S Steenbergen-Langeveld, S Sleijfer, R Debets
Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, Netherlands
We have previously designed and validated a GMP protocol for retroviral
gene transduction and expansion of human T-cells. Here, we have revisited
the choice for optimal medium (composition) and tested different and newly
available media for their effects on yield as well as phenotypical and functional properties of receptor-engineered T-cells. GMP-defined (serum-free)
media that were tested included AIMV and Optimize (Invitrogen), Xvivo15
(Lonza), CellGro (CellGenix) and TexMACS(Miltenyi). We tested
these media with or without supplementation of 2% plasma. In addition, we
tested 3 blended media (i) AIMV (20%)+RPMI(80%)+2% plasma (EMC
standard); (ii) AIMV(50%)+RPMI(50%)+5% human serum; and (iii) Xvivo15(20%)+RPMI(80%)+2% plasma. First experiments used CAR-engineered
T-cells.The non-supplemented media performed the least with respect to
T-cell yield, transduction efficiency and function of CAR T-cells. The best
performing media with respect to these parameters included 3 plasmasupplemented media (AIMV, Xvivo15 and TexMACS) and the 3 blended
media, including the EMC standard. Five of these media, supplemented with
IL15+IL21, were re-tested in parallel to generate MC2/A2 TCR T-cells. In
terms of T-cell yield, transduction and function, AIMV+2% plasma performed the best, whereas Xvivo+2% plasma performed the least. Notably,
with regard to the preservation of early T-cell differentiation this sequence
was reverse. Taken together, this study shows the impact of culture media on
yield and phenotypical and functional properties of human T-cells. Importantly, the medium resulting in highest T-cell yield led to more differentiated
T-cells, and vice versa. This study provides important information with
respect to medium selection to gene-engineer and process T-cells for clinical
studies.
32
PRECLINICAL
CHARACTERIZATION
OF
DUOC-01,
A
CANDIDATE CELL THERAPY PRODUCT DERIVED FROM
HUMAN BANKED UMBILICAL CORD BLOOD INTENDED FOR
USE IN TREATMENT OF DEMYELINATING DISEASES
J Kurtzberg1, S Buntz1, T Gentry1, R Storms1, DA Wenger2, P Noldner1,
J Zhou1, A Ozamiz1, B Rusche1, A Balber1
1
Robertson Clinical & Translational Cell Therapy Program, Duke
Translational Medicine Institute, Durham, North Carolina, United
States, 2Neurology, Jefferson Medical College, Philadelphia, Pennsylvania,
United States
Allogeneic umbilical cord blood [CB] transplantation can slow or reverse
progression of central nervous system demyelination in inherited metabolic
diseases. Clinical observations suggest that several months are required after
transplant for donor derived cells in the brain to provide benefit. We are
developing DUOC-01 as a bridging cell product administered intrathecally to
patients early post-transplant to provide therapeutic effects prior to CNS
engraftment by cells from the CB transplant. DUOC-01 is manufactured
under cGMP conditions from the 20% compartment of the same CB unit
used for systemic transplantation by a modification of a previously described
method. We performed preclinical characterization of DUOC-01. Time lapse
imaging showed that the cultures evolve into attached, motile, highly active
cell populations resembling macrophages. The cells express characteristic
myeloid macrophage markers including CD45, CD11b, and Iba1. Cells had
activities of 11 disease-relevant lysosomal enzymes similar to wild type blood
leucocytes. All DUOC-01 batches secreted IL-6 and IL-10. Some secreted
TGF-b, IL-1b, INF-g, TNF-a or very low amounts of IL-12 or IL-2. IL-4,
IL-5 and IL-13 were not detected. Peripheral blood mononuclear cells
[MNC] proliferated and released cytokines in response to DUOC-01. CB
MNC did not respond to DUOC-01 made from the same unit, and DUOC01 did not proliferate in response to mismatched MNC. Following intrathecal
injection DUOC-01 cells were targeted to and persisted in brains and spinal
cords of newborn NOD/SCID-IL2Rgnull mice for up to 56 days. Brains of
NOD-SCID mice injected intrathecally or intracerebrally with DUOC-01
showed no tumors, ectopic tissue growth, or gross clinical abnormalities
during 56 days of observation. DUOC-01 accelerated remyelination in NOD/
SCID-IL2Rgnull mouse brains following curpizone feeding. Thus, preclinical
studies suggest that DUOC-01 has promise as a candidate cell therapy for
demyelinating diseases.
33
TREATMENT OF CEREBELLAR ATAXIA WITH MESENCHYMAL
STEM CELLS: A PHASE I/II TRIAL
B Soong2,4, O Lee1,3,5
1
Director, Stem Cell Research Center, National Yang Ming University,
Taipei, Taiwan, 2Prof. & Chairman, Dept. of Neurology, Taipei Veterans
General Hospital, Taipei, Taiwan, 3Deputy Dean, Office of International
Affairs, National Yang Ming University, Taipei, Taiwan, 4Prof. & Chairman,
Dept. of Neurology, National Yang Ming University, Taipei, Taiwan, 5Prof.,
Institute of Clinical Medicine, National Yang Ming University, Taipei,
Taiwan
Spinocerebellar ataxias (SCA), are determined rare diseases by the Office of
Rare Diseases Research at the National Institutes of Health. SCA causes
progressive difficulty with coordination and gait which interferes in performing
normal daily functions. SCA patients die from respiratory failure, aspiration
pneumonia, or severe infection within 20 years of onset. There are no approved
therapeutics for treating SCA (spinocerebellar ataxia). PolyQ SCAs are caused
by an extensive CAG sequence repeat which encodes for expanded polyQ
residues within the mutated protein. All polyQ SCA patients clinically present
limb and gait ataxia because the same ataxia interactome is shared among subgroups. Extensive polyQ in cells, including Purkinje neurons, leads to cell
dysfunction and triggers cell apoptosis. Loss of Purkinje cells leads to the
symptoms and disease outcomes of SCA. Our pre-clinical research has achieved
pre-clinical evidence suggesting adipose tissue-derived mesenchymal stem cell
(ADMSC) transplantation ameliorates motor function deterioration of SCA in
SCA2 transgenic mice by rescuing cerebellar Purkinje cells (Journal of
Biomedical Science 2011, 18:54; Chang, et al). The infusion of ADMSCderived Stemchymal MSCs into SCA patients may be safe and may demonstrate evidence of ameliorating motor function deterioration by arresting
continued loss of Purkinje cells to premature apoptosis caused by oxidative
stress from excessive PolyQ expression. Our trial design includes a single 7
x107 Stemchymal cells infusion into seven patients with 12 months follow up.
Primary outcome measures for safety include vital signs, clinical lab tests and
adverse events. Secondary outcome measures for early evidence of efficacy
include changes in the scale for the assessment and rating of ataxia (SARA)
score, changes in sensory organization test (SOT) score, changes in adaptation
test (ADT) scores and changes in electronystagmogram (ENG). At 10 months,
Phase I / II safety and early efficacy data supports the feasibility of using
allogeneic Stemchymal(TM) Cell Therapy in the treatment of SCA patients.
Longer term follow-up and larger, well-controlled clinical trials will be
required to get to reach a definitive conclusion for Stemchymal treatment
of SCA.
S17
34
FUCOSYLATION OF EX VIVO EXPANDED CORD BLOOD (CB)
CELLS IMPROVES ENGRAFTMENT IN NOD/SCID MICE
I McNiece1, H Yang1, M Thomas1, J Jun1, L Miller2, P Zweidler-McKay1,
E Shpall1
1
Stem Cell Transplantation and Cellular Therapy, The University of Texas
MD Anderson Cancer Center, Houston, Texas, United States, 2American
Stem Cell Inc, Floresville, Texas, United States
CB is as an alternate stem cell source for patients receiving high dose chemotherapy, however the time to neutrophil and platelet engraftment is delayed
compared to BM or PBPC products. We have demonstrated faster engraftment
with CB products expanded in MSC co-culture leading to an ongoing phase 3
trial. Also studies have demonstrated that fucoyslation of unmanipulated CB also
results in faster engraftment in patients. Therefore we hypothesized that fucosylation of expanded CB may provide faster engraftment. CBs were expanded
using co culture on MSCs and expanded cells were fucoyslated with fucosyltransferase (FT) VI. CD34+ cells were isolated from the same CB as a control
cell population. Immunodeficient (NSG) mice were injected with CD34+ cells,
expanded CB cells, or fucosylated expanded cells. Expansion resulted in an increase of 9 fold of TNC and 116 fold increase in CD34+ cells. The expanded
cells resulted in faster engraftment of human (hu) CD45+ cells when injected
into NSG mice compared to animlas injected with CD34+ cells. The levels of
huCD45 in recipient mice was dose dependent with higher cell doses (140 M)
resulting in earlier engraftment of huCD45+ cells with a maximum level of 25%
huCD45+ cells in the PB, compared to a lower dose (50 M) which engrafted at
less than 1% of huCD45+ cells. Treatment with FTVI resulted in an increase of
fucosylation from 72% to 98% CLA+ cells. Animals injected with fucosyalted
expanded cells achieved up to 25% huCD45+ cells in the PB compared to non
fucosylated expanded cells and the engraftment of human cells was more rapid.
These data suggest that fucosylation of expanded CB cells may provide enhanced
engraftment in the setting of low cell doses such as CB transplantation. Further
studies are being performed to support evaluation of fucosylated expanded CB
cells in clinical trials in the near future.
35
EX-VIVO EXPANSION OF CORD BLOOD HEMATOPOIETIC
STEM AND PROGENITOR CELLS FOR TRANSPLANTATION
USING AN ANTIOXYDANT-SUPPLEMENTED MEDIUM AND A
CYTOKINE COCKTAIL INDUCING HYPOXIC-LIKE CELLULAR
RESPONSE
P Duchez1,2, J Chevaleyre1,2, B Dazey1, M Vlaski1,2, Z Ivanovic1,2
1
Aquitaine-Limousin, Etablissement Français du sang, Bordeaux,
France, 2UMR 5164, CNRS/University Segalen, Bordeaux, France
We recently developed a clinical grade ex vivo cord blood (CB) expansion
procedure enabling a massive amplification of hematopoietic progenitors
without any loss of stem cell potential as revealed on the basis of serial
engraftment of Nod/SCID mice (Ivanovic et al, Cell Transplant. 2011). This
procedure, in line with our concept “Oxygen Stem Cell Paradigm” (Ivanovic, J
Cell Physiol 2009), is based on Day 14 liquid cultures of CB CD34+ cells, in
medium Macopharma HP01 (containing several antioxidant molecules and
nicotinamide) and in presence of Stem Cell Factor - SCF (100 ng/ml), fmsRelated Tyrosine Kinase 3 e Ligand - FLT3-ligand (100ng/ml), Megakaryocyte Growth and Developmental Factor - MGDF (100 Ng/ml) (these two
cytokines acting in favor of HIF-1alpha transcript stabilization) and Granulocyte e Colony Stimulating Factor -G-CSF (10 Ng/ml). This cocktail had to
be modified due to the commercially unavailability of clinical grade MGDF
molecule. So, MGDF was replaced by Thrombopoietin - TPO in five-fold
lower dose (20 ng/ml) and culture time was reduced to 12 days (Duchez et al
Cell Transplant 2012). That way, a mean expansion fold of 400, 80, and 150
was obtained for total cells, CD34+ cells and Colony Forming Cells e CFC,
respectively. This amplification was associated with a slight enhancing effect on
stem cells (Scid Repopulating Cells - SRC). These are the ultimate pre-clinical
modifications of a clinical grade expansion protocol which is already employed
in an ongoing clinical trial (adult allogeneic context) started in 2010). The
preliminary results are encouraging e rapid and durable hematopoietic
reconstitution after injection of one ex vivo expanded CB unit only.
S18
Poster Abstracts
Poster Abstracts
36
WILL NOT BE PRESENTED
37
38
DEVELOPMENT OF SERUM-FREE DIFFERENTIATION MEDIUM
TOWARDS CD1A SUBSET OF DENDRITIC CELLS
K Kuwahara, J Ni
Irvine Scientific, Santa Ana, California, United States
Dendritic cells have garnered interest in recent years due to their ability
to combat cancer. These dendritic cells once programmed can generate
cancer vaccines and recruit the aid of T-cells which will later attack
cancerous cells. However dendritic cells are not readily available in large
numbers. In many cases, dendritic cells are either isolated or derived from
monocytes. Currently monocyte derivation is the preferred method due to
the availability of monocyte and its obtainability from an autologous
source. Further research has shown the media to be a key component in
deriving the dendritic cells into the proper phenotype for cancer vaccines.
Irvine Scientific (IS) has developed a serum free media that is specifically
for deriving dendritic cells from monocytes. Through rational design of
its formulation, this IS media favors derivation of the CD1a positive
subset of dendritic cells which are now to activate the immunogenic
response against cancer by T-cells. Comparison data between competitor’s
media and its serum added counterpart show higher amounts of the CD1a
positive subset of dendritic cells produced with IS media. Functionality
data is generated to reflect this CD1a positive DC phenotype. In this
study, we demonstrate how the serum-free formula of IS media can
provide the proper phenotypic subset of dendritic cells with cancer vaccine capability.
39
BORTEZOMIB INCREASES THE SENSITIVITY OF MULTIPLE
MYELOMA CELLS TO THE CYTOTOXICITY OF GAMMADELTA T CELLS
J Cui, W Li, S Hao, H Jin, C Niu, G Wang
Cancer Center, the First Hospital of Jilin University, Changchun, China
Objectives: Multiple myeloma (MM) is by far not curable plasma cell malignancy, although the new drugs such as bortezomib, achieve an attractive results.
This study is to investigate the possible synergistic cytotoxic effects against
MM cells between gdT cells and bortezomib, and its mechanism, with the aim
of providing a novel therapeutic strategy for MM.
Methods: Cytotoxicity of gdT cells against MM cell line (U266 cells) was
measured by calcein assay. The expression of NKG2D ligands and TRAIL
receptors on U266 cells before and after treatment with bortezomib was
detected by flow cytometry (FCM).
Results: It was shown that highly activated gdT cells were amplified from
peripheral blood mononuclear cells (PBMCs) of MM patients with a median
percentage of 90% (76% to 98%). The expanded gdT cells were significantly
more cytotoxic against U266 cells pretreated with 10.0nmol/L bortezomib
compared to untreated cells. Compared with the untreated group, the
expression of NKG2D ligands ( ULBP1 ,ULBP2, ULBP3, MICA/B) and
TRAIL receptors (DR4, DR5) on U266 cells increased after 8 hours’ preincubation with bortezomib. After 16 hours’ treatment with bortezomib, the
expression of ULBP2, MICA/B, and DR4 was significantly increased, but the
expression of ULBP1, ULBP3, DR5 showed no alteration. After 24 hour’s
treatment, the expression of NKG2D ligands (ULBP1, ULBP2, ULBP3,
MICA/B) and TRAIL receptors (DR4, DR5) appeared downregulated. It is of
great significance to choose the right time point with the combination
strategy.
Conclusion: Bortezomib not only promotes apoptosis of MM cells, but also
increases the immunogenity of MM cells. And its mechanism may be associated
with upregulation of NKG2D ligands (ULBP1,ULBP2,ULBP3, MICA/B) and
TRAIL receptors (DR4, DR5) on the cell surface. This study provided a
promising therapeutic option for MM, and it is worthwhile to study this
combination strategy in the clinical settings.
40
PROCESS TRANSFER OF DCVAX-L TO EUROPE AND
INITIATION OF A PHASE III CLINICAL TRIAL IN UK AND
GERMANY
G Schmiedeknecht1, K Kebbel1, C Sonnabend1, M Wagner1, M Gryczka1,
M Stella2, K Ganjei2, M Bosch3, LF Powers3
1
Fraunhofer Institute for Cell Therapy and Immunology IZI, Leipzig, Free
State of Saxony, Germany, 2Cognate BioServices, Inc., Memphis, Tennessee,
United States, 3Northwest Biotherapeutics Inc., Bethesda, Maryland, United
States
DCVax-L is an autologous immunotherapy applicable for treatment of a wide
range of cancers. It consists of Dendritic Cells (DC’s) loaded with tumor
lysate generated from patient’s own tumor tissue. DC’s are manufactured in
an eight-day process by using a leukapheresis product as starting material.
After finishing promising Phase I/II studies in the US, Northwest Biotherapeutics Inc. is now under way with a 312-patient double blind, randomized Phase III study in both the US and Europe. Most small US biotech
companies do not undertake their clinical trials in Europe as well as the US e
especially for a complex product such as an autologous cell therapy. In order
to bring this Phase III trial to Europe, and reach more patients, Northwest
Biotherapeutics and its manufacturer, Cognate Bioservices Inc., have started
in 2011 a process transfer of the entire GMP manufacturing process to
Germany (to Fraunhofer IZI). After a 15 month tech transfer, a DCVax-L
specific manufacturing authorization according to x13 German Drug Act was
granted to Fraunhofer IZI. This period of time was necessary to create all
documents, to establish the methods and train the Fraunhofer personnel both
in Germany and in the US, to set-up the supply chain, to qualify the suppliers
and to perform a process validation as well as a validation of the analytical
methods. In parallel, it was necessary to qualify the tumor procurement
centers in order to comply with 2006/17/EC requirements, and to qualify the
apheresis units for ensuring compliance with 2002/98/EC. After creating an
Investigational Medicinal Product Dossier (IMPD) clinical trial applications
were filed to MHRA (UK) and Paul-Ehrlich-Institut (Germany). After successful approval in both countries, the clinical trial launched in Europe (in the
UK) in the late spring 2013 and is currently being expanded by including
further clinical trial centers.
41
42
A NOVEL AUTOMATED PROCESS GENERATES VIRUSSPECIFIC T LYMPHOCYTES FOR IMMUNOTHERAPY THAT
MAINTAIN THEIR IN VIVO PHENOTYPE AND FUNCTIONAL
COMPETENCE
A Richter, L Preussner, V Traska, M Peters, A Oysal, A Kaiser,
M Assenmacher
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Adoptive transfer of virus-specific T cells is an option to treat severe infections after allogenic stem cell transplantation. To generalize this
approach, a cGMP-compliant process for enrichment of virus-specific CD4+
and CD8+ T cells is required. Here, we use a newly established automated
manufacturing process for rapid ex vivo isolation of multi-virus- or CMV
pp65 peptide-specific T cells. During this process, first 109 white blood cells
from healthy donors were stimulated with either a combination of peptide
pools (CMV pp65, EBV EBNA-1, BZLF1, and LMP2, and ADV hexon) or
with a pp65 peptide for 4 hours. Subsequently, virus-specific T cells were
magnetically enriched according to IFN-g secretion. To examine, if cell
processing influence the phenotype, virus-specific T cells were analyzed
before and up to 4 days after the selection process. Isolated T cell products
consist of 86.6% IFN-g+ CD8+ multi-virus-specific T cells and 75.5% IFNg+ pp65 peptide-specific CD8+ T cells. However, after a resting phase in
vitro, IFN-g production decreased drastically. In addition, we detected an
upregulation of CD69 and CD137 in T cell products directly and 24 hours
after selection, respectively. The transient nature of activation could again be
confirmed, as both, CD69 and CD137 were downregulated later on. To test
functionality we re-incubated 3-days rested cells with antigens. Induction
of IFN-g in up to 100% of T cells was observed demonstrating cells
maintained their in vivo imprinted physiological role. We also monitored
CD45RA, CD28, and CCR7 expression on pp65-peptide specific CD8+
T cells before and directly after the enrichment. The enrichment process did
not induce phenotypic changes. Thus, the newly developed automated
manufacturing process provides a product, where the original phenotypic and
functional characteristics of virus-specific T cells are conserved. Hence this
cellular product is expected to fight viral infections effectively upon adoptive
transfer.
43
CHARACTERISTICS OF PRODUCTS FOR NK-CELL INFUSION
PREPARED BY CD3 DEPLETION FOLLOWED BY CD56
ENRICHMENT: REQUIREMENTS FOR RARE EVENT ANALYSIS
C Keever-Taylor1, LA Jones2, S Heimfeld3, BM Sandmaier3,4, P Hari1,
MS Thakar1
1
Medicine/Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin,
United States, 2Seattle Cancer Care Allliance, Seattle, Washington, United
States, 3Fred Hutchinson Cancer Research Center, Seattle, Washington,
United States, 4University of Washington, Seattle, Washington, United States
NK cell immunotherapy delivered early after HLA-haploidentical BMT can
prevent relapse. Such products require processing that rigorously depletes
T cells to reduce GVHD risk while sparing NK cells. Here we describe
characteristics of 18 products processed by two labs supporting a multicenter clinical trial. Marrow donors underwent a single non-mobilized
apheresis day 6 post-BMT with next day processing by a 2-step method of T
cell depletion/NK cell enrichment using the Miltenyi CD3/CD56 CliniMACS system. Starting products and all fractions were characterized by
flow for T(CD3+CD56-), NK-T(CD3+CD56+), NK(CD3-CD56+), & B
cells(CD19+) & monocytes (CD14+). T, B, & NK cells were assessed after
gating on a low side scatter population negative for dead cells (7-AAD),
monocytes and myeloid cells (CD33) to reduce background staining.
Starting products did not differ between sites however, trial products did
have a higher % of T cells compared to DLI products (N¼17, p¼0.03).
CD3-depletion alone preserved NK cells (81.211.0% recovery) but
removed only 3.10.3 logs of T cells and 2.00.5 logs of NK-T cells.
Subsequent CD56-enrichment reduced T cells to 5.60.7 logs and NK-T
cells to 2.40.3 logs. Final NK cell recovery was 52.48.1% (39.5%
-66.7%) with B cells constituting only 0.20.2% of the final product. Both
labs produced final products with >5.0 logs of TCD and with similar NK
cell purity (91% and 84.1%) and recovery (52.9% and 52.7%). Monocytes
constituted 6.97.1%(range 1.2-28.2%) of the final product that did not
differ by lab. We conclude that using the Miltenyi system with a 2-step T
cell depletion/NK cell enrichment labs can consistently achieve good recovery of NK cells with extremely low T & B cell content allowing NK
infusions up to 5.0E6/kg. A consensus flow cytometry analysis method was
critical to detect rare events and ensure safe levels of T cells for infusion
across major HLA barriers.
44
INCREASED REACTIVITY OF CIRCULATING NATURAL KILLER
CELLS FOLLOWING ADOPTIVE TRANSFER OF EX VIVO
EXPANDED NATURE KILLER CELLS
X Deng1, H Terunuma1,2,3, A Terunuma1,2, T Takane1, M Nieda1
1
Biotherapy Institute of Japan, Tokyo, Japan, 2Tokyo Clinic, Tokyo,
Japan, 3Southern Tohoku General Hospital, Fukushima, Japan
Introduction: We have established large-scale ex vivo expansion kit of natural
killer (NK) cells from peripheral blood mononuclear cells (PBMCs). This study
was conducted to evaluate the change of circulating NK cells following the
transfer of expanded NK cells.
Methods: NK cells were expanded ex vivo using BINKITÒ (Biotherapy
Institute of Japan, Tokyo, Japan) from PBMCs of healthy volunteers after
written informed consent. The expanded autologous cells were administrated
intravenously. Cell surface markers, activating receptors, as well as IFN-g and
CD107 were measured on NK cells of PBMCs before and after days 1 7, and
14 of administration using flow cytometry. Simultaneously, cytotoxicity of
these cells was tested using Terascan.
S19
Results: A total of 0.9 to 5.7 109 (median 3.4 109) cells were harvested after 13 to 22 days of expansion. NK cells were 36.4% to 94.9%
(median 76.3%) within total expanded cells, and the number of expanded
NK cells were 0.7 109 to 5.1 109 (median 2.7 109) cells. Expanded
NK cells showed significantly higher expression levels of activating receptors, as well as IFN-g and CD107 when stimulated by K562 cells
compared to NK cells from PBMCs. In consequence, expanded NK cells
showed significantly higher cytotoxicity against K562 than PBMCs. There
were no severe adverse events related to the cell infusions. NK cells frequency in the PBMCs was increased after the infusions. These NK cells in
PBMCs showed higher expression of CD69, NKp44, IFN-g production,
and CD107 mobilization in response to K562 compared to the NK cells
before the infusions. As a result, the cytotoxicity of PBMC against K562
were enhanced after the infusions.
Conclusion: Intravenous infusions of autologous expanded NK cells was
well tolerated and enhanced the reactivity of circulating NK cells against
cancer.
45
SELECTIVE DEPLETION OF RECIPIENT-ALLOREACTIVE
T-CELLS WHILE RETAINING VIRAL-SPECIFIC AND MEMORY
T-CELLS ENABLES SAFE AND EFFICACIOUS HAPLOIDENTICAL HSCT
J Velthuis1, L De Jong1, RS Boumedine2, M Giard2, D Roy2, M Ruediger1
1
Kiadis Pharma, Amsterdam-Duivendrecht, Netherlands, 2Hôpital
Maisonneuve-Rosemont, Montreal, Quebec, Canada
Haplo-identical HSCT may resolve the lack of sufficient suitable completely
HLA-matched donors for treatment of end-stage blood-cancer patients with
a HSCT. Yet, current techniques do not allow for such procedure as
presence of T-cells will cause severe GvHD and absence of T-cells (i.e.
naked HSCT) will lead to occurrence of opportunistic infections. Kiadis
Pharma is developing ATIR, a T-cell immunotherapy based on a donor
lymphocyte preparation selectively depleted of host alloreactive T-cells,
through use of photo-dynamic therapy. In a dose-finding phase I/II clinical
study (CR-GVH-001), the product was shown to enable safe and efficacious
haplo-identical HSCT. Currently, a subsequent phase II clinical study (CRAIR-007) is ongoing. Using recently developed analytical methods, the
ATIR product from both studies was/is extensively characterized showing
that only recipient-alloreactive T-cells were selectively depleted from the
product while retaining reactivity against unrelated 3rd party antigens and
general potent T-cells. Additionally, ATIR was shown to have retained
viral-specific T-cells, preserved the presence of memory and naïve T-cells
and showed responsiveness to pathogens. Thereby, ATIR will provide
mature immune cells that offer immune protection without eliciting severe
GvHD. The in vitro characterization data are supported by clinical data
showing absence of TRM during 5-year follow-up (CR-GVH-001) over a
broad dose range and no occurrence of severe GvHD/infections in the
ongoing clinical study (CR-AIR-007). Together, these data show that using
ATIR as an adjunctive medication, haplo-identical HSCT can be safe and
efficacious.
46
GENERATION AND ADMINISTRATION OF AUTOLOGOUS
T CELLS TRANSDUCED WITH A 3RD GENERATION GD2
CHIMERIC ANTIGEN RECEPTOR FOR PATIENTS WITH
RELAPSED OR REFRACTORY NEUROBLASTOMA
CU Louis1, B Savoldo1, A Heczey1, E Yvon2, A Gee1, C Rooney1, H Heslop1,
G Dotti1, MK Brenner1
1
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston,
Texas, United States, 2Stem Cell Transplantation, MD Anderson Cancer
Center, Houston, Texas, United States
Background: Administration of T cells modified with a 1st generation chimeric
antigen receptor (CAR) targeting GD2 has been safe and can produce clinical
responses, including prolonged complete remissions. Although responses were
more frequent, and progression free survival was longer in patients in whom
CAR-T cells could be detected for 6 weeks after infusion, these cells were
only present at very low frequency in peripheral blood. Increasing the in vivo
proliferation of GD2 T cells after adoptive transfer may lead to improved
S20
Poster Abstracts
T cell to tumor cell ratios, increasing anti-tumor activity and improving patient
survival. We therefore incorporated the OX40 and CD28 co-stimulatory
endodomains into our GD2-CAR construct in the hope they would enhance in
vivo expansion and persistence of CAR T-cells in patients with relapsed/refractory neuroblastoma.
Methods: This is a Phase I, dose-escalating, safety trial administering
3rd generation GD2-CAR T cells to patients with relapsed/refractory
neuroblastoma.
Results: We have manufactured 4 autologous 3rd generation GD2-CAR
T cell products (patient ages: 4 e 23 years). Starting with a median of
20x106 PBMCs, we obtained a median of 450x106 CAR+ T cells (range
240x106 to 480x106, 18-fold expansion) after 103 days of culture. The
transduction efficiency was 795% by FACS. Phenotypically, the T cell
lines were 2711% CD4+ and 6710% CD8+, with 82% central memory
(CD45RO+CD62L+CCR7+), and 1210% naïve (CD45RA+CCR7+) cells in
the final products. 51Cr release assays showed the T cell lines produced
707% and 558% lysis of GD2+ targets (LAN-1 and CHLA255,
respectively) at a 20:1 effector:target ratio. One patient has been treated, so
far without significant toxicity. Clinical and immunological results are
pending.
Conclusion: Generation of autolgous 3rd generation CAR T cells for the
treatment of high-risk neuroblastoma appears feasible and the safety and antitumor activity of the approach are now being assessed.
47
THERAPEUTIC
EFFICACY
OF
EX
VIVO
EXPANDED
ALLOGENEIC
NATURAL
KILLER
CELLS
IN
MOUSE
NEUROBLASTOMA MODEL
O Lim, J Her, S Kang, Y Hwang
Cell Therapy, Mogam Biotechnology Research Institute, Yongin, Republic
of Korea
Natural killer (NK) cells are specialized lymphocytes that provide a first line
of defense against viral infections and cancer. The therapeutic role of allogeneic NK cells has been shown in the setting of haploidentical hematopoietic stem cell transplantation. In the present study, we assessed anti-tumor
efficacy of ex vivo-expanded allogeneic NK cells in mouse model to investigate the potential of therapeutic application of allogeneic NK cells from
MHC-mismatched unrelated donor. Splenocytes from C57BL/6 mice were
stimulated and expanded in the presence of IL-15 for 6 days. Compared with
resting NK cells, these enriched NK cells displayed activated phenotype,
efficiently produced cytokines, such as IFN-g and TNF-a, and showed
potent cytoxicity against various tumor cell lines. Therapeutic efficacy of
expanded NK cells was evaluated in A/J mice injected with Neuro-2a mouse
neuroblastoma cells. The progression of subcutaneously transplanted neuroblastoma was significantly suppressed by intratumoral injection of ex vivoexpanded NK cells. Of note, recurrence after surgical resection of tumor mass
was efficiently controlled by intravenous injection of ex vivo-expanded allogeneic NK cells. In conclusion, ex vivo-expanded allogeneic NK cells
demonstrated potent anti-tumor efficacy and have the potential for therapeutic application to treat measurable tumor burden and minimal residual
disease as well.
48
SIMULTANEOUS INDUCTION OF TRI-ANTIGEN SPECIFIC
CYTOTOXIC
T
LYMPHOCYTES(CTLS)
USING
DCS
TRANSFERRED WITH WT1, SURVIVIN AND TERT MRNA FOR
ADOPTIVE T CELL THERAPY
H Sohn1, H Lee1, H Kim2, H Cho4, H Park4, H Cho1,3, Y Kim2, S Cho2,
T Kim1,4
1
Catholic Hematopoietic Stem cell bank, College of medicine, The Catholic
university of Korea, Seoul, Republic of Korea, 2Catholic Blood and Marrow
Transplantation Center, Seoul St. Mary Hospital, Seoul, Republic of
Korea, 3Cancer Research institute, The Catholic University of Korea, Seoul,
Republic of Korea, 4Department of Microbiology and Immunology, College of
Medicine, The Catholic University of Korea, Seoul, Republic of Korea
Adoptive T cell therapy is a promising approach for treating cancers,
including hematopoietic malignancies. For the successful tumor immunotherapy, several immune escaping by tumor must be overcome. One of
them is immune editing caused by antigenic heterogeneity and variation.
Here we describe a novel approach for the simultaneous generation of
tumor-reactive T cells specific to three tumor associated antigens (TriTAAs) of wilms tumor 1, survivin and telomerase reverse transcriptase,
which have been known as universal tumor antigens. The polyclonal T cell
lines from normal healthy donors were generated by three rounds of
stimulation with dendritic cells transfected with mRNA of whole Tri-TAAs
to overcome the limitation of known HLA restricted epitopes. The T cell
lines were composed of 67.62.8% CD8+ T cells (mainly CD45RO+/
CCR7/CD62L), 26.92.4% CD4+T cells, 2.60.3% NKT cells, and
10.3% NK cells. In IFN-g EliSpot assay, T cell lines stimulated with TriTAAs showed similar IFN-g positive T cell frequency to each antigen in
comparison with that of individual single-TAA stimulated T cells. More
importantly, the T cell lines revealed potent antigen-recognition and
cytotoxicity against partially HLA-matched primary leukemic blasts. These
results indicate that our approach could circumvent a potential antigenic
variation of tumors, and broaden a clinical application for the treatment of
many cancers.
49
MANUFACTURING OF CMV-SPECIFIC T CELLS FROM G-CSF
MOBILISED DONORS FOR ADOPTIVE IMMUNOTHERAPY
THAT PRESERVE STRONG ANTI-VIRAL AND CYTOTOXIC
ACTIVITY
L Beloki1, ER Samuel2, N Ramírez1, E Olavarría1, M Lowdell2
1
Oncohematology Research Group, Navarrabiomed-Miguel Servet
Foundation, Pamplona, Navarra, Spain, 2Department of Haematology,
University College London, London, United Kingdom
CMV reactivation remains an important risk after stem cell transplantation
but can be controlled by adoptive transfer of donor derived CMV-specific T
cells (CMV-T), that are usually obtained from PBMC collected prior to GCSF mobilisation. Our aim was to assess the feasibility of CMV-T generation
from G-CSF mobilised PBMC in comparison with conventional non-primed
PBMC, contrasting the anti-viral function of obtained product. We isolated
and expanded CMV-T from mobilised and non-mobilised donors (n¼6)
based on CD137 expression following CMVpp65 stimulation. CD137+ cells
were sorted by magnetic enrichment and expanded in short-term culture.
Expanded CMV-T were analysed for surface marker expression, cytokine
production, cytotoxicity and proliferation after CMVpp65 re-challenge. In
HLA-A*02:01 donors, CTL specificity was determined by HLA-multimer
staining. In G-CSF mobilised PBMC, mean yield following CD137+ selection
was 27.12%7.70 and mean purity in the positive fraction 68.32%14.45.
They were mainly central and effector memory T cells. Following CMVpp65
re-challenge of CMV-T from mobilised PBMC, mean CD137+ expression
was 55.64%17.59, they secreted high levels of IFN-g/Granzyme B, low
levels of IL-2/TNF-a and no IL-4/IL-5/IL-10, lysed CMV-loaded PHAblasts (mean 60.41%18.13 calcein release) and proliferated after CMVloaded PBMC addition (mean 66.43%2.44). No significant differences were
observed between G-CSF mobilised and non-mobilised CMV-T. Finally,
CTLs expressed the TCR specific for the immunodominant peptide
NLVPMVATV, with 81.60%10.75 Pentamer+CD8+. In conclusion, we
successfully manufactured highly specific, functional and cytotoxic CMV-T
from mobilised PBMC, with anti-viral activity equivalent to non-mobilised
CMV-T. We confirm that the use of an aliquot from G-CSF mobilised
samples is suitable for CMV cellular therapy manufacture and thereby abrogates the need for successive donations and ensures availability for patients
with unrelated donors.
50
EPITHELIAL
TO
MESENCHYMAL
TRANSITION
(EMT)
INCREASES IMMUNOMODULATORY PROPERTIES OF CANCER
CELLS
M Ricciardi1, M Zanotto1, G Malpeli2, G Bassi1, F Bifari1, O Perbellini1,
M Chilosi3, M Krampera1
1
Department of Medicine, Section of Hematology, Stem Cell Research
Laboratory, University of Verona, Verona, Veneto, Italy, 2Department of
Surgery, Section of General Surgery, University of Verona, Verona, Veneto,
Italy, 3Department of Pathology and Diagnostics, Section of Pathological
Anatomy, University of Verona, Verona, Veneto, Italy
S21
Epithelialemesenchymal transition (EMT) plays a central role in cancer
progression and metastatic dissemination, and may be induced by local
inflammation. Cell lines of lung adenocarcinoma (a549), breast cancer
(MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B
and NK cells before and after EMT induced by either inflammatory cytokines (interferon-gamma and tumor necrosis factor-alpha) or transforming
growth factor-beta. The onset of EMT triggered multiple immunosuppressive mechanisms in cancer cells resulting in NK and T cell apoptosis,
inhibition of lymphocyte proliferation and stimulation of regulatory T and
B cells. Indoleamine 2,3-dioxygenase (IDO) pathway was the main
responsible for these effects, as shown by the use of specific inhibitors.
Thus, EMT induced by inflammatory stimuli confers to cancer cells
immunomodulatory properties resembling those shown in mesenchymal
stromal cells, which could be a cue for cancer progression and metastatic
dissemination.
51
ANTI-CD20 CHIMERIC ANTIGEN RECEPTOR (CAR) MODIFIED
EXPANDED NATURAL KILLER (NK) CELLS SIGNIFICANTLY
MEDIATE RITUXIMAB SENSITIVE AND RESISTANT BURKITT
LYMPHOMA (BL) REGRESSION AND IMPROVE SURVIVAL IN
HUMAN BL XENOGRAFTED NSG MICE
Y Chu1, A Yahr1, J Ayello1, C van de Ven1, M Barth2, M Czuczman3,
M Cairo1,4
1
Pediatrics, New York Medical College, Valhalla, New York, United
States, 2Pediatrics, State University of New York at Buffalo, Buffalo, New
York, United States, 3Medicine & Immunology, Roswell Park Cancer Institute,
Buffalo, New York, United States, 4Medicine, Pathology, Microbiology and
Immunology, and Cell Biology & Anatomy, New York Medical College,
Valhalla, New York, United States
Background: The prognosis for children and adolescents with relapsed/
resistant BL is very dismal. Novel therapies are deperately needed. Natural
Killer (NK) cells play an important role in tumor surveillance post allogeneic
stem cell transplantation.
Objective: To examine the anti-tumor effect of anti-CD20 CAR modified
expanded PBNK (exPBNK) against CD20+ BL in vitro and in xenografted
NSG mice.
Methods: exPBNK was expanded with inactivated K562-mbIL15-41BBL
and nucleofected with Anti-CD20-4-1BB-CD3z mRNA. CAR expression
was detected by flow cytometry. exPBNK cytotoxicity was assessed by
europium release assay. Raji or Raji-2R expressing luciferase was i.p. or s.c.
injected into NSG mice. NK therapy was given to each mouse once a week
for 3 weeks.
Results: 50 to 95% exPBNK was detected to express CAR at 16 hrs after
CAR mRNA nucleofection. CAR+ exPBNK has significantly enhanced in
vitro cytotoxicity compared to CAR- exPBNK against CD20+ rituximab
Figure 1. Anti-CD20 CAR Enhances expanded PBNK In-vitro
Cytolytic Activity against Rituximab sensitive and resistant cells.
Figure 2. Anti-CD20 CAR exPBNK cells significantly inhibit Raji
cells growth in xenografted mice.
sensitive Raji and resistant Raji-2R and Raji-4RH (p<0.001) (Fig.1). In Raji
mice model, the luciferase signals measured in CAR+ exPBNK treated mice
were significantly reduced than that in the control mice (P¼ 0.0087) and the
CAR- exPBNK treated mice (P¼ 0.0128) (Fig.2A). The CAR exPBNK
treated Raji mice had significantly extended survival time with median 40 days
compared to the untreated mice (29 days, P<0.001) and the CAR- exPBNK
treated mice (30 days, P<0.001) (Fig.2B). Similarly, in the Raji-2R mice
model, the luciferase signals were also significantly reduced in the CAR+
exPBNK treated mice compared to the CAR- exPBNK treated mice
(P<0.01). And the tumor size measured in the CAR+ exPBNK treated mice
was significantly smaller than that in the untreated (P¼ 0.0175) and the CARexPBNK treated mice (P¼ 0.0122).
Conclusion: CAR+ exPBNK inhibits BL cells growth in vitro and in xenografted mice. These results indicate therapeutic potential of multiple injections of CAR mRNA modified exPBNK cells against BL in patients.
52
THE CYTOTOXICITY OF CYTOKINE INDUCED KILLER CELLS
SEEMS TO BE INDEPENDENT BY LYTIC DEGRANULATION
AND IS FULLY RETAINED BY THE CD56+ CELL FRACTION
K Chieregato1,2, S Castegnaro1, C Zanon1, F Rodeghiero1,2, G Astori1
1
Department of Cellular Therapy and Hematolo, San Bortolo Hospital,
Vicenza, Italy, 2Hematology Project Foundation, Vicenza, Italy
Introduction: Cytokine induced killer cells (CIK) are mainly constituted by
two fractions: the CD56+ subset represented by NK-T cells (CD3+CD56+)
and NK cells (CD3-CD56+), and the CD56- subset characterized by T lymphocytes (CD3+CD56-). While the CIK antitumor effect seems to be mainly
restricted to the CD56+ fraction, the CD56- fraction is thought to be
responsible of CIK alloreactivity. We have investigated the CD56+ subset
cytotoxicity and alloreactivity by exploring its lytic degranulation and cytokine
secretion profile.
Methods: CIK were obtained by stimulating PB-MNC with IFN-gamma
(day 0), IL-2, anti-CD3 monoclonal-antibody (day 1) and IL-2 every 3 days
(n¼6). At day 21, CD56+ and CD56- fractions were sorted by immunomagnetic labeling. Cell degranulation was evaluated by CD107a expression,
and Th1 (IFN-gamma, TNF-alfa, IL2) and Th2 (IL4, IL6, IL10) cytokines
released in the supernatant after MLR and cytotoxic assay were quantified by
flow cytometric analysis. The alloreactivity of the CIK bulk and the sorted
populations was tested by mixed lymphocyte reaction (MLR) and cytotoxicity
against K562 cells by calcein-release assay.
Results: The CIK cytotoxicity was fully retained by the CD56+ cell subset,
and appeared to be independent by lytic degranulation as confirmed by the
absence of differences in the expression of CD107a on NK, NK-T and by the
cytokines released in the supernatant. This suggests that cytotoxicity could be
mediated by an alternative mechanism probably dependent on FAS-L or CD3
S22
Poster Abstracts
pathways. Alloreactivity was mainly restricted to CIK bulk and CD56- subset as
confirmed by the Th1/Th2 cytokines released in the supernatant.
Conclusion: In the contest of a clinical application, the immunomagnetic
purging of the CD56- cell fraction by the CIK bulk could limit the risk of Graft
Versus Host Disease maintaining at the same time the antitumor effect
restricted to the CD56+ cell subset.
53
HTERT/SURVIVIN
MRNA-LOADED
DENDRITIC
CELL
VACCINATION COMBINED WITH EX-VIVO EXPANDED T
CELL TRANSFER IN STAGE IV MELANOMA PATIENTS SHOW
A LONGER OVERALL SURVIVAL IN PATIENTS WITH
SUSTAINED IMMUNE RESPONSES AGAINST HTERT
I Bigalke1, SB Torhaug2, M Lundby1, C Mollatt1, E Inderberg-Suso1,
A Kolstad3, G Gaudernack4, A Rasmussen4, S Aamdal2, G Kvalheim1
1
Department of Cellular Therapy, Oslo University Hospital, Oslo,
Norway, 2Department of Clinical Cancer Research, Oslo University Hospital,
Oslo, Norway, 3Department of Oncology, Oslo University Hospital, Oslo,
Norway, 4Department of Immunology, Oslo University Hospital, Oslo,
Norway
Up to now immune responses following therapy of malignant melanoma with
dendritic cells (DCs) appear to be only transient. We investigated the possibility to prolong the immunological effect by treating melanoma stage IV
patients with a combination of hTERT/Survivin mRNA loaded autologous
DCs and expanded autologous T cells in a clinical phase I/II study. Patients
who showed immune responses against hTERT and/or Survivin after initial
treatment with DCs were offered additional treatment with ex-vivo expanded T
cells. T cells were enriched with the Elutra cell separator from leukapheresis
and expanded with CD3/CD28 Dynabeads for 10 days in a WAVE bioreactor.
Tregs were depleted prior to expansion. 3x1010 T cells were transfused fresh.
DC vaccination was continued the day after T cell transfer. Three patients who
showed immune responses either already before start of DC vaccination or 3-5
months after the first vaccination were treated with the DC/T cell combination. Patients one and two had a sustained response against hTERT peptides
after treatment with T cells. In patient one this response dropped 20 months
after beginning of DC-vaccination correlating with progression of the disease.
In patient two the response dropped 29 months after start of DC-vaccination
and was combined with an up-coming immune response against Survivin and a
progression of disease from week 31 on. In patient three the response against
hTERT peptides vanished at the time of T cell transfer and was combined with
a strong response against Survivin peptides. The disease progressed after 11
months. Patients without additional T cell treatment had a progression free
survival (PFS) of 3 to 13 months. These data indicate that immune responses
can be prolonged by additional T cell transfer. Prolonged responses against
hTERT seem to result in longer PFS. The role of responses against hTERT
and Survivin and their impact on clinical outcome in melanoma patients has to
be further investigated.
54
ADENOVIRUS (ADV) SPECIFIC T CELLS (CYTOVIRÔ ADV)
DEVELOPED AS AN ADVANCED THERAPY MEDICINAL
PRODUCT (ATMP), FOR THE TREATMENT OF ADV
INFECTIONS FOLLOWING AN ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTAT (ALLO-HSCT)
A Stansfield1, A Mitra1, S Thomas1, A Skulte1, W Qasim2, W Ip2,3,
A Friedetzky1, K Newton1
1
Cell Medica, London, United Kingdom, 2UCL Institute of Child Health,
London, United Kingdom, 3Great Ormond Street Hospital, London, United
Kingdom
ADV is a cause of significant morbidity and mortality after paediatric
HSCT. Antiviral drug therapy is only modestly effective for ADV infection
in HSCT patients, and is associated with significant toxicity. Although
antiviral drug therapy may attenuate viraemia in the short term, T cell
reconstitution is required for sustained viral clearance. Hence, adoptive virus-specific immunotherapy is one option for improved control. Here we
present a new approach to generating ADV-specific T cell therapy that
results in a therapeutic T cell dose from at least 90% donors, corresponding
to the percentage of the population that has been exposed to ADV. Lymphocytes for the expansion process are sourced from the transplant donor
and may be obtained from peripheral blood or a 5 ml aliquot of the original
stem cell donation. The one-touch rapid expansion process for generation of
this ADV cell therapy is based on the exposure of T cells to overlapping
peptides covering the entire hexon V protein and a 10 day expansion in the
presence of cytokines without further intervention until the cells are harvested and cryopreserved. The final product (Cytovir ADV), a formulation
of naturally occurring antigen-specific T cells, is formulated in cryopreserved doses of 1x10e4 and 1x10e5 CD3+ T cells per kg body weight
(BW) of the recipient in 4.5% human serum albumin (HSA) and 10%
dimethylsulfoxide (DMSO) with 1x10e2 ADV-specific T cells per kg BW
of recipient. The ATMP manufactured in this way is currently being studied
in children undergoing allogeneic HSCT at Great Ormond Street Hospital
London, UK. The study is designed to assess the safety and tolerability of
the ADV cellular therapy with results being expected in 2015. We conclude
that our one touch rapid expansion process for production of Cytovir ADV
is an efficient and robust process, suitable for GMP manufacturing to support further clinical development of this product in patients with ADV
infection post HSCT.
55
LONG TERM AND STABLE SUPPLY OF RECONSTITUTED
NEUTROPHILS
ACHIEVED
BY
REDUCED
INTENSITY
CONDITIONING WITH ALLO-BMT INDUCED REMISSION OF
INFAMMATORY STATUS IN X-CGD WITHOUT PROCEDURE
RELATED COMPLICATION
Y Takeuchi1,3, E Takeuchi2, D Ishii3, K Yoshida3
1
Internal Medicine, Kitasato University School of Medicine, sagamihara,
Japan, 2Immunology, Kitasato University school of Medicine, sagamihara,
Japan, 3Renal Transplant Unit, Kitasato University school of medicine,
sagamihara, Japan
Background: X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency of phagocytes, resulting in recurrent infection and
in development of granuloma formation by chronic inflammation. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the
curable procedures. However, this approach has been reluctantly offered
because of 1) procedure related toxicity, 2) graft failure, and 3) graft-versushost disease (GVHD). We have established the reduced intenstity conditioning with allogeneic bone marrow transplantation (allo-BMT) for
achievement of durable mixed chimerism without GVHD. This less toxic
regimen was attempted to mouse model of human X-CGD for cure without
complications.
Method: X-CGD male mice (H-2b/d) were given 3 Gy of TBI on day -1
and one injection of anti-CD40L mAb on day 0 with allo-BMT from BCF1
(H-2b/k) donor. Donor chimerism was analyzed by FCM. Body weight was
measured as a hallmark of procedure related complication. Reconstituted
neutrophils with reactive oxygen species (ROS) production was assessed
using APF dye, a noble probe for ROS. Therapeutic effect was assessed by
the development of granuloma formation against broken product of
Aspergillus fumigatus.
Results and Conclusion: X-CGD F1 mice treated with BMT regimen
showed durable mixed chimerism of lymphocytes and stable supply of functional reconstituted neutrophils within one year. The development of granuloma formation was suppressed in all chimeric mice, but it was apparent in
non-chimeric mice. Thus, long-term and stable supply of functional reconstituted neutrophils accompanied with durable mixed chimerism achieved by
reduced intensity BMT regimen remitted inflammation reaction in X-CGD
mice without procedure related complication.
56
EX-VIVO
TOLERANCE
INDUCTION
TO
TOLL-LIKE
RECEPTOR (TLR7) IN DONOR LYMPHOCYTES AS A
PROPHYLACTIC TOOL OF EXPERIMENTAL ACUTE GRAFT VS
HOST DISEASE (AGVHD)
N Zogas1,2, G Karponi1, F Iordanidis3, S Malasidis1, V Paraskevas1,2,
Z Scouras2, A Anagnostopoulos1, E Yannaki1
1
Gene and Cell Therapy Center-Hematology-BMT Unit, G. Papanicolaou
Hospital, Thessaloniki, Greece, 2Department of Genetics, Development and
Molecular Biology, School of Biology, Aristotle University, Thessaloniki,
Greece, 3Pathology Department, G. Papanicolaou General Hospital,
Thessaloniki, Greece
AGvHD is a life-threatening complication after allogeneic hematopoietic cell
transplantation (alloHCT) to which many conventionally treated patients are
resistant. TLRs are known to interact with pathogen-derived products and
endogenous self-antigens. Moreover, in-vivo TLR tolerance induction by TLR
agonist injections was shown to prevent experimental autoimmunity. In order
to address whether TLR tolerance induction could also prevent alloreactivity
and aGvHD by a clinically applicable approach, we investigated the ex-vivo
TLR tolerance induction to donor lymphocytes (DLs).We first tested the invitro TLR2,4,7 tolerance induction after exposure of mouse splenocytes
(mSPLCs) to repeated low-dose TLR-specific agonists followed by a high-dose
challenge and determined the optimal dose/duration for tolerance induction
by TNFa quantification in the mSPLCs culture supernatants. TLR7 “desensitization” in immunomagnetically-separated hPBMCs subpopulations
demonstrated that the TLR7 tolerance is mostly mediated by T-lymphocytes.
We subsequently examined the ex-vivo TLR4,7 DL tolerance induction in a
mismatched transplantation mouse model. The groups tested received: T-cell
depleted bone marrow cells (TCD-BM) alone or with mSPLCs (Groups-I,II)
and TCD-BM+TLR4- or TLR7-tolerized mSPLCs (Groups-III,IV).The
clinical assessment of aGvHD was based on a 10-point murine GvHD scoring
system. No differences were observed regarding survival and aGvHD rates
between Groups-I,IV. No animal from Groups-II,III survived beyond day25
post-transplantation, whereas Groups-I,IV showed significantly lower aGvHD
score and higher survival rates. Upon sacrifice, increased IFNg mRNA levels in
PBMCs and severe histological aGvHD lesions in affected tissues were
observed in Groups-II,III contrary to Groups-I,IV. Conclusively, the ex-vivo
induced TLR7 tolerance to DLs may serve as a novel and clinically applicable
form of cellular therapy that could prevent aGvHD and improve the outcome
of alloHCT.
57
CYTOTOXIC POTENTIAL OF INTERLEUKIN-15 STIMULATED
CYTOKINE INDUCED KILLER (CIK) AGAINST EPITHELIAL
CANCER CELL LINES
P Iudicone1, D Fioravanti1, E Cicchetti1, G Bonanno2, A Pandolfi1,
R Scocchera1, L Pierelli1,3
1
Immunohaematology and Transfusion Medicine - Stem cell and Cell Therapy
Unit, A.O. S. Camillo-Forlanini, Rome, Italy, 2Protection of Woman’s health
and child-adolescent life, Catholic University, Rome, Italy, 3Experimental
Medicine, University of Rome Sapienza, Rome, Italy
Cytokine Induced Killer (CIK) cells are T derived effector cells co-expressing
the CD3 and CD56 molecules, endowed with a non-MHC-restricted antitumor activity. CIK are induced by in vitro priming with IFN-g followed by
the activation with anti-CD3 and expansion with IL-2. In this study we
investigated the in vitro cytotoxic potential of CIK expanded with IL-2 (IL-2CIK) or with IL-15 (IL-15-CIK). CIK were generated from mononuclear
cells of three healthy donors and cultivated for 28 days in serum free medium
(Biowhittaker) additioned with IL-2 (Miltenyi) 300 U/ml or with IL-2
replaced by IL-15 (Miltenyi) 25 ng/ml by the 7th day of culture. Cell counts
and CIK phenotype were performed before starting the culture and at 7, 14,
21 and 28 days. At the end of culture CIK were also analyzed for the KIR
surface receptors and TCR ab and gd. Cytotoxicity was performed on 21 and
28 days of culture against the target cell lines HeLa (cervix adenocarcinoma)
and MCF-7 (breast adenocarcinoma) using flow-cytometry CFSE based assay.
In comparison with the protocol of CIK generation (IFN-g+anti-CD3+IL-2),
the replacement of IL-2 with IL-15 by the 7th day of culture allowed to reach
a lower amounts of CIK cells. Conversely, the results of cytotoxicity against
the epithelial tumor cells showed a higher killer activity of IL-15-CIK in
respect to IL-2-CIK, and the difference was more relevant at lower E/T
ratios: - 21 day % lysis IL-2-CIK vs IL-15-CIK / E/T 10/1 25% vs 34%,
5/1 13% vs 26%, 2/1 3.6% vs 11.3% as mean value; - 28 day % lysis IL-2CIK vs IL-15-CIK / E/T 10/1 16% vs 35%, 5/1 8% vs 21%, 2/1 L-2 2.6%
vs 12.6% as mean value. These preliminar data suggest that IL-15 is able to
generate CIK with greater cytotoxic potential against cancer cells of epithelial
origin.
58
INDUCTION OF ANTIGEN SPECIFIC T CELLS USING
PEPTIVATOR-PULSED DENDRITIC CELLS
M Takahara1, S Goto2, K Miki1, S Saiwaki1, K Nagaoka1, H Matsushita1,
T Kondo1, H Bohnenkamp3, T Yoshimoto4, R Maekawa1, T Kamigaki2
S23
1
Medinet Medical Institute, MEDINET Co., Ltd., Tokyo, Japan, 2Seta Clinic
Group, Tokyo, Japan, 3Miltenyi Biotec, GmbH, Bergisch gladbach,
Germany, 4Institute of Medical Science, Tokyo Medical University, Tokyo,
Japan
The PepTivator is a peptide pool consisting mainly of 15-mer sequences with
11 amino acids overlap and covering the complete sequence of target protein.
In this study, we used PepTivator ovalbumin protein (PepTivator OVA), and
evaluated the inducing activity of antigen specific CD8+ and CD4+ T cell
responses in a mouse model. In a clinical study we investigated the immunological response induced by PepTivator Wilms’ tumor gene 1 (PepTivator
WT1) and zoledronate (Zol) co-pulsed DC vaccines in solid tumors, and also
investigated the feasibility and safety. PepTivator OVA-pulsed DCs were
cultured with OT-I CD8T cells derived from OT-I transgenic mouse or OTII CD4T cells derived from OT-II transgenic mouse. The proliferation of
OT-I CD8T cells and OT-II CD4T cells cocultured with PepTivator OVApulsed DCs seemed to be similar to the proliferation of the cells cocultured
with OT-I epitope peptide (OVA257-264aa) and OT-II epitope peptide
(OVA323-339aa) pulsed DCs. As for cytokines secretion, no difference was
observed in concentration of IL-2 and IFN-gamma when PepTivator OVA
was compared with OT-I or OT-II epitope peptide, respectively. In a clinical
prospective study, five patients with WT1-positive solid tumor (ovary: 4, lung:
1) were enrolled. PepTivator WT1-pulsed Zol-DC was administered to patients subcutaneously for six times every two weeks. In this study no serious
adverse events were observed in three patients who had completed all DC
vaccines. In order to examine the specific immune responses to WT1 antigen,
PBMCs were in vitro restimulated with PepTivator WT1-pulsed Zol-DCs. In
two of three patients, the antigen specific IFN-gamma-producing cells were
detected in CD8+ T cells from PBMCs after therapy using ELISpot assay.
We found that PepTivator WT1-pulsed Zol-DC vaccines are feasible and
safe, and can induce antigen specific T cells without restriction to HLA. We
need more extensive investigation for the immunological monitoring and
clinical outcome.
59
TESTING CAR-T CELL ACTIVITY IN HUMAN HEMATOCHIMERIC MOUSE MODEL WITH THE NORMAL RANGE OF
ANTIGEN EXPRESSION NOT RESCTRICTED TO THE
TUMOUR ONLY
A Dolnikov1,2,3, A Chitranjan3, S Shen1,2, G Klamer2,3, T O’Brien1,2,3
1
Cord and Marrow Transplant Laboratory, Sydney Children’s Hospital,
Randwick, New South Wales, Australia, 2Faculty of Medicine, UNSW,
Randwick, New South Wales, Australia, 3Children’s Cancer Institute Australia
for Medical Research, Randwick, New South Wales, Australia
Immunotherapy using T cells modified to express CD19-directed chimeric
antigen receptor (CAR) has shown clinical efficacy in treatment of B cell leukaemias and lymphomas. To explore the impact of normal tissue expression of
the target antigen (Ag) we developed a ‘humanised’ mouse model to investigate
the effect of anti-human CD19 (hCD19)-specific CAR T cells against a normal
hCD19+ B cells. Ex vivo generated CAR-T cells targeting hCD19 were infused
to NSG mice transplanted with human haematopoietic stem cells generating all
essential immune subsets in the host including hCD19+B cells. Selective
elimination of CD19+ B lymphocytes but not other cell subsets was registered
in mice received CAR-T cells. Rapid deletion of CAR-T cells in mice reconstituted with large numbers of donor B cells was identified as a mechanism
impairing anti-tumour activity of CAR-T cells. We hypothesised that direct
antigen recognition on a number of host target cells over a minimal threshold
triggers CAR-T cell inactivation and justifying the use of lymphodepleting
conditioning prior to CAR-T cell infusion. We have shown that after
exhaustion of CAR-T cells, mice efficiently reconstitute B cells indicating the
preservation of stem cell pool. CAR-T cells composed of central and effector
memory cells showed low xeno-reactivity against the host antigens compared to
control mock-T cells that induced acute Graft-Versus-Host Disease suggesting
that donor engineered CAR-T cells combine rapid effector function with
reduced allo-reactive potential providing important advantages when used in
allogeneic stem cell transplantation. This humanised hemato-chimeric mouse
model can be used to test the efficacy and on-target toxicity of human CAR-T
cells to provide information that may prove pertinent to the design of future
CAR- based clinical trials and define additional strategies to enhance therapeutic efficacy of CAR-T cells.
S24
Poster Abstracts
60
STANDARDIZATION AND CHARACTERIZATION OF ATIR
CELL THERAPY PRODUCT: APPLYING QBD TO BOTH
PROCESS AND ASSAY DEVELOPMENT
Ld Jong1, J Velthuis1, J Darwiche2, M Corrieveau2, M Ruediger1, R Preti3,
D Roy2
1
Kiadis Pharma, Amsterdam, Netherlands, 2Hopital Maisonneuve-Rosemont,
Montreal, Quebec, Canada, 3PCT Progenitor, Allendale, New Jersey, United
States
Kiadis Pharma is developing ATIR, a T-cell immunotherapy based on a donor
lymphocyte preparation selectively depleted of host alloreactive T-cells to
reduce risks and improve outcomes of haploidentical stem cell transplantation
for blood cancer patients. Laboratory evaluations of ATIR have shown photodepletion of recipient-reactive cells and immune reactivity retention. ATIR
product demonstrated favorable anti-infection and anti-leukemia properties
without causing severe graft-versus-host disease in a Phase I clinical trial. In
2011, the company terminated a next-phase clinical trial, because the product
did not resemble ATIR, but contained dead and apoptotic cells. A root cause
failure investigation confirmed that the process should be re-engineered. A
Quality-by-Design (QbD) approach was taken towards both process and assay
development, aiming at (i) development of a standardized manufacturing
process for a phase II clinical study, and (ii) development of new cellular potency assays that accurately measure critical process and product attributes.
Experiments were designed to test single modifications in full ATIR runs.
Ultimately, comparability of the new process/product to the original process/
product was demonstrated in a head-to-head comparison, using the fixed and
qualified assays, currently used for release testing in ongoing clinical study CRAIR-007. This approach has resulted in a set of qualified assays fulfilling
predefined requirements, enabling methodical process characterization during
development. The knowledge gained about critical process parameters will be
highly valuable during next phase development steps. The standardized, robust
and transferable process resulting from the QbD approach is currently used for
manufacturing ATIR at CMO’s in North America and Europe for ongoing
clinical studies.
61
ANTI-CANCER VACCINATION USING MRNA-LOADED CMRF56 IMMUNOSELECTED BLOOD DENDRITIC CELLS
PD Fromm1,2, S Anguille3, M Papadimitrious1, C Bryant1,2, F Kupresanin1,
GJ Clark1,2, E Newman4, KF Bradstock1,5, ZN Berneman3, DN Hart1,2
1
Dendritic Cell Biology and Therapeutics Group, ANZAC Research Institute,
Sydney, New South Wales, Australia, 2Sydney Medical School, University of
Sydney, Sydney, New South Wales, Australia, 3Centre for Cell Therapy and
Regenerative Medicine, Antwerp University Hospital, Antwerp, Belgium,
4
Haematology Ambulatory Care Unit, Concord Repatriation General
Hospital, Sydney, New South Wales, Australia, 5Bone and Marrow Transplant
Services, Westmead Hospital, Sydney, New South Wales, Australia
Anti-cancer vaccination using mRNA-antigen-loaded monocyte-derived (mo-)
Dendritic Cells (DC) has shown promising clinical results in early trials. These
in vitro mo-DC differ from pre-formed blood Dendritic Cells (BDC) in terms
of migration and function. We used our BDC isolation platform to test
whether GMP-grade human-chimeric CMRF-56 isolated cells can be transfected with antigen-encoding mRNA and elicit antigen specific T cell responses. Chimeric anti-CMRF-56 isolated BDC nucleofected with mRNA
encoding the enhanced green fluorescent protein, displayed stable transgene
expression level over 48 hrs post-nucleofection, without compromising their
viability or ability to migrate to CCL21 in vitro. Protein expression was
highest in CD141+ DC, followed by the CD1c+ DC and residual monocytes
and B cells. DC loaded with mRNA coding for the influenza virus matrix
protein were capable of inducing autologous influenza-specific T cell responses that was not further enhanced by pre-activation of DC with GM-CSF
prior to nucleofection. Stimulation of CMRF56+ BDC loaded with Wilms
tumour antigen (WT1) mRNA was able to induce primary autologous CD4+
T cell responses but did not result in significant Interferon gamma (IFNg)
production by allogeneic WT1 126-134 specific CD8+ T cell clones. Nucleofection with WT1-mRNA containing a signal sequence for lysosomal associated membrane protein, resulted in dramatic increases in intracellular
transgene expression and the induction of WT1 126-134 specific IFNg production in some T cell clones but did not significantly increase autologous
IFNg CD8+ T cell responses over unmodified WT1 IVT-mRNA. Our data
shows that primary blood DC can be loaded efficiently with antigen using
mRNA nucleofection, resulting in high expression and the generation of
functional T cell responses. This new finding should encourage the investigation of primary human blood DC as a potentially superior DC source for
therapeutic vaccination.
62
DEVELOPMENT OF COL-TREG, AN ANTIGEN SPECIFIC TREG
BASED IMMUNE-CELLULAR THERAPY FOR JOINT AND EYE
INFLAMMATORY DISEASES
N Clerget-Chossat1, H Asnagli2, P Plence3, V Brun1, N Belmonte2,
M Forte1, C Jorgensen3, A Foussat2
1
Clinical, TxCell, Valbonne Sophia-Antipolis, France, 2R&D, TxCell,
Valbonne Sophia-Antipolis, France, 3Inserm U844, Montpellier, France
Treg cells are ideal tools for antigen-specific suppressive treatment of
inflammation. The objectives were to evaluate the therapeutic potential of
Collagen-II specific type1 Treg (Col-Treg) in murine models of Arthritis and,
based on this technology, to generate and characterize Col-Treg from PBMCs
of refractory Rheumatoid Arthritis (RA) patients.
Methods: Col-Treg were isolated, cloned, selected and expanded from
Collagen-II specific TCR transgenic mice and from RA patients’ PBMCs
exposed to collagen-II. Col-Teg therapeutic potential was evaluated after
adoptive transfer in collagen-antibodies and collagen-induced arthritis mice
models or through analysis of their suppressive function in vitro.
Results: A single Col-Treg cell-infusion led to a significant decrease of
incidence and clinical severity in both acute and chronic preclinical arthritis
models. Col-Treg migrated to the inflamed joints suggesting that the suppressive activity of Col-Treg is triggered locally by local collagen-II. Longterm tracking of Col-Treg in mice did not show any tumorigenicity or
unlimited proliferation. Production of Col-Treg was successful in RA patients
whatever the disease history and immunosuppressive or anti-inflammatory
treatment. Col-Treg from RA patients showed surface molecule expression and
cytokine secretion consistent with Type1 Treg identity. Consistent in vitro
suppressive activity of patients-Col-Treg on CD4+ T-cell proliferation was
observed.
Conclusion: We demonstrated efficacy and safety of Col-Treg infusion in 2
mice models of arthritis. Feasibility of translation of this model to the human
settings was documented by the successful production of functional suppressive
Col-Treg from RA patients. Collagen-II, the specific antigen for Col-Treg
cells is naturally present in joints and eyes. These results suggest that triggering
autologous Col-Treg activation is feasible and has a potential for treatment in
articular and ophthalmic inflammatory diseases.
63
THIRD GENERATION AUTOLOGOUS MYELOID-DERIVED
DENDRITIC CELLS DEVELOPED FROM PATIENTS WITH
CMML AND MDS DEMONSTRATE PHENOTYPIC PROPERTIES
OF MATURE FUNCTIONAL DENDRITIC CELLS
K Anderson1, I Dybedal2, I Bigalke1, D Schendel3, O Chowdhury4, P Woll4,
S Jacobsen4, G Kvalheim1
1
Department of Cellular Therapy, The Radium Hospital, Oslo University
Hospital, Oslo, Oslo, Norway, 2Department of Hematology, Rikshospitalet,
Oslo University Hospital, Oslo, Oslo, Norway, 3Deutsches Forschungszentum
für Gesundheit und Umwelt (GmbH), Helmholtz Zentrum München,
Neuherberg, Germany, 4Haematopoietic Stem Cell Biology Laboratory,
WIMM, Oxford University, Oxford, Oxford, United Kingdom
Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders of the elderly characterized by inefficient
haematopoiesis with frequent progression to AML. Chronic myelomonocytic
leukaemia (CMML) is a clonal haematopoietic malignancy characterised by
features of both MPN and MDS, including leukocytosis and monocytosis. It
has poor prognosis and with the exception of allogeneic bone marrow transplantation no curative treatments options. Active immunotherapy (AIT) using
dendritic cell (DC) vaccines have become an exciting concept for treating these
patients. Our purpose was to find out (1) whether DCs generated from CMML
and MDS patients acquire phenotypic and functional characteristics of mature
DCs; (2) how many patients could be potential candidates for DC vaccine
treatment taking in account their clinical characteristics and general condition.
We investigated 10 CMML patients, 5 MDS patients and 5 healthy controls.
Median age of CMML vs. MDS patients was 69 and 68. Mean monocyte count
was 4,0x109/l and 1,0x109/l for CMML vs. MDS patients. Mean haemoglobin
was 13 and 12 g/dl for CMML vs. MDS patients. Two CMML patients were
previously treated with either HU or Aza, while all MDS patients were under
active treatment at the time of sampling. All patients had ECOG status 0-1.
DCs were developed by a novel protocol consisting of 2 days of culturing
monocytes with GM-CSF and IL-4 followed by one day of maturation using
GM-CSF, IL-4, TNF, INF-g, PGE2, IL-1b and R848. We were able to
generate mature functional DCs in 9/10 CMML cases and all MDS patients.
There were insignificant variations in down-regulation of CD14 and expression
of CD80, CD86, CD40, CD83, CD274, CCR7 and HLA-DR for patients
compared to normal controls. Thus, all but one patient in the CMML group
and all patients in MDS group could be relevant candidates for AIT with DC
vaccine within a novel clinical protocol where DCs will be transfected with
WT1 and PRAME mRNA.
64
TENFOLD
EXPANSION
OF
REGULATORY
T
CELLS
HOMOZYGOUS FOR THE CCR5 GENE VARIANT D32 AFTER
CD3/CD28
ACTIVATION
IN
THE
PRESENCE
OF
EXOGENOUSLY ADDED RECOMBINANT IL-2 IN VITRO
K Pavelcova, M Trunda, J Chalupnikova, J Jencik, R Klubal, J Bodor
Czech Gene Bank, Prague, Czech Republic
The unprecedented power of the hematopoetic stem cell transplantation of the
CCR5D32/D32 cells resistant to HIV has been proved to cure HIV infection in
the case of leukemic patient (‘Berlin patient’) reported two years ago. Since
then another two cases which proved to cure HIV infection after hematopoetic
stem cell transplantation of the CCR5D32/D32 cells were reported from
Brigham and Women Hospital in Boston. Viral entry into CD4+ T cells is
mediated by the interaction with a cellular chemokine receptor, the most
common of which are CCR5 and CXCR4. The major barrier to viral eradication in patients receiving antiretroviral therapy is the establishment of HIV
reservoirs. Cells of persons homozygous for the CCR5 gene variant D32
(CCR5D32/D32) are naturally resistant to infection with CCR5-tropic HIV
strains (R5 HIV) because of the lack of functional CCR5 cell-surface expression. Our data based on the general population in Czech Republic show a
frequency of approximately 20% heterozygous persons. Four homozygous
persons bearing D32 mutation (CCR5D32/D32) were identified out of 709
individuals tested based on the genotyping of CCR5 D32 deletion. Here we
report a more than tenfold increase in the frequency of regulatory T cells
(Tregs) following CD3/CD28 co-stimulation within a week of in vitro cultivation of human Tregs, irrespective of their genotype. Importantly, similar the
treatments, which lead to the activation of Treg function in humans e e.g. antiCD3/CD2/CD28 stimulation simultaneously drove expansion of Tregs e.g.
using anti-CD3/CD28 and/or IL-2. Our study demonstrates a useful tool for in
vitro evaluation of Treg function and facilitates further understanding of the
mechanisms of immunological self-tolerance, which may also provide insights
into how strong immune responses, such as graft rejection, can be restrained
and engraftment of HIV resistant cells in HIV patients with AIDS lymphoma
or leukemia can be augmented.
65
A PRODUCTION PLATFORM FOR ANTIGEN-SPECIFIC TREGULATORY (AG-TREG) IMMUNOCELLULAR THERAPY OF
AUTOIMMUNE AND INFLAMMATORY DISEASES
N Belmonte1, H Asnagli1, V Brun1,2, N Clerget-Chossat2, M Forte2,
A Foussat1
1
R&D, TxCell, Valbonne-Sophia Antipolis, France, 2Clinical, TxCell,
Valbonne-Sophia Antipolis, France
Antigen-specific Treg (Ag-Treg) cells have shown tolerability and potential
efficacy in first in man clinical trials in inflammatory diseases such as
Crohn’s Disease. A robust production platform is required to exploit their
potential.
Methods: Human PBMCs are activated with a selected antigen (Ag)
depending on the condition to treat and the Ag localization in the target tissues. Ag-T cells are cloned and expanded in-vitro. Clones showing Treg
identity based on IL-10 secretion are selected, expanded and frozen. Mouse
Ag-Treg cells were produced from splenocytes. The Ag Ovalbumin (Ova),
S25
Collagen-type II (Col-II), Myelin-Oligodendrocyte-glycoprotein (MOG),
Dust-mite antigen Derp1 and Heat-shock Protein-60 (HSP60) have been used
in mice and/or in humans for Ag-Tregs production to treat inflammatorybowel disease, joint/eye inflammatory diseases, central nervous system inflammatory diseases, dust mite allergy and other autoimmune diseases,
respectively.
Results: Ag-Tregs for different antigens were successfully produced using
the production platform. Adoptive transfer of mouse Ova-Tregs, Col-Tregs
and MOG-Tregs leads to inhibition of inflammation in animal models of inflammatory colitis, Rheumatoid Arthritis and Multiple sclerosis, respectively.
Ag-Treg cells migrated in inflamed tissues within 24hours. Ova-Treg and ColTreg were produced from the blood of healthy donors and Crohn’s Disease or
Rheumatoid arthritis patients, respectively with comparable surface markers
expression and cytokine profile. Human Ag-Treg cells were successfully produced with HSP60 and Derp-1 demonstrating Ag-Treg production platform
can address other inflammatory diseases.
Conclusion: We developed a production platform for Ag-Treg cells
allowing treatment of multiple inflammatory conditions according to the Treg
specific antigen. This technology triggers Treg cell suppressive activity leading
inflammation inhibition by targeting Ags localized in the inflamed tissues.
66
MESENCHYMAL STEM/STROMAL CELL-DERIVED MICROPARTICLES SHOW ANTI-INFLAMMATORY ACTIVITY IN AN
ANIMAL MODEL OF ULCERATIVE COLITIS
A Del Fattore, R Luciano, A Fierabracci, M Muraca
Regenerative Medicine Area, Children’s Hospital Bambino Gesù, Rome, Italy
Exogenous Mesenchymal Stem/Stromal Cells (MSCs) contribute to the recovery of tissue injury both by improving tissue regeneration and by modulating the immune response. The latter effect led to clinical applications for
the treatment of disorders such as GVHD and inflammatory bowel disease.
At experimental level, exogenous MSCs administration in rodent models of
ulcerative colitis improves the pathologic condition, with reduced expression
of inflammatory cytokines. Recently, it was shown that the regenerative
stimulus of MSCs can be mediated by the release of microparticles (MPs). We
showed that MSC-MPs exhibit immunomodulatory activity both on B and T
cells in vitro. Here, we evaluated the immunomodulatory ability of MPs in
vivo in a model of ulcerative colitis induced in C57BL/6J mice by administration of Dextran Sulfate Sodium (3%) for 5 days. Results are expressed as
MeansSD, n¼5. When compared to vehicle-treated controls, mice injected
daily with MPs intraperitoneally showed improved disease activity index
(2.30.1 vs. 1.90.2, p<0.05. Index 1:Normal to semisolid stools, no blood;
2: Semisolid to fluid stools with definitive evidence of blood; 3: Bloody fluid).
A less severe reduction in colon length was observed in MP-treated vs.
vehicle-treated animals (6.700.25cm vs. 5.740.3cm; p<0.05). Real time
RT-PCR analysis performed on RNA extracted from colon tissue revealed a
strong inhibition of the induction of inflammatory cytokines with respect
to vehicle-treated animals: (IL-1b 57.0042.55 vs. 146.6262.00,p<0.05;
COX2 0.830.23 vs. 1.340.24, p<0.05). These results suggest that MSCMPs exhibit anti-inflammatory activity also in vivo and might thus have
therapeutic potential.
67
IMMUNOREGULATORY EFFECTS OF MESENCHYMAL STEM
CELL-DERIVED MICROPARTICLES ON T LYMPHOCYTES
A Del Fattore, R Luciano, F Capolunghi, R Carsetti, B Goffredo, E Giorda,
A Fierabracci, M Muraca
Children, Rome, Italy
The immunomodulatory activity of mesenchymal stem cells (MSCs) is largely
mediated by paracrine factors. We have recently shown that the immunosuppressive effects of MSCs on B lymphocytes in Peripheral Blood Mononuclear Cell (PBMC) culture can be fully reproduced by membrane
microparticles (MPs) isolated from MSC culture supernatants. Here, we
investigated the effect of bone marrow-derived MSC-MPs on T cells in 5-day
PBMC cultures stimulated with aCD3/aCD28 beads. Results (n. events in
20000 lymphocytes) are MeansSD; n¼6. Stimulation significantly increased
the number of proliferating CD3+ cells (6410.004175.00 vs. 91.1769.81,
p<0.05) as well as of CD4+/CD25+/CD127low T regulatory cells (Tregs)
(1340.00523.80 vs. 5.674.68, p<0.05). Stimulation did not affect the
S26
Poster Abstracts
number of apoptotic cells (255.00105.90 vs. 121.4081.38, p¼n.s.). Coculture with MSCs did not affect apoptosis, but a reduced proliferation of
both CD3+ cells and Tregs was observed. Addition of MSC-MPs to PBMC
cultures on days 0 and 1 did not affect proliferation of CD3+ cells
(101534799 vs. 64104175, p¼n.s.), but induced a 3-fold increase of
apoptosis in this cell population (826.20549 vs. 255.00105.90, p<0.05).
Moreover, addition of MPs induced a 2-fold increase of Treg proliferation
(3018.00933.90, p<0.05). Similar effects were observed with MSCs and
MSC-MPs isolated from umbilical cord. The activity of indoleamine 2,3dioxygenase (IDO), an established mediator of MSC immunosuppressive effects, was increased in supernatants of PBMCs co-cultured with MSCs, but
was not affected by the presence of MSC-MPs. In conclusion, MSC-MPs
demonstrate immunomodulatory effects on T cells in vitro. However, at
variance with previous observations on B cells, both these effects and the
underlying mechanisms appear to be different from those exhibited by the
parent MSC cells.
68
CYTOKINE MILIEU IN THE LYMPHATIC FLUID AND SERUM
OF CHRONIC PATHOLOGY PATIENTS WITH BANCROFTIAN
LYMPHATIC FILARIASIS.
S Magapu1, M Pandian2, Parakkal3, S Chandran2, Neog2, G Nagarajan2
1
Department of Applied Biotechnology, Ministry of Higher Education,
College of Applied Science, Sur-Sultanate of Oman, Oman, 2Department of
Biotechnology, Rajalakshmi Engineering College,Thandalam, Chennai,
India, 3Department of Immunology,NIH-ICER-NIRT, National Institute for
Research in Tuberculosis, Chennai, India
Aims: Lymphatic Filariasis (LF), a chronic infection caused in humans by the
nematode parasites Wuchereria bancrofti, Brugia malayi & B. Timori
was studied with an aim at understanding the cytokine milieu in CP patients
of filarial subjects and comparing the paradigm with cancer subjects
to comprehend the immunopathological mechanism of lymphoedema
formation.
Methods: Clinical spectrum in LF has3 kinds of patients; Endemic
normal (EN), Chronic pathology (CP) & Microfilariamics (MF), eliciting
Th1,Th1/Th2&Th2 respectively. Samples of Lymphatic fluid & corresponding Serum were collected from CP patients sequential analysis was
done.GroupA:10 SkinCancer & 13 filarial CP patients. GroupB & GroupC:8
& 15 filarial CP patients,respectively. Cytokine analyses were done in lymph
& serum of the CP patients using (1) Bio-Plex Cytokine Bio-Assay instrument (BioRad, USA); (2) antibody analysis in lymph & serum against Bancroftian filariasis, (3) the antigenic analysis using Trop-Bio Kit for
circulating filarial antigen in the lymph & serum of CP patients compared
with EN.
Results: ManeWhittney analysis was done. Level of expression for IL1b: same in both patients with variability for filarial subjects unlike the
cancer subjects. TNF- a level:low for both cancer & filarial subjects.IL-6
levels:high in cancer than the filarial subjects. Group B showed skewed up
levels of GM-CSF than IFN-gamma. TNF-Alpha was lower than IL-1
lower, than IFN-gamma. IL-10, IL-13, GM-CSF, IFN-Gamma were
skewed up in Group C.
Conclusion: The cytokine paradigm in LF CP patients exhibits both Th1 &
Th2 immune response thus showing a mixed response, as analyzed in lymphatic
fluid & serum of the same individuals. The expression profile of IgG responses,especially specific IgG4 responses in lymph & serum of CP patients,
compared to EN samples were shown in group C.The Ag analysis showed
significant presence of parasite specific Ag.The pro-inflammatory cytokines
play a pivotal role in the disease progression in CP like in cancer & LF
lymphoedema.
69
THE WAVE BIOREACTORÔ 2/10 SYSTEM CAN PRODUCE
CLINICALLY RELEVANT CELLS
K Wernersson1, M Janas2, C Mölleryd1, B Mark2
1
GE Healthcare, Uppsala, Sweden, 2GE Healthcare, Cardiff, United Kingdom
Lymphocytes, expanded for clinical use, often consist of a small selected
starting population, which requires multiple rounds of replication to achieve
therapeutic doses. By using perfusion culture with the WAVE Bioreactor 2/10
System, high cell density cultures which are sufficient for therapeutic doses, can
be generated. The CellbagÔ bioreactors, used together with the WAVE system, are functionally closed, single-use bioreactors that are delivered presterilized and suitable for cGMP production. Perfusion is automatically
maintained by the WAVE system, which removes metabolites through an internal filter while supplying the culture with nutrients, thus keeping the culture
in a steady state. The handling of only one culture using the WAVE system,
compared to having to manipulate multiple T-flasks, simplifies the sampling
process. Cells produced for use in clinical trials often need to meet specific
release criteria, which can include showing that the product contains low levels
of endotoxin and is sterile. Since the cells grown in the Cellbag bioreactor are
contained in one homogenous mixture, only one sample is required to get a
representative cell count or to analyze release criteria. Such samples can be
withdrawn from the Cellbag through the sample clave port, by connecting a
Luer syringe or other device, without the need of opening the system or
transferring it to a laminar air flow hood, thus keeping the system functionally
closed.
70
XENO-FREE SERUM REPLACEMENT SUPPLEMENT FOR EX
VIVO CULTURE AND EXPANSION OF T CELLS
K Schjetne, G Økern, S Kuligowski, M Bonyhadi, T Aarvak
Life Technologies Corp., Oslo, Norway
Background: The manufacture of a majority of clinical T cell products for
immunotherapy applications requires ex vivo T cell culture and expansion.
Commercialization of T cell manufacturing processes requires reagents that
meet regulatory guidelines and ultimately help reduce manufacturing cost of
goods. A key component in many T cell culture protocols is human serum,
which is expensive and may require testing prior to use for the manufacture of a
cGMP-compliant T cell product. To this end, we have developed a xeno-free
serum replacement supplement with defined components that can be used in
combination with several different cell culture media to support ex vivo
expansion of T cells.
Results: T cells activated ex vivo and expanded with DynabeadsÒ CD3/
CD28 CTSÔ and cultured in OpTmizerÔ CTSÔ, X-VivoÔM 15, or AIMVÒ CTSÔ supplemented with pooled human serum or serum free T cell
serum replacement showed similar growth kinetics, total fold expansion and
transduction efficiency after 2 weeks in culture. Numbers of expanded CD4+
and CD8+ T cells were comparable in the expanded cultures regardless in the
presence of human serum or the newly developed serum free T cell serum
replacement. Restimulated T cells expanded in serum free T cell serum
replacement show similar cytokine profile and proliferation as T cells expanded
in human serum.
Conclusion: This study shows that human serum may be replaced by a
xeno-free formulation in several commonly used cell culture media to support
ex vivo expansion and lentiviral transduction of polyclonal T cells. Culturing T
cells in serum free T cell serum replacement facilitates a favorable culture
profile and immune function. The serum free T cell serum replacement contains only fully tested human-derived or human recombinant proteins which
facilitates supply security for clinical large scale and commercial therapies.
71
SCREENING AND ENUMERATION OF VIRUS- AND TUMORASSOCIATED ANTIGEN-SPECIFIC CD8D T LYMPHOCYTES
USING
STREPTAMER
TECHNOLOGY
AND
SINGLEPLATFORM FLOWCYTOMETRY ASSAY
S Matko1, M Teichert1, M Odendahl1, M Wohsmann1, T Tonn1,2
1
German Red Cross Blood Donation Service North-East, Dresden,
Germany, 2University of Technology Dresden, Dresden, Germany
Adoptive cell therapy has become an important component of virus-refraction-treatment after allogeneic stem cell transplantations (ASCT). Enabling
direct visualization and efficient enrichment of antigen-specific cytotoxic
CD8+ T-Lymphocytes (CTLs) Streptamer technology may allow the identification of rare tumor-(WT1)-antigen specific CTLs as an option for
treatment of AML-relapse-patients. To examine quality and sensitivity of
Streptamer technology, a defined number of CMV pp65 A*0201
(NLVPMVATV) specific CTLs were titrated in blood without CMV-specific
CTLs. Single-platform flow cytometry and True Count tubes provided the
benefit to assess absolute cell numbers directly within the blood samples.
Intra-assay-deviation was calculated after application of CMV pp65 B*0702
(TPRVTGGGAM) Streptamers in ten separate stainings, using the same
donor-material. For WT1 donor-screening, we used a HLA A*0201 restricted
Streptamer, presenting the peptide RMFPNAPYL. Quality control through
spiking of antigen-specific CTLs showed a linearity of r2>0.99 and a
detection limit of 1 CMV pp65-specific CTL per microliter, correlating with
a frequency of 0.0320.0033. Intra-assay-deviation stated a frequency of
0.60.03 (SD 5%). Screening results for WT-1 specific CTLs in 88 HLA
A*0201 positive donors with a mean frequency of 0.003 and a SD of 0.005
corresponded to background noise. Also the maximum frequency of
0.0260.005 did not state a distinct population. In comparison CMV A*0201
pp65/B*0702 pp65 screening in the same group of donors showed a mean
value of 0.2190.899/0.7841.602 and a maximum frequency of
7.5960.899/5.1751.602. In conclusion, Streptamer technology is a reliable
technique with high sensitivity and reproducibility. Also, our data indicate
that the frequency of WT-1 specific CTLs among healthy donors is below the
detection limit of Streptamer technology.
72
IMMUNOTHERAPY WITH DONOR LYMPHOCYTES DEPLETED
OF ANTI-HOST REACTIVE CELLS RESULTS IN SAFE AND
EFFICACIOUS HAPLOIDENTICAL HSCT: INTERIM RESULTS
FROM A PHASE II TRIAL IN PATIENTS WITH HEMATOLOGIC
MALIGNANCIES
D Roy1, J Maertens2, I Walker3, R Foley3, P Lewalle4, D Selleslag5,
J Velthuis6, L Gerez6, K Reitsma6, E Wagena6, J Roy1, S Lachance1, S Mielke7
1
Hematology-Stem cell transplantation, Hopital Maisonneuve-Rosemont,
University of Montreal, Montreal, Quebec, Canada, 2Hematology, University
Hospital Leuven, Leuven, Belgium, 3Medicine, Juravinski Hospital and Cancer
Centre, Hamilton, Ontario, Canada, 4Experimental Laboratory of
Hematology, Institut Jules Bordet, Brussels, Belgium, 5Hematology, AZ St-Jan,
Bruges, Belgium, 6Kiadis Pharma B.V., Amsterdam,
Netherlands, 7Hematology-Oncology, Universitätsklinikum Würzburg,
Zentrum Innere Medizin (ZIM), Würzburg, Germany
Haploidentical hematopoietic stem cell transplant (HSCT) is an appealing
alternative for patients lacking an HLA-matched donor. However, haploidentical HSCT necessitates intensive T-cell depletion that results in
frequent and often lethal infectious complications and/or high relapse rates. In
an open-label multi-center phase 2 study, 23 patients with AML, ALL or MDS
will undergo an immunotherapeutic approach consisting of donor lymphocytes
selectively photodepleted of host alloreactive T-cells (ATIR) infused 28-32
days after a haploidentical CD34-selected HSCT. No post-transplant GVHD
prophylaxis is used.
Results: As of December 12, 2013, 7 patients have been treated with ATIR
(mean age 44, range 21-61), AML¼4, ALL¼3, CR1¼2, CR2¼5, with a mean
f-up of 4 months post HSCT (range 2-7 months). Two additional grafts have
been treated ex vivo and 3 patients are being prepared for HSCT. Stable
engraftment was achieved in all patients at a median of 11 days (8-18) for
neutrophils and 12 days (9-23) for platelets. No patient experienced graft
rejection. None of the patients developed acute GVHD (any grade), no severe
Figure 1A. : CFSE dilution pattern of ATIR cells stimulated with
3rd party cells. B: Representative donor cells (grey) and ATIR final
product (black) CFSE-based proliferation against donor, recipient,
and 3rd party cells as well as CD3/CD28 stimulation confirm selective depletion of alloreactive T cells and preservation of immune
reactivity.
S27
infections (grade 3 or 4) were reported, and no patient relapsed or died. ATIR
(9 batches) mainly consists of T-cells (>90%), with 10% B and NK cells.
Donor cell proliferation toward donor, recipient, and third party cells, as well
as CD3/CD28, measured by CFSE labelling, before photodepletion and in the
ATIR cell product show elimination of anti-host reactivity and preservation of
immune reactivity toward third-party cells and anti-CD3/CD28 (Figure 1).
Moreover, donor cytotoxic T-lymphocyte precursors (CTLp) toward the host
are decreased by more than 95%.
Conclusions: These interim data confirm that novel immunotherapy consisting of donor lymphocytes selectively photodepleted of alloreactive cells
(ATIR) can be manufactured consistently and reproducibly, and preserve antiinfectious activity without promoting GVHD in patients with hematological
malignancies.
73
PRODUCTION OF T CELLS FOR ADOPTIVE CELLULAR
IMMUNOTHERAPY USING THE XURI CELL EXPANSION
SYSTEM W25
R Ismail
Cell Biology, GE Healthcare, Cardiff, United Kingdom
Introduction: Immunotherapeutic products can be classified broadly into (1)
active immunotherapy (therapeutic vaccines), (2) adoptive cellular immunotherapy (transfer of immune cells, genetically modified T-cells or precursor
cells) or (3) passive immunotherapy (antibody or receptor ligand administration). Adoptive cellular immunotherapy products are the most recent to show
signs of benefit and therapeutic value to the patient population, and there is
now an emerging need to develop more effective manufacturing processes that
will require less user intervention, permit scale-out, minimise the risk of cell
contamination, achieve high cell densities, allow careful control of the expansion protocol and be suited for a regulated environment. The XuriÔ Cell
Expansion System W25, based on the well-known WAVETM rocking technology, offers many advantages in this regard over the commonly used static
systems. Xuri W25 is a functionally closed, single use bioreactor for working
volumes between 0.3 and 25L, which is suitable for use in a regulated environment and offers comprehensive process monitoring and remote control
capabilities. Data presented here compares the performance of Xuri W25 to
commonly used static bioreactors with regard to workflows, cell yield, cellular
phenotype, functional performance, cost and risk. The expanded T cells remain
biologically functional and can be re-activated post expansion to produce high
amounts of cytokines. The cytokine expression profile confirms the presence of
TH1 phenotype, naïve/early phenotype and low in senescence markers. This
data demonstrates that Xuri W25 is capable of expanding functional T-cells for
therapy in a safer, low risk and more cost effective way compared to the static
systems used in this study.
74
ENHANCED EXPRESSION AND ANTI-TUMOUR ACTIVITY OF T
CELLS ENGINEERED TO EXPRESS A CYSTEINE-MODIFIED
TCR TARGETING A FRAMESHIFT MUTATION IN MSI-HI
COLORECTAL CANCER
E Inderberg-Suso1, S Wälchli1, MR Myhre1, M Wang1, H Almåsbak1,
G Kvalheim1, G Gaudernack2
1
Dept. of Cellular Therapy, Oslo University Hospital-The Norwegian Radium
Hospital, Oslo, Norway, 2Section for Immunology, Oslo University HospitalThe Norwegian Radium Hospital, Oslo, Norway
T-cell receptor (TCR) transfer is an attractive strategy to increase the number
of cancer-specific T cells in adoptive cell therapy. However, recent findings
both in the clinic and in pre-clinical mouse models indicate that careful consideration of the target antigen is required to avoid on-target, off-site toxicity. Rather
than targeting tumour-associated self-antigens, it may be safer directing engineered T cells against mutated proteins such as frequently occurring frameshift
mutations. Furthermore, frameshift mutations result in novel polypeptides
allowing selection of TCRs from the non-tolerant T-cell repertoire avoiding the
problem of low affinity TCRs due to central tolerance. The transforming growth
factor b Receptor II frameshift mutation (TGFbRIImut) is found in hereditary
non-polyposis colorectal cancers and in around 15% of sporadic colorectal and
gastric cancers displaying microsatellite instability. The -1A mutation in an
adenine stretch of the TGFbRII gene gives rise to immunogenic peptides which
S28
Poster Abstracts
have been used for vaccination of MSI+ colorectal cancer patients in a Phase I
clinical trial. From a responding patient we isolated a CD8neg/CD4neg CTL
clone from which we cloned an HLA-A2-restricted TGFbRIImut-specific TCR.
We showed that both CD8+ and CD4+ T cells transiently redirected with this
TGFbRIImut-TCR recognised colon carcinoma cell lines harbouring the
frameshift mutation, indicating CD8+ co-receptor independency. Moreover, we
demonstrated that TCR-transfected T cells could reduce the growth of colorectal
cancer in a NOD/SCID xenograft mouse model. The TCR-expression vector was
designed by fusing the TCRa and b chains with the picornavirus-derived 2A selfcleaving peptide to promote parallell expressions of the chains. Although surface
levels of the TGFbRIImut-TCR in the redirected T cells were close to that of the
parental T-cell clone, we found that cysteine modifications could further improve
expression and in vitro function of the TCR.
75
ALTERNATIVE APPROACHES IN THE CLOSED SYSTEM
MANUFACTURING FOR DENDRITIC CELL VACCINES
AJ Dafferner1, Hc Britton1, P Warkentin2, J Talmadge1
1
Pathology and Microbiology, University of Nebraska Medical Center,
Omaha, Nebraska, United States, 2Biologics Production Facility, University of
Nebraska Medical Center, Omaha, Nebraska, United States
DC vaccines hold promise as cancer therapy; however, their manufacture
under GTP requires a closed system. We initially used an ElutraÒ (TerumoBCT), a device that allows semi-automatic enrichment of monocytes,
with a single-use, closed and cGMP compliant tubing set. However, some
mononuclear apheresis products from cancer patients had a high frequency of
hypodense myeloid cells, or myeloid-derived suppressor cells (MDSCs), that
interfered with monocyte isolation by elutriation. Despite their low density,
they have a granulocyte morphology and <1% bands or metamyelocytes.
Phenotypic analysis of some products revealed that >98% of the granulocytes
(FSxSS) were MDSCs i.e. lineage-(CD3,CD19,CD56)CD14dullCD13dullCD11b+CD33+. Because we could not separate MDSCs from monocytes
by Ficoll-Hypaque, elutriation or freeze-thaw, we investigated the use of
VuelifeÒ 72-AC bags (Saint-Gobain), cGMP compliant, 510(k)-cleared,
fluorinated ethylene propylene (FEP) containers designed for adherent culture. Studies of monocyte enrichment from an apheresis product with the
VuelifeÒ AC bags revealed yields of approximately 70%, and preliminary data
showed lower granulocyte and lymphocyte contamination than in plastic flask
enrichment. Adherence was undertaken at 1x106 cells/ml using serum-free
CellGenix DC media and a 30-minute incubation on stainless-steel racks in
5% CO2 37C incubators. Following a single wash adherent monocytes were
cultured in the same bags, resulting in DC purity >85%. The recovered DCs
(CD11c+DR+) were immature with 40-80% expressing CD14. The DCs were
transferred between bags by sterile docking including freezing in the
KryosureÒ 6-F FEP freezing bag (Saint-Gobain). The resulting DCs had a
high frequency of CD80 and CD86 expression and low CD83 expression
levels. In summary, VuelifeÒ AC bags, pumps, and sterile closure system(s)
allow DC manufacturing of MDSC products in a functionally closed and time
efficient manner.
76
GENERATION AND QUALIFICATION OF A GMP COMPLIANT
MASTER CELL STOCK OF CAR EXPRESSING ERBB2-SPECIFIC
NK-92 CELLS FOR CLINICAL TRIALS
P Nowakowska2, MK Odendahl1, WS Wels3, M Grez3, S Naundorf4,
H Bönig2, E Seifried2, T Tonn1,5
1
Experimental Transfusion Medicine, Institue for Transfusion Medicine
German Red Cross Blood Donation Service Desden, Dresden, Saxonia,
Germany, 2Institute for Transfusion Medicine and Immunohaematology,
German Red Cross Blood Donation Service Baden Württemberg-Hessen ,
Frankfurt a. Main, Saxonia, Germany, 3Chemotherapeutisches
Forschungsinstitut Georg-Speyer-Haus, Universität Frankfurt a. M., Frankfurt a.
Main, Hessen, Germany, 4EUFETS GmbH, Idar-Oberstein,
Germany, 5Transfusion Medicine, Medical Faculty Carl Gustav Carus, TU
Dresden and CRTD, Dresden, Saxonia, Germany
Introduction: The continuously growing NK-92 cell line is highly cytotoxic
against a broad spectrum of tumor targets and has completed phase I clinical
trials in humans. To further improve the anticancer activity towards ErbB2
expressing cancer cells NK-92 cells from an FDA-licensed master cell stock
(MCS) were transduced under GMP-compliant conditions with an ErbB2specific humanized CAR construct by lentiviral gene transfer.
Aim: Generation, qualification and release testing a GMP compliant MSC
of genetically modified CAR expressing NK-92 cells for clinical trials.
Methods: CAR expressing NK-92 cells were cultured and expanded in XVivo 10 medium supplemented with 5% heat inactivated human fresh frozen
plasma and 500U/ml of IL-2 in VueLife bags to a density of 5x10e5 cells/ml
and frozen. After thawing the identity and stability of CAR expression were
monitored by staining with ErbB2/IgG fusion protein and anti-human IgG
F(ab)2-APC. The potency was analyzed using FACS-based cytotoxicity and
methylcellulose assays coincubating CAR expressing NK-92 cells either with
ErbB2 positive or negative cancer cells. Final testing for identity and sterility of
the MSC was performed in accordance with the principles of GMP for qualification and release by Bioreliance.
Results: A MCS comprising 200 ampules of 2,5x10e7 CAR expressing NK92 cells was established and qualified to meet predefined specifications with
regard to identity, stability (CAR expressing cells >99%) and potency (specific
cytotoxicity >85%). Qualification and release testing confirmed the identity
(Karyology, Analysis of Isoenzymes, DNA fingerprinting) and sterility (absence
of microbial organisms, adventitious viral agents and retroviral reverse transcriptase activity) of the established MCS.
Conclusion: The GMP compliant MCS of a humanized CAR expressing
ErbB2-specific NK-92 cells meets all required specification (identity, stability,
potency, sterility) and may serve as a platform for future clinical trials.
77
A BISPECIFIC CHIMERIC ANTIGEN RECEPTOR TARGETING
ANTIGEN ESCAPE VARIANTS IN GLIOBLASTOMA
M Hegde1, A Corder1, Z Grada1, TT Byrd1, KK Chow1, VS Brawley1,
SS Krebs1, H Heslop1, S Gottschalk1, E Yvon2, N Ahmed1
1
Center for Cell and Gene Therapy, Pediatric Hematology Oncology, Baylor
College of Medicine, Houston, Texas, United States, 2Department of Stem Cell
Transplantation, MD Anderson Cancer Center, Houston, Texas, United States
Highly selective targeted immunotherapy using chimeric antigen receptor (CAR)
modified T cells is emerging as a safe and effective modality for the treatment of
cancer. We have observed, however, that targeting a single tumor antigen results in
antigen loss variants, culminating in relapse. We reasoned that simultaneous targeting of multiple tumor restricted antigens could result in a tolerance-proof
mechanism to offset antigen escape and lead to better therapeutic efficacy. We have
genetically engineered T cells from glioblastoma (GBM) patients’ to express a novel
bispecific CAR that incorporates two extracellular antigen-recognition domains, a
mutated IL-13 molecule to target IL13Ra2, and a HER2 specific scFv, based on the
HER2 monoclonal antibody FRP5, in tandem (TanCAR). TanCAR was designed
using systematic computational modeling, assembled on clone manager, synthesized, then sequence verified and force expressed on T cells using a moloneyderived retroviral system. Engagement of TanCAR by cognate ligands induced
activation of T cells, which had distinct effector activity against individual target
antigens, HER2 and IL13Ra2, and enhanced functionality upon simultaneous
encounter of both molecules. In addition, TanCAR T cells exhibit improved antitumor activity against autologous GBM cells demonstrating their potential for
therapeutic application.
78
USE OF A LOW-COST PASSIVE FREEZING DEVICE IN THE
EFFECTIVE CRYOPRESERVATION AND RECOVERY OF HUMAN
REGULATORY T-CELLS FOR USE IN A CELL THERAPY TRIAL
A Foussat2, R Rietze2, M Thompson1, B Schryver1, R Ehrhardt1
1
BioCision, Larkspur, California, United States, 2Tx Cell, Sophia Antipolis,
Valbonne, France
Regulatory T cell therapy represents a promising new frontier in the immunotherapy of autoimmune disorders, especially for patients refractory to available
treatments. Because of their intrinsic nature, cell therapy products can be highly
sensitive to variation in manufacturing procedures. The standardization of
Drug Product cryopreservation and storage steps are thus key to ensuring
consistent trial results. Researchers at TxCell are currently investigating the
use of an autologous antigen-specific Treg (Ag-Treg) cell-based immunotherapy for the treatment of patients with severe refractory Crohn’s disease.
In order to ensure a standardized and consistent Drug Product freezing and
to minimize batch-to-batch differences in cell recovery and viability postthaw, TxCell scientists tested the CoolCellÒ container, a passive freezing
device, as an alternative to the classical controlled rate freezer. Results
showed that by using a CoolCell freezing device, Ag-Tregs can be successfully cryopreserved and recovered in a standardized way acceptable to
the processing and manufacturing of cell therapies. TxCell now intends to
use the CoolCellÒ device in its phase IIb clinical study with its lead AgTreg cell product candidate, OvasaveÒ. Use of the CoolCellÒ passive
freezing device for cell therapy manufacturing of Ag-Treg represents a new
standardized method of cell therapy product cryopreservation. The ability
to develop and store functional Treg in a cost-effective and reproducible
manner is an important milestone in the ultimate use of these cells in the
clinic.
79
VALIDATION OF A NOVEL PORTABLE FREEZING DEVICE IN
THE
OPTIMAL
FREEZING
OF
PERIPHERAL
BLOOD
MONONUCLEAR CELLS FOR POTENTIAL CELL THERAPY USE
M Thompson1, Q Tang2, B Schryver1, R Ehrhardt1
1
BioCision, Larkspur, California, United States, 2Surgery, UCSF, San
Francisco, California, United States
Peripheral blood mononuclear cells (PBMCs) are used in fighting diseases
such as leukemia, cancer and infectious disease. They are also used as a
starting materials for the isolation of T cells, NK cells, monocytes and dendritic cells for therapeutic applications. High viability of PBMCs is essential
for efficient immunotherapy and consistent trial results. Therefore there is a
need to isolate and preserve PBMCs in a standardized, reproducible way.
Researchers at USCF are currently investigating the cryopreservation of
PBMCs with the intention of isolating regulatory T lymphocytes (Tregs) for
autologous cell-based immunotherapy treatment in patients with autoimmune
diseases or transplantation recipients. In the past, to successfully freeze
PBMCs, controlled-rate freezers were used. However they are expensive,
difficult to operate, prone to malfunction and logistically difficult to ensure at
all cell therapy collection sites. Using the CoolCellÒ container, a passive
freezing device, as an alternative to the electronically controlled rate freezer,
the researchers showed that PBMCs could be cryopreserved and recovered
with high viability and recovery as seen using controlled-rate freezers. The
incorporation of the CoolCell passive freezing device in cell therapy research
will overcome the hurdles of multiple clinical site collections due to its
portability, lack of necessary maintenance and ease of use. The ability to
develop and store PBMCs and potentially functional Treg in a cost-effective
and reproducible manner is an important milestone in the ultimate use of
these cells in the clinic.
80
MATURATION OF DENDRITIC CELLS FROM CD14+
MONOCYTES IN AN AUTOMATED FUNCTIONALLY CLOSED
HOLLOW FIBER BIOREACTOR SYSTEM
T Startz, K Nguyen, R Peters, B Nankervis, M Jones, R Kilian, N Frank,
B Vang, D Hill
Terumo BCT, Lakewood, Colorado, United States
Dendritic cells make up a scarce population of immune cells that process
antigen material in the peripheral blood, acting as a messenger between the
innate and active immunity. Their rarity in the human body and difficulty to
isolate has been a major obstacle in researching their genesis and development. It is only recently that their therapeutic value to present foreign and self
antigens to T-cells has been recognized due to advancements with in vitro cell
culture techniques. The large number needed for cellular therapy and their
relatively short life span in the mature state has shown a necessity to derive
these cells from a more abundant source and be delivered in an expedited
manner to the patient. CD14+ monocytes have been shown to differentiate
into mature dendritic cells with addition of GM-CSF, IL-4, TNFa and IL-1b
to culture media. In the Quantum System we have been able to transform a
large percentage of CD14 positive monocytes into mature dendritic cells in a
functionally closed environment that reduces the time and resources needed
to produce a therapeutic dose while reducing the risk of contamination by
eliminating open steps. Mature DC phenotype was confirmed by flow
cytometry as Lymphocyte lineage (CD3, CD14, CD16, CD19, CD20 AND
S29
CD56) negative and positive for HLA-DR, CD83, CD86, CD197 and
CD209. Dendritic Cell functionality was shown by the antigen uptake of
Alexa Flour 488 labeled Dextran and increased production of IL-6 and IL-12
p70 cytokines.
81
TOOLS FOR OPTIMIZATION OF ADOPTIVE CELLULAR
THERAPY
L Brix1, D Pan2, C Halgreen1, H Pedersen1, P Wallace2
1
Immudex, Copenhagen, Denmark, 2Roswell Park Cancer Institute, Buffalo,
New York, United States
Cell-based therapies using lymphocytes are promising approaches for immunotherapy. The transfusion of T lymphocytes - adoptive cell therapy - is an effective
treatment for viral infections in immune-compromised patients, and has induced
regression of cancer in early-stage clinical trials. Adoptive T cell therapy can be
optimized in several ways: - Patient stratification - only those that benefit from
therapy are treated, - Measurement of T-cell immunity pre and post treatment - to
evaluate if the desired immune response has been induced and additional therapy
is needed, - Quantitative quality control of the cellular product prior to infusion to ensure consistent cell number and quality MHC Multimer assays can be used to
stratify patients, measure T-cell immunity, and perform quality control on the
cellular infusion products. We used Dextramers to measure CMV-specific T-cell
responses in transplant patients. Multiple adoptive cell therapy trials are investigating the benefit of transferring CMV-specific T cells to transplant patients to
avoid reactivation of CMV. We used the Dextramer CMV assay, to quantitate
CMV-specific T cells in whole blood and processed cell samples. The reconstitution of CMV immunity was successfully followed in >90 transplant patients and
it was shown that induction of CMV-specific immunity upon adoptive transfer of
CMV-specific T cells could be reliably measured. Furthermore we showed
that CMV Dextramers can be used to characterize cellular products with
respect to CMV-specific T-cell composition upon in vitro expansion. Our
results demonstrate that the CMV Dextramer assay is a valuable tool that may
improve CMV-specific adoptive cellular therapy. The Dextramer assay allows Stratification e Identification of patients with low CMV-specific immunity, Immune monitoring e Determination of the induced cellular response in
patients, - Quality control - QC of manufactured cellular products.
82
CELLULAR IMMUNOTHERAPY WITH THE CONTINUOUSLY
GROWING NK-92 CELL LINE AS AN ALTERNATIVE TO
DONOR DERIVED BLOOD NK-CELLS
H Klingemann1,2, B Simon1
1
Conkwest Inc, Cambridge, Massachusetts, United States, 2Tufts University
Medical School, Boston, Massachusetts, United States
Introduction: Infusions with cytokine-activated NK cells obtained from blood
of MHC mismatched donors have shown some promising results especially in
patients with AML. There is also some evidence that infusions of KIR receptor
mismatched NK cells as part of a stem cell transplant result in lower relapse
rates.
Methods: Expanding NK cells form donors requires them to first undergo
leukapheresis with subsequent removal of CD3+ lymphocytes (to prevent
GvHD). In contrast, the continuously growing NK cell line NK-92 can be
easily expanded to clinical scale in bioreactors. The broad cytotoxicity of NK92 is due to the lack of most of the KIR receptors while expressing a range of
activating receptors.
Results: We report here results from concluded and ongoing clinical phase I
studies with NK-92 cells in patients with advanced cancer, that confirm their
safety profile. Anti-tumor responses were seen in some patients with advanced
hematological malignancies and solid tumors. NK-92 cells also provide a
platform for further tumor targeted engineering. A variant has been generated
that expresses a high affinity FcgIIIRA receptor that can augment ADCC of
monoclonal antibodies. NK-92 cells have also been engineered to express
CARs to make them targeted to melanoma, myeloma, leukemia and brain
cancer. Tracking studies show that NK-92 CAR home to the tumor sites and
video-lapse studies confirm that they are able to do “serial killing”.
Conclusion: The human clinical grade NK-92 cell line has advantages over
peripheral blood NK cells as a cell therapy product for cancer patients. NK-92
cells are now in phase II studies as an ‘off the shelf’ tumor targeted local and
systemic cell therapy.
S30
Poster Abstracts
83
RAPID SCREENING OF HEALTHY DONORS SUGGESTS
STRATEGIES FOR CLINICAL PURIFICATION OF VIRUSSPECIFIC T CELLS
L Lagerlöf, L Björkman, M Bemark
Clinical Immunology and Transfusion Medicine, Sahlgrenska University
Hospital, Gothenburg, Sweden
Transfer of virus-specific memory T cells is an emerging treatment for viral diseases in immunocompromised patients. One approach is to purify antigen-specific
cells based on their up-regulation of activation markers after short-term in vitro
stimulation with peptides. In order to rapidly screen the frequency of antigenspecific CD4 and CD8 T cells in potential donors we have modified a commercial
kit from Miltenyi so that we can simultaneously measure induction of IFN-gamma,
CD40L and CD137 on both cell types following peptide stimulation. In a prestudy using blood from healthy donors we found that the presence of IFN-gamma
producing cells following stimulation with EBV or CMV peptides correlated with
the presence of serum antibodies against the viruses. Simultaneous stimulation with
peptides derived from multiple proteins lead to activation of similar number of cells
as stimulation with separate peptides. Whereas it was relatively easy to identify
memory cells reactive to CMV and EBV, fewer donors maintained cells that could
be activated by peptides derived from JC and BK viruses. IFN-gamma producing
cells had increased expression of CD137, and CD4 cells also CD40L. However,
more cells up-regulated their expression of CD137 than IFN-gamma following
peptide stimulation and background levels of CD137 expression was higher,
demonstrating that care has to be taken when selecting activation marker for purification. Taken together, this pre-study to clinical purification of virus-specific
memory T cells shows that rapid screening of potential donors is possible, and
suggests strategies for activation and purification of virus-specific cells.
84
CELLULAR THERAPY USING TH9402 FOR THE EXPANSION OF
TREGS IN THE TREATMENT OF CHRONIC GRAFT VERSUS
HOST DISEASE
J Bastien1,3, V Dave1, É Cournoyer1,3, D Roy1,3,2
1
Hôpital Maisonneuve-Rosemont, Montreal, Quebec, Canada, 2Kiadis pharma,
Amsterdam, Netherlands, 3Universite de Montreal, Montreal, Quebec, Canada
Often the most potent immunosuppressive drugs fail to control graft-versus-host
disease (GVHD), which is the most frequent cause of transplant related mortality.
We previously reported that photodepletion (PD) of PBMCs using dibromorhodamine (TH9402) eliminates activated allo-reactive T cells from healthy donors,
while it spares resting T cells. In the present study, we identified PD conditions that
selectively eradicate a vast majority of host-reactive T cells in PBMCs isolated from
chronic GVHD patients, with concomitant preservation of a significant number of
CD4+CD25+FoxP3+ regulatory T cells (Tregs). PD resistant Tregs suppressed
proliferation of cGVHD cells in a cell contact and CTLA-4 dependent manner. We
also observed that co-culture of PD cells with cGVHD cells results in the induction
of functionally competent Tregs from CD4+CD25- cells. Moreover, this Treg
generation activity was enhanced when PD cells were co-cultured in the presence of
monocyte-derived plasmacytoid dendritic cells (pDC) suggesting a role for pDC in
this effect. Furthermore, we observed that the co-culture of non PD cGVHD cells
with PD cells was associated with an increase in the expression of Indoleamine 2,3
dioxygenase (IDO). Interestingly, the inhibition of IDO impaired anti-proliferative
activity and Treg generating property of PD cells. Hence, we also found that patients treated with TH9402 based cellular therapy displayed an increase in the
FOXP3+ Treg population and IDO induction by PD cells provides a possible
mechanism of Treg generation in these patients. In conclusion, these results identify
a novel approach to selectively eliminate host-reactive T cells while sparing and
expanding Tregs. These results could fill the need for novel therapeutic strategies
for cGVHD patients refractory to immune suppressors.
85
ESTABLISHMENT OF A BANK OF BLOOD DONOR DERIVED
EPSTEIN BARR VIRUS SPECIFIC CYTOTOXIC T CELL
LINES
FOR
TREATMENT
OF
POST-TRANSPLANT
LYMPHOPROLIFERATIVE DISEASE
M Turner1, N Robinson1, G Wilkie1, N Rivera1, N Fraser1, D Clark1,
D Turner1, V Robertson1, H Newlands1, P Flanagan2, T Haque3, M Vickers1
1
Scottish National Blood Transfusion Service, Edinburgh, United
Kingdom, 2New Zealand Blood Service, Auckland, New Zealand, 3Department
of Virology, Royal Free Hospital, London, United Kingdom
Epstein-Barr virus (EBV) driven lymphomas associated with immunosuppression
are a significant problem, particularly after transplantation. Conventional treatment (withdrawal of immunosuppression, administration of rituximab/chemotherapy) is often effective but risks organ rejection and causes significant side
effects. EBV specific cytotoxic T cells (CTL) generated in vitro from autologous
lymphocytes can be effective with few side effects, but take months to prepare so
are difficult to use in clinical practice. We have established a bank of EBV CTLs
derived from platelet / plasma donors for issuing ‘off the shelf product’ to
partially human leucocyte antigen (HLA) matched patients on a named patient
basis. Donors from the New Zealand platelet / plasma panel were sourced to
reduce the risk of Creutzfeldt-Jakob disease (CJD). The current panel of donors
selected was EBV positive, Blood Group O, and met all current requirements for
mandatory virology markers. The panel was also chosen to maximize the
probability of HLA class I and II matches and minimize the number of mismatches. We estimated that this initial panel of 25 donors should provide a
partial match for at least three (HLA-A, -B, -DRB1) loci for w80% of Caucasian
patients. So far, lymphoblastoid cell lines have been made from 25 donors ready
for use in stimulating CTLs and 18 CTLs have been manufactured and cryopreserved for clinical use. Since the bank was granted a ‘Specials’ license by the
MHRA for this Advanced Therapy Medicinal Product in January 2012, there
have been enquiries about 14 patients: 4 improved on conventional therapy
without requiring CTLs, 3 died before any cells could be issued and 6 cell lines
have been released. The indications for issue have been: 3 cases of post-transplant lymphoproliferative disorder (1 CD20- rituximab resistant 1 with
concomitant graft versus host disease precluding reduction of immunosuppression 1 unfit for chemotherapy) 2 cases with congenital immunosuppression as a
bridge to allogeneic haematopoietic stem cell transplant and 1 case of EBVassociated leiomyosarcoma post-cardiac transplant (related donor). Clinical
improvement has been seen in 4 out of 6 assessable patients. A 3rd party donorderived anti-EBV CTL cell bank can be operated under current legislation and is
a valuable addition to existing therapies for selected patients.
86
CD14D CELL ISOLATION USING MAGNETIC-ACTIVATED CELL
SORTER (CLINIMACS) FOR CANCER IMMUNOTHERAPY
F Chung, M de Jesus, K Semon, M Capulong, M Barile, A Lee, J Sy, E Flores,
M Fernandez
Makati Medical Centre, Makati, Manila, Philippines
We report here our Makati Medical Center experience with CliniMACS
(magnetic-activated cell separation system) in six adult cancer patients. Our aim
is to evaluate the purity, recovery and viability of CD14+ cells selected from
apheresis product post Magnetic-Activated Cell Sorting using CliniMACS. Six
adult patients with advanced cancer (3 breast cancer, 1 lung carcinoid, 1 adrenal
cancer, 1 pancreatic cancer) were given daily subcutaneous injection of granulocyte colony stimulating factor for 5 days. Leukapheresis was carried out for
peripheral blood stem cell (PBSC) harvest. The harvested PBSC were then
positively selected for CD14+ cells using the CliniMACS device (Milteny
Biotech, Germany). Our Lab parameters are set as follows: cell viability of 70 %
and cell purity of 80 %. Our mean (SD) CD14 % viability from cancer patient
is 89.5 (7.29), for recovery and purity, 43.2(19.7) and 91.8(4.80), respectively. Our findings suggest that positive selection of CD14+ cells using magnetic
separation technology by CliniMACS results in acceptable parameter based on
the standards set by the Lab. Isolation of CD14+ cells for cancer immunotherapy
may be obtained through Magnetic-Activated Cell Sorting (MACS).
87
TRAIL EXPRESSING MESENCHYMAL STEM CELLS AS A NOVEL
CELLULAR THERAPY FOR MALIGNANT MESOTHELIOMA
B Sage, K Kolluri, K McNulty, A Gaingreco, S Janes
University College London(UCL), London, United Kingdom
Background: Malignant pleural mesothelioma (MPM) is an aggressive fatal
cancer with no effective treatments. Mesenchymal stem cells (MSCs)
migrate and incorporate into tumour stroma making them good vehicles for
the delivery of anti-cancer therapies. TNF-related apoptosis inducing ligand
(TRAIL) selectively induces apoptosis in malignant cells without affecting
healthy tissues. This study aimed to test whether MSCs modified to express
TRAIL (MSCTRAIL) could be a successful cell therapy for MPM.
Methods: Human MSCs were transduced with a lentiviral vector containing
TRAIL IRES-GFP under the control of a tetracycline dependent promoter.
Successful transduction was measured using flow cytometry and TRAIL
S31
HPL formulation. In our project, we compared media containing the “new”
HPL to standard endothelial cell culture media supplemented with FBS and
media supplemented with “standard” HPL to identify optimal culture condition
for ECFCs. We found no significant differences in ECFC morphology, proliferation, colony formation, surface phenotype, and in vitro cord-forming
ability; although, a slight reduction of CD34 expression was noted in the ECFC
cultured with the “new” HPL. Further in vivo vessel forming assays will need to
be performed before validation of “new” HPL cultured ECFCs is complete and
access to HPL is commercially available for future cell studies.
89
LENTIVIRUS-MEDIATED WWOX EXPRESSION SUPPRESSES
UROTHELIAL CARCINOMA CELL SURVIVAL
C Liu1, T Tsai1, C Tsai1, P Chiang2, L Chang2,3
1
School of Pharmacy, Kaohsiung Medical University, Kaohsiung,
Taiwan, 2Departmet of Occupational Therapy, I-Shou University, Kaohsiung,
Taiwan, 3Department of Pharmacy, E-DA Hospital, Kaohsiung, Taiwan
Figure 1. In vivo delivery of MSCTRAIL resulted in a significant
reduction in MPM tumour growth. The longitudinal bioluminescent signal shows a significant reduction in tumour burden in animals treated with MSCTRAIL(*p<0.05, two-way RM ANOVA
with Bonferroni’s multiple comparision test; n¼7 per group).
expression was confirmed by ELISA. The biological activity of MSCTRAIL was
determined using co-culture experiments and cells were stained with Annexin V
and DAPI to detect apoptosis and death respectively on flow cytometry. To test
the effect of MSCTRAIL in vivo a bioluminescent tumour model was established. MPM cells were transduced with a lentiviral vector containing firefly
luciferase, YFP and hygromycin resistance genes. A pure population was
generated using hygromycin selection. 80,000 MPMLuc cells were injected into
the pleural cavity of NOD/SCID mice and their growth was assessed using an
IVIS Lumina system to detect bioluminescence. 1 million MSCTRAIL cells
were delivered via tail vein injections on days 5, 9, 12, 15 and 18 post tumour
inoculation and bioluminescence was measured twice weekly.
Results: MSCs were successfully transduced with TRAIL with 96% efficiency and TRAIL production was confirmed by ELISA. Seven human MPM
cell lines were tested with 6/7 (86%) being sensitive to MSCTRAIL. In vivo
delivery of MSCTRAIL resulted in a significant reduction in MPM tumour
growth (Figure1).
Conclusions: Delivery of TRAIL via MSCs causes a significant reduction in
MPM tumour growth and is a potential novel cellular therapy for this incurable
disease.
88
COMPARISON OF HUMAN PLATELET LYSATE AND FETAL
BOVINE SERUM CONTAINING MEDIA FOR OPTIMAL
CULTURE CONDITIONS OF ENDOTHELIAL COLONYFORMING CELL EXPANSION
C Shelley, I Tsung, M Yoder
Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana,
United States
Therapeutic vascularization remains a challenge in tissue repair strategies.
Patients suffering from the consequences of vascular diseases such as peripheral
vascular disease would benefit greatly from a cell therapy with angiogenic capabilities. Human endothelial colony-forming cells (ECFC) are cells which have
been shown to be a potential solution, as they are highly proliferative and possess
in vivo vessel-forming capacity. Advancements in therapies harnessing ECFCs
are currently hindered by the use of fetal bovine serum (FBS) in culture. Several
novel culture media formulations lacking xenogeneic compounds have been
developed and demonstrated to yield ECFCs with similar phenotypic and
functional characteristics as those cells cultured in traditional FBS-containing
media. Of these formulations, human platelet lysate (HPL) has emerged as a
promising substitute for FBS; however, the high cost and limited availability of
HPL has constrained extensive ECFC studies. We have been provided a new
Lentiviral vectors (LVs) are promising tools for long-term gene transfer to
nondividing tissues for human gene therapy. Although LVs have been strongly
developed in design, in biosafety and in their ability of transgene expression,
the applications of LVs in urothelial carcinoma of bladder treatment are rare.
The purpose of this study was to investigate whether LVs-mediated local
overexpression of tumor suppressor gene could serve to diminish tumor growth
of bladder cancer in vitro. WW domain-containing oxidoreductase (WWOX)
is a tumor suppressor gene and loss or down-regulation of WWOX was
identified in lung, liver and bladder cancers. This study investigated the antiproliferative activity of lentivector-mediated WWOX overexpression in rat
bladder cancer cells. Rat bladder cancer cell lines (AY-27) cell was used to
investigate the effects of tumor suppression and gene expression under
WWOX overexpressed situation. Full-length WWOX cDNA was subcloned
into pLKO_AS2.puro (a mammalian expression lentivector) to form LVWWOX. Lentiviral particles were produced by transient transfection of
LV-WWOX to 293T cells; then transfected into AY-27 cells (denoted as AY27-LV-WWOX) to overexpress WWOX protein. The LV-WWOX was
transduced into the AY-27 cells, and the effect of WWOX overexpression on
the biological characteristics was analyzed by a series assays. The stable overexpression of WWOX obviously resulted in up-regulation of TNF-a expression demonstrated by co-localization of immunofluorescence images.
LV-WWOX suppresses urothelial carcinoma cell survival by measuring cellular
number, morphology and performing MTS assay. LV-WWOX also inhibit
cell migratory by wound healing assay. These results provide potential application of LV-WWOX in orthotopic superficial bladder cancer gene therapy.
90
LENTIVECTOR-MEDIATED APOBEC3G GENE TRANSFER
TRIGGERS TUMOR SUPPRESSION IN HUMAN HEPATOCELLULAR CARCINOMA CELLS
P Wu1, T Kuo1, C Liu2, T Yu3, P Chiang3, L Chang3,4
1
Department of Kinesiology, National University of Kaohsiung, Kaohsiung,
Taiwan, 2School of Pharmacy, Kaohsiung Medical University, Kaohsiung,
Taiwan, 3Departmet of Occupational Therapy, I-Shou University, Kaohsiung,
Taiwan, 4Department of Pharmacy, E-DA Hospital, Kaohsiung, Taiwan
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC)
cytidine deaminases, which convert cytosine to uracil during RNA editing and
retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. An APOBEC mutagenesis pattern is pervasive in human cancers
and correlates with APOBEC mRNA levels. APOBEC3G (A3G) belongs to the
mammalian APOBEC family and exhibits deoxycytidine deaminase activity to
protect against viral infection. Recent study showed evidence for APOBEC3B
mutagenesis in multiple human cancers. APOBEC3B-catalyzed genomic uracil
lesions are reported to responsible for a large proportion of both dispersed and
clustered mutations in multiple distinct cancers. However, the effect of A3G in
tumor cell behavior modulation is still obscure. To investigate the role of A3G in
hepatocellular carcinoma cells (HCC), A3G cDNA was subcloned into a
mammalian expression lentivector (LV), pLKO_AS2.puro, to form LV-A3G.
LV-A3G was then transfected into the human HCC cell line Hep 3B. Hep3B
contains only a single copy of integrated Hepatitis B virus (HBV) genome. Our
results indicated the inhibitory effects of lentivector-mediated A3G overexpression on cellular growth and migration of Hep 3B. In conclusion, our results suggest that A3G might exert tumor suppressive effects in hepatoma cells.
S32
Poster Abstracts
91
MODIFIED ADIPOSE MESENCHYMAL PROGENITORS TARGET
EWING’S SARCOMA
G Grisendi1, C Spano1, N D’Souza1, V Rasini1, E Veronesi1, S Piccinno1,
G De Santis1, E Horwitz2, P Conte3, P Paolucci1, M Dominici1
1
Department of Medical and Surgical Sciences for Children & Adults,
University Hospital of Modena and Reggio Emilia, Modena, Italy, 2Division of
Oncology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania,
United States, 3Department of Surgery, Oncology and Gastroenterology,
University of Padova, Istituto Oncologico Veneto IRCCS, Padova, Italy
Ewing’s sarcoma (ES) is the second most common bone tumor in children
and young adults. The real ontogeny of ES is debated and evidences suggest
it may derive from a transformation of precursors identified within mesenchymal stromal/stem cells (MSC). Recent studies on ES microenvironment
additionally indicated that MSC take active part in generation of a supportive
stroma (1). ES initially responds to chemotherapy, however 30% of patients
relapse and up to 80% of patients with metastases die within 5 years from
diagnosis (2,3). Based on this knowledge, we conceived to use modified
adipose MSC as “trojan horse” to deliver an anticancer molecule against ES.
We have previously demonstrated that a pro-apoptotic agent named TRAIL
may be delivered by MSC into cancers. For the first time, we investigated
the impact of gene modified MSC expressing TRAIL against ES. In 24
hours, MSC-TRAIL induced apoptosis in more than 80% of ES cell lines
(p<0.001). Cell death was specifically associated with caspase-8 activation in
ES cells after only 8 hours of co-culture with MSC TRAIL (p<0.001). An in
vivo model further challenged the approach and, when injected into ES
burden, MSC TRAIL persisted within its stroma causing massive apoptosis
by Tunel-assay combined with a size reduction (p<0.03). Conclusively, our
data indicate that MSC, as cellular vehicle for TRAIL, could open novel
therapeutic opportunities for mesenchymal tumors still characterized by very
poor prognosis. The work was supported by AIRC, Ministero Salute-Giovani Ricercatori 2008 & ASEOP. 1 Burns JS et al. Cancer Lett. 2012. 2 Grier
HE et al. N Engl J Med. 2003. 3 Helman LJ, Meltzer PNat Rev Cancer.
2003.
92
DEVELOPMENT OF A NEW MINIPIG MODEL OF TRANSIENT
GENE THERAPY TO TREAT CUTANEOUS RADIATION
SYNDROME
D Riccobono1, F Forcheron1, H Scherthan2, V Meineke2, M Drouet1
1
Radiobiology, French Military Biomedical Research Institut (IRBA), Bretigny
sur Orge cedex, France, 2Radiobiology, Bundeswehr Institut fur Radiobiology,
Munich, Germany
Cutaneous radiation syndrome (CRS) characterized by incomplete wound
healing and poor revascularization is the delayed consequence of localized
skin exposure to high doses of ionizing radiation. Adipocyte derived Stem
Cells (ASCs) have been demonstrated to favor tissue regeneration in animal
models of CRS via secreted factors. Thus optimizing their secretome i.e.
“transient gene therapy strategy” may increase their therapeutic potential.
Here Sonic Hedgehog (Shh), a secreted protein involved in cell proliferation
and neoangiogenesis has been tested as a first candidate. This preliminary
study aimed at establishing the feasibility of transient gene therapy in an
animal model close to human. ASCs were isolated from minipigs’ subcutaneous fat tissue 30 days before irradiation and nucleofected (Amaxa) using a
biscistronic PIRES2 plasmid coding for Shh and GFP. GFP cells represented
about 30% of alive cells (FACS analysis). Shh synthesis/secretion was evaluated in short term cultures using western blot analysis. Intracellular Shh
content decreased from day 2 to day 9 post transfection and conversely Shh
increased in culture medium. Göttingen minipigs were locally irradiated using
a 60Co gamma source at the dose of 50 Gy (entry area) and received Phosphate Buffer Salin (n¼8), autologous ASCs (4 local injections of 50x10<6>,
n¼5) or autologous Shh-ASCs (4 local injections of 25+/-7 x10<6> n¼1). All
PBS animals exhibited a typical clinical evolution of CRS with final persistent
necrosis. A wound healing was observed in stem cells injected minipigs in four
out of five grafted animals. The sonic hedgehog animal, albeit injected with a
lower number of transfected stem cells, presented a very similar evolution of
skin healing without final necrosis or uncontrollable pain. Globally this preliminary report establishes the feasibility of transient Sonic hedgehog therapy.
Further studies will establish whether a clinical benefit may result from this
strategy.
93
CYTOKINE-INDUCED KILLER (CIK) CELLS ENGINEERED
WITH CHIMERIC ANTIGEN RECEPTORS (CARS) BY SLEEPING
BEAUTY SYSTEM
C Magnani1, N Turazzi1, F Benedicenti2, S Tettamanti1, GG Attianese1,3,
V Rossi1, E Montini2, L Cooper3, A Aiuti2, A Biondi1, E Biagi1
1
Centro Ricerca Tettamanti, Clinica Pediatrica, Università Milano Bicocca,
Osp. San Gerardo/Fondazione MBBM, Monza, Italy, 2San Raffaele Telethon
Institute for Gene Therapy (HSR-TIGET), Ospedale San Raffaele, Milan,
Italy, 3MD Anderson Cancer Center, University of Texas, Houston, Texas,
United States
Adoptive immunotherapy of T cells engineered with Chimeric Antigen Receptor (CARs) has been recently proved to be effective in the treatment of
Acute Leukemias associated with poor prognosis and high rate of relapse.
Since the efficacy, safety and feasibility of cell manufacturing and gene therapy
by viral vectors still remain major concerns, we explored here the use of
Sleeping Beauty (SB) Transposon. With nucleofection and an optimized
clinical-grade stimulation protocol, we genetically modified cytokine-induced
killer (CIK) cells to express two distinct 3rd generation CARs specific for
myelogenous leukemia (AML) CD123+ or acute lymphoblastic leukemia
(ALL) CD19+ blasts. The nucleofection minimally affected the phenotype of
CIK cells, and the optimized protocol was effective in inducing T-cell
expansion, with a fold increase of 33.97 (CD123-CAR) and 32.56 (CD19CAR) within 3 weeks, sufficient for clinical translation. Modified CIK cells
displayed stable expression of CD123-CAR (51.43%, n¼15) or CD19-CAR
(48.78%, n¼7), and exerted efficient lysis of leukemic cell lines and primary
blasts. Interestingly, CAR triggering promoted specific and CAR-restricted
cytokine secretion and proliferation. The loss of the expression of transposase
during the differentiation was assessed by absolute quantification to assure the
genome stability of the cellular product. Finally, preliminary insertion-site
analysis by LAM-PCR confirmed that the integrations in the genome of SB
system do not correlate with the genes-enriched regions. In conclusion, SB
system and an optimized method of differentiation efficiently expand modified
CIK cells, redirect their activity towards leukemic cells, and retain a safe
pattern of integrations in the genome. The development of an easy-clinical
grade adoptive cell/gene therapy protocol will be fundamental to improve the
range of applications of immunotherapy to control relapse in leukemic
patients.
94
QUANTITATIVE STUDY OF EFFECTS OF FREE CATIONIC
POLYETHYLENIMINE
CHAINS
ON
INTRACELLULAR
TRAFFICKING OF DNA/POLYMER POLYPLEXES
1
1,2
J Cai , C Wu
1
Chemistry, The Chinese University of Hong Kong, Hong Kong, Hong
Kong, 2Chemical Physics, The Hefei National Laboratory of Physical Science
at Microscale, Hefei, China
Knowledge on the cellular uptake, intracellular routing and nuclear import
mechanisms of polyplexes made of anionic DNA and cationic polymer chains
is of great importance for optimizing a non-viral gene delivery system. It has
been generally known that most of anionic DNA chains are complexed with
cationic chains, such as polyethylenimine (PEI), to form small polyplexes
(w102 nm) when N/P w 3, where N/P is the molar ratio of nitrogen from
PEI to phosphate from DNA, but the in-vitro gene transfection efficiency is
w102fold higher when N/P 10. Previously, we showed that it is those
cationic PEI chains free in the solution mixture that dramatically promote the
gene transfection because they block the intervesicular fusion and prevent the
transport of the polyplexes into the lysosomes. In the current study, we
investigated how those free cationic polymers help the polyplexes in HepG2
cells to overcome the intracellular barriers, including cellular uptake, lysosomal escape and nuclear import by using PEI chains with different topologies (linear or branched) and lengths (Mw w 2 or 25 kg/mol) as free chains
while fixing the complexation of the pDNA encoding the luciferase (pGL3)
with high-molar mass branched PEI chains due to their better protection of
DNA from degradation. Our preliminary results from quantitative analysis of
three-dimensional confocal images, flow cytometry and TaqMan real-time
PCR revealed that: (i) free PEI chains help the cellular uptake little, ruling out
its possible effect on the gene transfection efficiency; (ii) free PEI chains reduce
the entrapment of the polyplexes inside the lysosomes from 75% to 30% of all
ingested DNA chains except for the short branched PEI chains (Mw w 2 kg/
mol) that have no such reduction; (iii) free PEI chains promote the nuclear entry
of DNA so that pGL3 inside nuclei increases from 4.4 103 up to 3.3 104
copies per cell after 6 h; and (iv) pGL3 is released from the polyplexes before
they enter the nuclei.
95
96
RAPID AND EFFECTIVE ISOLATION OF T CELL POPULATIONS
BY A NEW METHOD AVOIDING MAGNETIC BEADS
KB Weiss1,2, H Stadler1, S Przibilla1, S Dreher2, C Stemberger1,
L Germeroth1, DH Busch2
1
STAGE cell therapeutics, Göttingen, Germany, 2Inst. f. Med. Microbiology,
Immunology and Hygiene, MIH, TUM, Munich, Germany
In the last two decades, conventional isolation of therapeutic cells has been
carried out using high affinity antibodies and magnetic bead technology.
Although good purity and reasonable yields can be obtained in many cases,
major disadvantages remain comprising biological interference of nonreversible selection reagents (e.g. stimulation, receptor blockade etc.), the
difficulty to purify complex multi-parametric cell populations by positive
selection, time consuming protocols as well as limitations in highthroughput processing. We report here on the development of a new nonmagnetic and fast cell selection technology applying immune affinity chromatography. Therefore, a matrix consisting of beads coated with Streptactin
and low-affinity recombinant Fab-fragments (Fab-Streptamers) directed
against defined T cell surface antigens has been generated. Stable and specific target cell retention is achieved by passing whole blood or PBMCs over
the affinity matrix. After binding and washing, target cells can be gently
retrieved by D-biotin administration. Eluted cells are then passed over a
second matrix removing D-biotin and free Fab-Streptamers, subsequently
yielding in a label-free authentic cell population for further use. Most
importantly, total cell processing times can be kept extremely short;
depending on the sample size even down to several minutes. Sequential
isolation steps are possible and allow positive selection of complex cell
populations like regulatory T cells or central memory T cells defined by
several markers in high yields and purities. We are currently integrating this
approach into a fully closed separation device for clinical cell purification. In
addition, high throughput cell selection for diagnostic or basic research
applications can be achieved by embedding the matrix into pipette tips and
the use of suitable pipetting robots.
S33
MSC as control. IL-6 transcript and protein levels were determined using Real
Time qPCR and ELISA assay respectively. Transfected MSC were subjected to
viability, immunophenotyping and trilineage differentiation studies. Multiple
myeloma cells, U266 (3x102), were co-cultured with supernatant collected from
MSC expressing IL-6 siRNA. The level of IL-6 production and growth of U266
cells post co-culture were determined on days 3 and 5 respectively using ELISA
and MTS assays.
Results: IL-6 mRNA and protein were significantly suppressed by 5.9-fold and
63% relative to untransfected MSC by 72 hours. The suppression was sustained
up to 120 hours with 2.1-fold mRNA reduction and 71.8% protein reduction. IL6 siRNA transfection did not affect the viability, surface markers and trilineage
differentiation capacity of MSC. U266 co-culture results showed cell growth and
IL-6 production when co-cultured with MSC expressing IL-6 siRNA were
inhibited by 2-fold compared to untransfected co-culture wells up to 5 days.
Conclusion: When co-cultured together, MSC expressing IL-6 siRNA
inhibited U266 cell growth and IL-6 production significantly supporting the
potential usage of MSC for siRNA-mediated IL-6 gene silencing to treat multiple
myeloma.
98
GENETIC ENHANCEMENT OF MESENCHYMAL STEM CELLS
USING MINICIRCLE-BASED VEHICLE SHOWS SUPERIOR
THERAPEUTIC BENEFIT IN A MOUSE MODEL OF ACUTE
LUNG INJURY
SH Mei1,2, J Zhang1, Y Deng1, DJ Stewart1,2
1
Canada, 2Department of Medicine, University of Ottawa, Ottawa, Ontario,
Canada
97
INHIBITION OF U266 CELL GROWTH BY HUMAN
MESENCHYMAL
STROMAL
CELL-MEDIATED
SMALL
INTERFERING RNA SILENCING OF INTERLEUKIN-6
H Teoh1,2, P Chong2, Z Sekawi2, M Abdullah2, C Leong3, S Cheong1,4
1
PPUKM-MAKNA Cancer Centre, Universiti Kebangsaan Malaysia Medical
Centre, Kuala Lumpur, Malaysia, 2Faculty of Medicine & Health Sciences,
Universiti Putra Malaysia, Selangor, Malaysia, 3Faculty of Medicine, Universiti
Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 4Faculty of Medicine &
Health Sciences, Universiti Tunku Abdul Rahman, Selangor, Malaysia
Introduction: Acute lung injury (ALI) results in increased pulmonary
vascular inflammation and permeability, leading to high mortality in critically ill patients. Cell-based therapy with mesenchymal stem cells (MSCs)
produces partial improvement in experimental ALI, which can be markedly
enhanced by transfection with the vascular protective factor, angiopoietin-1
(ANGPT1). However, plasmid bacterial sequences can activate Toll-like
receptors and induce inflammation, which may be detrimental in the context
of ALI. We explored whether minicircle-DNA (MC), containing no bacterial
backbone, can provide a safer and more effective therapeutic platform to
treat ALI.
Methods: ALI was induced by intratracheal instillation of LPS, followed
by treatment with null- or ANGPT1-transfected MSCs.
Results: Nucleofection of MC-ANGPT1 resulted in a 5-fold increase in
ANGPT1 protein at 24 hrs (vs. plasmid-ANGPT1 [pANGPT1]). MSCs
(empty MC- or plasmid-transfected) reduced bronchoalveolar lavage (BAL)
neutrophil counts following LPS injury by 19% or 25%, respectively.
Administration of pANGPT1 or MC-ANGPT1-transfected MSCs reduced
BAL neutrophil counts by 50% and 70%, respectively (p<0.01), and albumin
and IgM in BAL fluid, parameters of pulmonary vascular permeability
(p<0.01). While both albumin and IgM were significantly reduced by
ANGPT1-enhanced MSCs (p<0.01), MC-ANGPT1 transfected MSCs were
much more effective in reducing BAL neutrophil counts (p¼0.06), albumin
levels (p¼0.02), and IgM levels (p<0.001) than pANGPT1-transfected MSCs.
Finally, pANGPT1-transfected MSC treatment significantly reduced levels of
inflammatory cytokine (TNF-a, IFN-g, IL-6, MIP-2 and MCP-1) compared
to LPS-injured animals, while MC-ANGPT1 transfected MSC treatment
showed a significantly enhanced benefit compared to pANGPT1-transfected
MSCs (p<0.01).
Conclusions: Overexpression of ANGPT1 using MC significantly
enhanced therapeutic efficacy of MSCs in a model of ALI to a greater extent
than plasmid DNA.
Background: Studies demonstrated that mesenchymal stromal cells (MSC)
from bone marrow stroma produced high concentration of interleukin-6 (IL-6)
that promoted multiple myeloma growth in a paracrine manner. In view of the
failure of IL-6 blocking antibodies to demonstrate substantial clinical responses
in early clinical trials, more effective methods are needed to disrupt the favourable microenvironment provided by the bone marrow. In this study, we evaluated
the silencing of IL-6 in MSC post siRNA transfection and the efficacy of these
MSC on multiple myeloma cell growth and IL-6 production inhibition.
Methods: MSC (1x105) were transfected with 100 pmol IL-6 siRNA
(SASI_Hs01_00155911) using 5mL Lipofectamine 2000 with untransfected
99
POTENCY OF PIGGYBAC TRANSPOSON-MEDIATED CD19SPECIFIC T-CELLS AGAINST DRUG-RESISTANT PHILADELPHIA
CHROMOSOME-POSITIVE ACUTE LYMPHOBLASTIC LEUKEMIA
S Saito1, Y Nakazawa1, M Tanaka1, R Yanagisawa1, A Sueki2, K Matsuda2,
MH Wilson3, C Rooney4, K Koike1
1
Department of Pediatrics, Shinshu University School of Medicine,
Matsumoto, Japan, 2Department of Laboratory Medicine, Shinshu University
Hospital, Matsumoto, Japan, 3Department of Medicine, Vanderbilt University
S34
Poster Abstracts
School of Medicine, Nashville, Tennessee, United States, 4Department of
Pediatrics, Baylor College of Medicine, Houston, Texas, United States
Background: To develop an alternative treatment for Ph+ALL resistant to
tyrosine kinase inhibitors (TKIs), we evaluated anti-Ph+ALL activity of T-cells
nonvirally engineered to express a chimeric antigen receptor (CAR) targeting
CD19.
Methods: A CD19-CAR gene was delivered into mononuclear cells
from healthy donors using piggyBac transposons and 4D-Nucleofector
System. Cells were stimulated with anti-CD3/CD28 mAbs, magnetically
selected for CAR, and cultured in serum-free TexMACS Medium with
interleukin (IL)-15 for 21 days (CAR-T cells). To evaluate the potency, we
perfomed no cytokine-coculture with 7 Ph+ALL cell lines including 3
TKI-resistant (T315I-muatated) ones at an effector/target ratio of 1:5 or
lower.
Results: We obtained 1.28 0.27 10E8 CAR-T cells from 10-ml blood.
In 7-days coculture, we observed complete eradication of Ph+ALL at 1:5 and
1:10 by FCM and at 1:5 by quantitative PCR. We found substantial reduction
of Ph+ALL even at 1:50 and 1:100. Kinetic study in 1:5 coculture showed a
significant proliferation of CAR-T cells to w16 times 10 days after 1st
coculture, and to w37 times 10 days after 2nd coculture, with almost complete
clearance of Ph+ALL within 24 hours. Interestingly, the intensity of CD19CAR and TRAIL expressions on CAR-T cells started increasing on day 1 after
1:5 cocuture, reached the maximum on day 3, returning to basal levels on day
10. We also detected abundant, but transient IL-2 production of CAR-T cells
from next day after 1st and 2nd cocultures.
Conclusions: We successfully generated an adequate number of CAR-T
cells from 10-ml blood using piggyBac transposons, 4D-Nulclofector, and
serum-free, tumor cell/virus-free culture method to reduce a potential risk and
facilitate cGMP approval. CAR-T cells exhibited marked cytotoxicity against
Ph+ALL regardless of TKI-resistance and subsequent proliferation by autocrine IL-2 secretion. Our CD19-specific T-cell therapy may be an effective and
safe option for relapsed/refractory Ph+ALL.
100
CLINICAL
STUDY
OF
UMBILICAL
CORD-DERIVED
MESENCHYMAL STEM CELL FOR TREATMENT OF NINETEEN
PATIENTS WITH STEROID-RESISTANT SEVERE ACUTE
GRAFT-VERSUS-HOST DISEASE
G Chen, T Yang, M Qiao, M Miao, D Wu
the First Affiliated Hospital of Soochow University, Suzhou, China
To evaluate the safety profile and efficacy of umbilical cord-derived
mesenchymal stem cell infusion in patients with steroid-resistant, severe,
acute graft-versus-host disease (aGVHD). A total of 19 patients with steroidresistant severe aGVHD received mesenchymal stem cell infusion treatment.
We analyzed the treatment response, transplantation-related mortality,
events associated with infusion and relapse rate. Two patients with grade 2, 5
patients with grade 3 and 12 patients with grade 4 aGVHD received a total of
58 infusions of mesenchymal stem cell. The mean total dose of mesenchymal
stem cell was 2.13106 (range 0.6-7.2106) cells per kg bodyweight. 7 patients received one infusion, 2 patients received two infusions, and 10 patients received three or more infusions. 11 patients had a complete response
and 4 had a partial response and 4 had no response. No patients had sideeffects during or immediately after infusions of mesenchymal stem cell and
no ectopic tissue was detected to date. 11 patients survived and 8 died, 4 for
aGVHD, 1 for infection and 2 for aGVHD with concomitant infection and 1
for underlying leukemia relapse. The cell viability of freshly prepared
mesenchymal stem cell is 93% (92%-95%) by trypan blue staining. The cell
viability of controlled-rate freezed and thawed cells mesenchymal stem cell is
72% (70%-74%). Infusion of umbilical cord-derived mesenchymal stem cell
expanded in vitro is an effective therapy for patients with steroid-resistant,
severe aGVHD without negative impact on relapse. Freshly prepared
mesenchymal stem cells are superior to freezed and thawed cells in terms of
cell viability.
101
FINE T CELL ANALYSIS IN A PATIENT SUCESSFULLY TREATED
WITH HAPLOIDENTICAL TRANSPLANTATION AND ADD BACK
OF DONOR LYMPHOCYTES EXPRESSING A SUICIDE GENE
H Hashimoto1, S Kitano2, R Ueda1, A Itoh1, K Tada2, D Tomura3,
I Nukaya3, J Mineno3, Y Heike2
1
Hematopoietic Stem Cell Transplantation Division, National Cancer Center
Hospital, Tokyo, Japan. Hematopoietic stem cell transplantation, National
Cancer Center, Tokyo, Japan, 2Immunotherapy Research Field, Translational
Research Group, and Translational Medicine Department, Phase 1 Group,
Exploratory Oncology Research & Clinical Trial Center National Cancer
Center, 3Center for Cell & Gene Therapy, Takara Bio Inc
Background: Haploidentical hematopoietic stem cell transplantation with
add back of donor lymphocytes expressing the Herpes Simplex Virus
Thymidine Kinase suicide gene (TK-cells) is a promising strategy. However,
the immunological status of TK-cells and donor T cells after the transplantation has not been well characterized yet. We performed fine immunological analysis of add backed TK-cells (labeled with LNGFR (CD271)PE and donor T cells in a patient separately. CASE: A 46-year-old female
with Ph-ALL was enrolled in our phase I clinical trial to receive haploidentical transplantation with add back of TK-cells. She underwent total
infusions of 2.1107 /kg TK-cells. Grade3 of aGVHD was developed
simultaneously with immune reconstituition by add back of TK-cells’ help,
which was rapidly resolved by ganciclovir administration. She has been in CR
and had no recurrent episode of GVHD for more than 32 month after
transplantation. The frequency of TK-cells was about 30% in peripheral
blood lymphocytes for over 2 years.
Results: Comparing proliferation rate of T cells at aGVHD onset with that
after its remission by ganciclovir administration, the frequency of Ki-67+
CD4+/CD8+ TK-cells were reduced from 65.2%/72.6% to 11.7%/12.0%
respectively. After ganciclovir administration ICOS+CD4+TK-cells were
reduced from 27.2% to 1.14%. Higher frequencies of cytotoxic (GranzymeB+/
IFNg+) TK-cells than those in non TK-cells continued to exist in this patient’s
peripheral blood for more than 32 months after transplantation. T cell receptor Vb chain repertoire was dramatically changed after ganciclovir
administration.
Conclusion: We confirmed the selective effect of ganciclovir on proliferating TK-cell, thought to cause aGVHD. Interestingly, TK-cells after a
restoration of donor-derived immune system continued to have stronger
cytotoxic capacity. T cell repertoire changes after add back of TK-cells might
explain why activated TK-cells did not induce recurrent GVHD in this
patient.
102
AN ISBT 128 IMPLEMENTATION EXPERIENCE AT TUMCELLS
INTERDISCIPLINARY CENTER FOR CELLULAR THERAPIES
T Perisic1, A Slobodianski2, M Hildebrandt2
1
Plastic Surgery and Hand Surgery, Klinikum Rechts der Isar, TU Munich,
Munich, Germany, 2TUMCells Interdisciplinary Center for Cellular
Therapies, Faculty of Medicine, TU Munich, Munich, Germany
In the last two decades, we are witnessing an enhanced development of cellbased therapies and a steep rise in the number of registered cellular therapy
products (CTP). Due to the biological nature of CTP, compatibility between
donor and recipient is a key factor and only an unequivocal labeling of
products can ensure safe application in patients. Establishment of the ISBT
128 international coding system for identification and labeling of medical
products of human origin (including blood, cell, tissue, and organ products)
has demonstrated multiple advantages. The terminology is published on
behalf ICCBBA and it is based on broad definitions (Classes) and modifying
characteristics (Attributes), which are used as building blocks for product
codes to describe each CTP. Since September 2013, TUMCells is registered
with ICCBBA as cellular therapy facility. The first attempt to apply the ISBT
128 labeling standard was made on a gene therapy medicinal product
(GTMP) manufactured from cultivated primary fibroblast isolated from split
skin and transfected with plasmids encoding angiogenic growth factors.
However, the great majority of attributes in the group of CTP appear not to
be suitable for the characterization of our GTMP and the new attributes need
to be requested. The CTP-terminology is been continuously updated by
adding new classes and attributes. However, the process involves timeconsuming reviews by appropriate advisory groups. In addition, in spite of
application of numerous cell culturing techniques in the production of CTP/
GTMP, the cell culture attributes are underrepresented in the ISBT 128. It
seems that the use of ISTB 128 labeling standard is not the first choice for
CTP which do not fall in the category of blood-based products. Thus, we
wish to express our encouragement to other cellular therapy facilities in
implementing the ISTB 128 standard and becoming a part of this global labeling system.
103
OPTIMIZATION OF THE ROCKING SPEED AND ANGLE OF A
PERFUSION BIOREACTOR FOR T CELL CULTURE
V Sauvage, M Janas, S Stone, A Marenghi, S Stubbs, B Mark
GE Healthcare Ltd, Little Chalfont, United Kingdom
The XuriÔ Cell expansion W5 bioreactor enables the culture of T cells to
high cell densities in an automated and closed system, making it an attractive
option for the manufacture of T cells for adoptive cell therapy. Using bioreactor settings of 10 rpm rocking speed at a 6 angle, cell concentrations of
greater than 1107 cells/mL are consistently reached in a 1L volume. In the
study presented here, we investigate the influence of rocking speed and angle
on the cell growth kinetics in order to identify those settings that optimize T
cell growth, thereby allowing for faster expansion. Studies, with rocking speed
varying between 2 rpm and 18 rpm and angle varying between 2 and 9 , were
conducted over a 14 day culture period. The growth kinetics of experiments
was compared in terms of fold expansion. Following data analysis, it was
predicted that the optimum conditions for cell growth were 15 rpm at 6 and
this was subsequently tested experimentally. A comparative experiment between a XuriÔ W5 system rocking at (10 rpm; 6 ) and another one rocking at
(15 rpm; 6 ) was carried out over 10 days. The viable cell number obtained at
day 10 with the platform rocking at 15 rpm was 24% higher than that of the
instrument rocking at 10 rpm, confirming improved cell growth kinetics at
15 rpm.
104
IL-21 EXPRESSING MESENCHYMAL STEM CELLS CAN
PREVENT LETHAL B-CELL LYMPHOMA THROUGH EFFICIENT
DELIVERY OF IL-21 WHICH REDIRECTS THE IMMUNE
SYSTEM AGAINST TUMOR
N Kim1, J Lim1, K Im1, E Lee1, Y Nam1, E Kim1, E Chae1, S Cho1,2
1
Laboratory of Immune Regulation, Convergent Research Consortium for
Immunologic Disease, The Catholic University of Korea, Seoul, Korea,
Republic of, 2Catholic Blood and Marrow Transplantation Center, Seoul St.
Mary’s Hospital, The Catholic University of Korea College of Medicine,
Seoul, Korea, Republic of
Mesenchymal stem cells (MSCs) are emerging as vehicles for cancer gene
therapy due to their inherent migratory abilities toward tumors. Whether
MSCs themselves have anti-tumor effects is still controversial; however,
MSCs are an ideal delivery vehicle to deliver anti-tumor substances directly
to the tumor site. The present study was performed to investigate whether
MSCs modified to highly express interleukin (IL)-21 (IL-21/MSCs) could
treat disseminated B-cell lymphoma in a murine model. For tumor induction, BALB/c mice were injected intravenously with syngeneic A20 B cell
lymphoma cells. One week following tumor induction, the tumor-bearing
mice were treated with IL-21/MSCs weekly, three times. Systemic infusion
of A20 cells lead to hind-leg paralysis as well as severe liver metastasis in the
control group. The IL-21/MSCs treated group showed delayed tumor
incidence in the mice as well as improved survival, whereas MSCs treated
group did not show significant difference from the non-treated mice. These
therapeutic effects were associated with high levels of IL-21 delivered to the
liver, which prevented the formation of tumor nodules in the liver.
Furthermore, the infusion of IL-21/MSCs lead to the induction of
CD3+CD56+ natural killer T (NKT) cells while potently inhibiting immune
suppressor cells such as myeloid derived suppressor cell (MDSC), regulatory
T cells (Tregs), and also IL-10 secreting CD4+ T cells. These results
demonstrate that MSCs highly expressing IL-21 have therapeutic potential
to induce potent anti-tumor effects against disseminated B-cell lymphoma
through localized IL-21 delivery to tumor site. It is also suggested that IL21/MSCs therapy can be improved by combining other anti-tumor substances to further enhance tumor immunity.
105
COMBINING
CD23
CHIMERIC
ANTIGEN
RECEPTOR
IMMUNOTHERAPY AND LENALIDOMIDE AS A NOVEL
S35
THERAPEUTIC STRATEGY FOR CHRONIC LYMPHOCYTIC
LEUKEMIA
M Bertilaccio1, S Tettamanti2, GG Attianese2, G Galletti1, S Arcangeli2,
T Rodriguez1, C Magnani2, F Barbaglio1, L Scarfò3,4,5, M Ponzoni5,6,
A Biondi2, F Caligaris-Cappio1,4,5, P Ghia1, E Biagi2
1
Laboratory of Lymphoid Malignancies, Division of Molecular Oncology,
Istituto Scientifico San Raffaele, Milano, MI, Italy, 2Centro Ricerca
Tettamanti, Clinica Pediatrica, Università Milano Bicocca, Osp. San Gerardo/
Fondazione MBBM, Monza, MB, Italy, 3Laboratory of B-cell neoplasia,
Division of Molecular Oncology, Istituto Scientifico San Raffaele, Milano, MI,
Italy, 4Università Vita-Salute San Raffaele, Milano, MI, Italy, 5Unit of
Lymphoid Malignancies, Department of Onco-Hematology, Istituto
Scientifico San Raffaele, Milano, MI, Italy, 6Pathology Unit, Istituto Scientifico
San Raffaele, Milano, MI, Italy
Lenalidomide is an immunomodulatory agent (IMID) able to induce significant long-lasting responses in Chronic Lymphocytic Leukemia (CLL)
patients. Lenalinomide was found to modulate CLL tumor microenvironment down-regulating critical cytokines, to activate immune effector cells,
and to reverse defects in immunological synapse between T and CLL cells.
Chimeric antigen receptors (CARs) are emerging as a powerful tool to
redirect T-cell specificity against leukemia. In order to revert in vivo the
acquired T cell defects in CLL patients, it becomes very intriguing to explore
a CAR-based immunotherapy combined with low doses of lenalidomide, to
maximize the effect of the immune attack. Using the Rag2-/-gc-/–xenograft
model of human CLL we performed experiments where mice were injected
with CAR.CD23+T cells from CLL patients together with lenalidomide at
low concentrations, uneffective in monotherapy. We observed a decreased
percentage of CD19+leukemic cells in all lymphoid and non-lymphoid tissues of mice after 20 days of treatment, as compared to controls treated with
CAR.CD23+T cells or lenalidomide alone. This combination resulted also in
improved survival of the treated cohort (NT+lenalidomide vs CAR+lenalidomide: p<0.03, n¼7). The effect of the combination with low dose lenalinomide was more effective also when compared to the addition of hIL-2 as
in traditional settings. In accordance to the in vivo efficacy, CAR.CD23+T
cells were observed in all leukemic sites suggesting an ability to migrate and
home in vivo. Moreover, CD23.CAR+T cells purified from bone marrow
were still able to mount tumor specific cytotoxic response in vitro, reaching
more than 50% of tumor lysis in both the conditions with lenalidomide and
hIL-2, compared to 20% exerted by unmanipulated T cells. These results
conceivably support the use for CLL immunotherapy of low doses lenalidomide to improve CAR cytotoxic response and avoid the potential
impairment of an effective immune response.
106
HUMAN MESENCHYMAL STEM CELLS PROVIDE PROTECTION
AGAINST
RADIATION-INDUCED
LIVER
INJURY
BY
ANTIOXIDATIVE PROCESS, VASCULATURE PROTECTION,
HEPATOCYTE DIFFERENTIATION AND TROPHIC EFFECTS
S Francois1,2, M Mouiseddine1,2, B Allenet-Lepage1, J Voswinkel3, L Douay2,
MM Benderitter1, A Chapel1,2
1
PRO-HOM, IRSN, Fontenay aux roses, France, 2Service d’Hematologie et de
Therapie Cellulaire, AP-HP, Paris, France, 3Equipe Proliferation et
Differenciation des Cellules Souches du Pr Luc Douay: Application à la
Therapie Cellulaire, UPMC UMRS_938, Paris, France
To evaluate the potential therapeutic effect of the infusion of hMSCs for the
correction of liver injuries, we performed total body radiation exposure of
NOD/SCID mice. After irradiation mir-27b level decreases in liver,
increasing the directional migration of hMSCs by up-regulating SDF1a.
Significant increases in plasmatic transaminases levels, apoptosis process in
the liver vascular system, and oxidative stress were observed. hMSC injection induced a decrease in transaminases levels and oxidative stress, a total
disappearance of apoptotic cells, an increase in Nrf2, SOD gene expression,
which might ROS production in the injured liver. Engrafted hMSCs
expressed cytokeratin CK18, CK19 and AFP genes indicating possible hepatocyte differentiation. The presence of hMSCs expressing VEGF and
Ang-1 in the peri-vascular region, associated with an increased expression of
VEGFr1, r2 in the liver, can confer a role of secreting cells to MSCs in
order to maintain the endothelial function. To explain the benefits for the
liver of hMSC engraftment, we find that hMSCs secreted NGF, HGF and
anti-inflammatory molecules (IL-10, IL1-RA) contributing to prevention of
S36
Poster Abstracts
apoptosis, increasing cell proliferation in the liver (increase of PCNA) gene
expression) which might correct liver dysfunction. MSCs are potent candidates to repair and protect healthy tissues against radiation damages.
107
TUMOR-SPECIFIC
DELIVERY
AND
ACTIVATION
OF
THERAPEUTIC GENES BY MESENCHYMAL STEM CELLS (MSC)
FOR THE TREATMENT OF ADVANCED ADENOCARCINOMA
OF THE GASTROINTESTINAL TRACT
R Huss, C Guenther
apceth GmbH & Co. KG, Munich, Germany
Current immunotherapies against cancer comprises of target specific antibodies,
cellular therapies like activated immune cells (e.g. DCs or NK cells), vaccination
strategies, the use of tumor-specific infectious agents like oncolytic viruses or a
combination thereof including conventional chemotherapies. All of those approaches attempt to target cancer specific pathways or certain immune response
mechanisms, which have to be activated or at least present in the individual patient. This requires the identification of predictive biomarker and / or the
execution of large clinical trials. Recent reports have shown that most clinical
responses to targeted therapies are still quite unpredictable and yield in large
clinical trials to demonstrate any benefit over conventional therapies. We have
developed a cell-based gene therapy approach using modified MSCs as a pharmaceutical delivery tool for drugs or therapeutic genes. Those pharmaceutical
grade MSCs delivering a gene-carrying cells directly into the cancer and its
environment (inflammatory tumor microenvironment (iTME) where cytotoxic
agents or prodrugs are activated only in the context of the tumor or its metastasis.
This significantly enhances the local anti-tumor efficacy and reduces unwanted
off-target toxicities. A first clinical trial (TREAT-ME1) targeting adenocarcinomas of the gastrointestinal tract is currently being conducted and first results of
this phase I/II study are quite promising. The clinical implementation of cellular
therapeutics represent a new challenge for clinical scientists and any pharmaceutical applicant with regard to the current regulatory requirements for
manufacturing, quality control, clinical trial approval and execution.
108
POTENT POLYCLONAL T CELL ACTIVATION AND EXPANSION
THROUGH GMP-GRADE TRANSACT NANO-MATRICES
D Mauer, N Mockel-Tenbrinck, K Drechsel, C Lehmann, I Johnston,
H Bohnenkamp, M Assenmacher, A Kaiser
Research & Development, Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany
The clinical success of adoptive T cell transfer therapy is resulting in growing
enthusiasm as indicated by the increasing number of clinical trials and the
involvement of large pharmaceutical companies. The stimulation reagents
commonly used to activate T cells are soluble anti-CD3 anti-CD28 antibodies that require accessory cells such as antigen presenting cells (APCs).
Resulting T cell expansions are often variable and dependent on the quality of
the “feeders” used. Alternatively, large beads (2-5 mm) coated with agonistic
anti-CD3/CD28 antibodies are used successfully. However, cell manufacturing
processes using such reagents entail manipulations that are suboptimal to
implement cell therapy for large numbers of patients. Aiming to streamline
clinical manufacturing of T cells, we have developed a cGMP compliant
stimulatory reagent, TransAct, a colloidal reagent consisting of iron oxide
crystals embedded into a biocompatible polysaccharide matrix with a diameter
of about 100 nm. Agonistic anti-CD3 and anti-CD28 antibodies are coated
onto two separate nanomatrices. This clinical grade TransAct fulfils the requirements for automated T cell preparations in that it is soluble and can be
removed by washing (ease of use), biodegradable, sterile filtered (safety) and
suitable for potent T cell activation, gene-modification and expansion. We have
compared the stimulatory potential of TransAct with known large beads.
TransAct was able to efficiently stimulate enriched T cells and resulted in
highly polyfunctional T cells which were comparable to those generated with
large beads. Additionally, the TransAct reagent was shown to prime naïve T
cells more efficiently. Finally, this novel cGMP reagent has also been successfully implemented into automated processes for the preparation of genemodified T cells using the CliniMACS ProdigyÒ platform, making this
product highly suitable for future clinical application in large patient groups
and commercial-scale manufacturing.
109
SPACER DESIGN INFLUENCES THE IN VIVO EFFICACY OF
CD19-CAR REDIRECTED T CELLS
H Almåsbak1, E Walseng2, A Kristian3, MR Myhre1, ES Inderberg1,
L Munthe4, M Wang1, G Kvalheim1, G Gaudernack5, J Kyte1
1
Section of Cell Therapy, The Norwegian Radium Hospital, Oslo University
Hospital, Oslo, Norway, 2Dept. of cancer biology, Scripps Research Institute,
La Jolla, Florida, United States, 3Dept. of tumor biology, Oslo University
Hospital, Oslo, Norway, 4Centre for Immune Regulation, Faculty of Medicine,
Oslo University Hospital, Oslo, Norway, 5Section for Immunology, Oslo
University Hospital, Oslo, Norway
Immunotherapy with CAR-expressing T cells is an alternative approach for
hematological cancers resistant to conventional treatment. After years with
disappointing clinical results, studies now demonstrate impressive antitumor
activity in various B-cell malignancies with CD19-CAR expressing T cells.
Although critical elements of the CAR are becoming more clearly understood and engineered for optimal function, there are still remaining questions concerning how different CAR-configurations other than the
signalling and recognition domains affect the in vivo efficiency of the
redirected T cells. Some CARs incorporate a spacer to provide flexibility
and accessibility for the binding moiety. The immunoglobulin domains
from the Fc-regions of antibodies, such as the IgG1 or IgG4-based “hingeCH2-CH3” are commonly used as spacers. We have compared T cells
redirected with CD19-CARs bearing full-length and truncated IgG1-based
spacers. Despite excellent in vitro antileukemia activity, we report that T
cells expressing CARs with full-length spacers were essentially inefficient in
a xenograft mouse model and moreover, that the CAR T-cells caused severe
and partly CD19-independent toxicity. We demonstrate that the toxicity
and lack of efficiency is related to the presence of the CH2-domain, which
harbours the binding site for Fcg-receptors (FcgR) as well as complement
proteins. There is a substantial crosstalk between human immunoglobulins
and murine FcgR and binding of CAR T cells through motifs in the spacer
to murine immune cells expressing FcgR could in theory cross-activate both
T cells and innate effector cells, leading to T-cell elimination by AICD or
direct killing. It is further suggested that bulky spacers might disturb the
optimal dimensions in the immunological synapse; however, such mechanisms would be expected to show impaired efficiency also in vitro. We are
currently conducting studies to verify the mechanisms leading to the lack of
efficacy and the toxicity.
110
INDUCTION OF MIXED CHIMERISM USING COMBINATORY
CELL-BASED IMMUNE MODULATION WITH MSCS AND
TREGS IN EARLY POST-TRANSPLANT PERIOD
K Im1, N Kim1, J Lim1, Y Nam1, E Lee1, E Chae1, E Kim1, S Cho1,2
1
Laboratory of Immune Regulation, Convergent Research Consortium for
Immunologic Disease, The Catholic University of Korea College of Medicine,
Seoul, Korea, Republic of, 2Catholic Blood and Marrow Transplantation
Center, Seoul St. Mary’s Hospital, The Catholic University of Korea College
of Medicine, Seoul, Korea, Seoul, Korea, Republic of
Establishment of mixed chimerism is an ideal approach to induce donorspecific tolerance expanding its potential in various clinical settings. Despite
the developments in partial conditioning regimens, improvements are still
needed in reducing toxicity and BMT related complications. Recently,
cell-based therapies including mesenchymal stem cells (MSCs) have been
incorporated in establishing noncytoreductive mixed chimerism protocols;
however, its efficacy is only partial and show reversed immunosuppressive
properties. The present study demonstrates a novel approach to induce mixed
chimerism and tolerance through combinatory cell-based immune modulation
(CCIM) of MSCs and regulatory T cells (Tregs). We hypothesize that the
interaction between these cells may lead to greater inhibition of host immune
responses. Compared to single cell therapy, CCIM induced a higher
engraftment rate and robust donor-specific tolerance to skin allografts across
full MHC barriers. These regulatory effects were associated with inhibition of
NK cell cytotoxic activity and CD4+IL-17+ cells with increased frequencies of
CD4+Foxp3+ cells and myeloid-derived suppressor cells (MDSC). Furthermore, CCIM was able to regulate mortality in a GVHD model through
reciprocal regulation of Treg/Th17. Taken together, we suggest CCIM as a
clinically applicable strategy for facilitating the induction of mixed chimerism
and permanent tolerance.
111
CGMP-COMPLIANT, CLINICAL SCALE, NON-VIRAL PLATFORM
FOR EFFICIENT GENE EDITING USING CRISPR/CAS9
L Li, P Natarajan, C Allen, MV Peshwa
Maxcyte, Gaitherburg, Maryland, United States
Gene editing offers a promising method for developing potential treatments for
monogeneic diseases. It is widely accepted that non-viral approaches to gene
editing are more advantageous than viral vector based approaches. However,
results of non-viral approaches to date result in low transgene expression and
higher cytotoxicity, and have thus limited effective translational development.
We have demonstratedthat loading of messenger RNA (mRNA) encoding
nucleases offers efficient transgene expression with low cytotoxicity in cell lines
and primary cells. MaxCyte’s automated, cGMP-compliant, scalable, FDA
Master Files supported, flow electroporation platform has already been
reviewed and allowed for engineering cells in over a dozen of clinical trials. We
investigated whether MaxCyte’s Flow Electroporation platform could be
employed for loading mRNA encoding CRISPR/Cas9 for targeted, efficient
gene editing in multiple cell types, including primary cells. The CRISPR reagents (Cas9/gRNA) targeting specific AAVS1 safe harbor site with doublestrand break (DSB) were purchased from Washington University Genomic
Engineering Center (St. Louis, MO). The mRNA for Cas9 and gRNA were
transcribed in vitro using mMESSAGEmMACHINEÒ T7 Ultra Kit from
Ambion (Austin, TX). It was found that the genomic DNA editing can be
efficiently achieved in human differentiation-terminated cells, such as the nonactivated resting peripheral blood lymphocytes (PBL) and human fibroblast; or
proliferated human cells, such as MSC and K562 cell line. The efficiency of
genomic DNA editing, measured by Cel-1 assay for determination of Nonhomologous End Joining (NHEJ) was 40%9 in non-activated resting PBL.
The viability of all transfected cells maintained 85% relative to control cells.
These results indicate that loading CRISPR/Cas9 as mRNA using MaxCyte’s
Flow Electroporation platform will permit translational development of efficient gene editing in human blood and hematopoietic stem cells.
112
CHIMERIC ANTIGEN RECEPTOR (CAR) MESSENGER RNA
(MRNA) LOADED FRESHLY ISOLATED PERIPHERAL BLOOD
LYMPHOCYTES AS A POINT-OF-CARE TARGETED TUMOR
IMMUNOTHERAPY
L Li, C Allen, P Natarajan, MV Peshwa
Maxcyte, Gaitherburg, Maryland, United States
CAR is a validated approach to enhance specific anti-tumor activity. Initial clinical
trials using lentivirus vectors encoding anti-CD19 CAR demonstrated durable
clinical responses. However, the ability to translate these findings to target other
antigens in solid tumors has met with limited successes because of clinical toxicities due to ‘on-target off-tumor’ activity of viral-vector-modified CAR T-cells.
Using mRNA encoding CAR permits both control of toxicity to normal tissue
and immunotherapy against solid tumors. In preliminary human studies, mRNA
encoding anti-mesothelin CAR was reported to be safe and efficient in reducing
tumor burden in two patients [Beatty, G.L., et. al. Cancer Research Immunology,
2013; in press]. In this study, mRNA CAR loading into ex vivo expanded T-cells
(mRNA-CAR-Expanded-T) was achieved using the MaxCyte GT System; an
automated, US FDA Master File supported cGMP compliant closed system for
clinical scale cell processing. In this report, we evaluated the anti-tumor potency
of mRNA CAR loaded into freshly isolated PBL (mRNA-CAR-PBL). We hypothesized that if anti-tumor activity of mRNA-CAR-PBL was similar to that of
mRNA-CAR-Expanded-T, it would permit commercial development of mRNACAR-PBL at point-of-care utilizing existing transfusion medicine infrastructure.
Our results indicate that mRNA CAR can be effectively loaded into freshly isolated PBL obtained from therapeutic apheresis with w90% cell viability and
>90% efficiency. In in vitro antigen-specific cytotoxicity assays, mRNA-CARPBL exhibited similar dose response to mRNA-CAR-Expanded-T from the same
donor. In animal studies using localized and disseminated tumors, mRNA-CARPBL demonstrated anti-tumor activity and survival in dose-dependent manner.
We have scaled-up mRNA-CAR-PBL manufacture to process an entire Therapeutic Apheresis product (w1e10 cells) in w10 minutes and are working to
translate this process into human clinical trials.
113
S37
114
LOCALISATION OF STEM CELL AND OTHER CELL THERAPIES
USING
CELL-IN-A-BOXÔ
FOR
MICROENVIRONMENT
CONTAINMENT IN PATIENTS: A CLINICALLY PROVEN
ENABLING CELL ENCAPSULATION TECHNOLOGY
B Salmons
Austrianova, Singapore, Singapore, Singapore
We have developed Cell-in-a-BoxÔ, a novel, clinically proven cell encapsulation technology. Cells producing therapeutic products such as enzymes,
growth factors, antibodies etc are encapsulated in polymers of cellulose sulphate and implanted in the body thereby achieving long term release of the
therapeutic. The cellulose sulphate is inert and biocompatible and the capsules
are robust and stable. The capsule protects the cells from the immune system
(even if they are from a different species) and localises them by physically
confining them but is porous so that therapeutic products can be released. The
capsule also protects the body from release of cells (such as stem cells) that may
cause pathology if they become lodged at non target sites. Safety and efficacy
has been demonstrated in clinical trials to treat pancreatic cancer, using a cell
based suicide gene/prodrug strategy (Lohr et al., Lancet 357, 1591-2) and later
stage clinical trials are now planned. Around 20 different cell types have been
successfully encapsulated, including stem cells which can be confined and thus
give local effects while at the same time reducing the number of cells required.
Encapsulated cells have been successfully implanted at various sites in the body
e.g. subcutaneous, intra-peritoneal, intramuscular, into blood vessels, peri-tumoral etc. The technology can be used to treat diseases as diverse as cancer,
diabetes , cardiovascular and neurodegenerative diseases, enzyme deficiencies
and other metabolic diseases and data supporting a number of different uses of
the technology will be presented.
115
VASCULAR PROTECTIVE EFFECT OF EXENDIN-4 IN
EXPERIMENTAL MODELS OF OXIDATIVE STRESS
P Li1, S Fan1, K Lee2, C Chen2, C Wang1, L Chang1
1
Departmet of Occupational Therapy, I-Shou University, Kaohsiung,
Taiwan, 2Department of Biological Science, National Sun Yat-Sun University,
Kaohsiung, Taiwan
Glucagon-like peptide-1 (GLP-1) is a gut-derived hormone, namely the
incretin family, secreted by small intestine under a fine-tune regulation in
response to glucose concentration. GLP-1 exerts stimulatory effect on in-
sulin release through a binding to GLP-1 receptor (GLP-1R) on pancreatic
beta cells and the analogues has been clinically employed for treating patients
with Type 2 diabetes. However, the mechanism underlying pleiotropic effect
of GLP-1 and its agonist remains to be elucidated, particularly the investigation into the protective effect of GLP-1 in the vascular endothelial injury
is very limited. After arteriovenous (AV) fistula, blood flow in the venous
limb of an AV fistula is also determined by the surrounding draining veins,
blood pumped from the arterial site is the most important factor in maintaining sufficient fistula blood flow. The reduced blood flow and impaired
endothelium-dependent relaxation response of AV fistula in diabetic rats was
S38
Poster Abstracts
derived from enhanced activity of pro-inflammatory genes and generation of
superoxide anions in the remodeled vasculature. Our results showed that
treatment with exendin-4 (a GLP-1 analogue) in cultured human umbilical
vein endothelial cells (HUVECs) improved the cell viability following
exposure to H2O2 in a concentration-dependent fashion. There is currently
no previous studies reported the application of GLP-1 analogues in the
protection of vascular endothelial function and mobilization of EPCs in
maturation of AV fistula of subjects with diabetes. Logistically extended from
these findings, the applicants hypothesize that Exendin-4 attenuates oxidative damage of vascular endothelial cells and mediates vascular protective
effect in function AV fistula of animals with diabetes. Administration of
GLP-1 analogue may also increase the mobilization of EPCs in the peripheral circulation, and may thus enhance the proangiogenesis in subjects
with hyperglycemia.
116
INVESTIGATION OF HYPOTHERMOSOLÒ AS A DELIVERY
MEDIUM FOR CARDIOMYOCYTE PROGENITOR CELLS
M Herías
Process Development and Research, PharmaCell, Maastricht, Netherlands
Cell preservation is an important issue in cell therapy trials. Cell growth
rescue after thawing is an important consideration to avoid functional
impairment or cell growth aberrations. PharmaCell participates in the
Dutch Bio-Medical Materials program SmartCare that investigates the potential of cardiomyocyte progenitor cells (CMPCs) on bioactive microtissues
for heart tissue regeneration. PharmaCell investigated the use of HypoThermosolÒ (BioLife solutions) to deliver cells of both fetal and adult origin
for in vivo trials that would preserve these cells from the time of harvesting,
preparation and transportation to the time of surgery. Cell number, viability,
morphology and cell marker expression (FACS) were tested at different time
points (0, 4, 8, 24, 48, 72 hrs). In addition, cells were grown after 72 or 96 h
suspension in HypoThermosolÒ to test if their proliferative capacity was
maintained. Cell numbers remained stable for both cell types. A modest
(15% decrease) in cell viability was noted for fetal cells (after 96hrs) and
16% (after 72 hrs) for the adult cells. Protein expression in fetal cells
remained within our specifications for cardiac markers NKX2.5, Gata-4, aActinin, while adult cells presented a substantial decrease in a-Actinin after
48hrs. Both type of cells were able to proliferate after HypoThermosolÒ
exposure, but their population doubling times increased. The results support
the use of HypoThermosolÒ as a preservation medium for fresh cells and
addresses differences between CMPCs of different origins. This research
forms part of the Project P1.04 SMARTCARE of the research program of
the BioMedical Materials institute, co-funded by the Dutch Ministry of
Economic Affairs. The financial contribution of the Netherlands Heart
Foundation is gratefully acknowledged.
117
CELL THERAPY FOR PERIPHERAL ARTERY DISEASE:
PRODUCT CHARACTERIZATION AND STABILITY
M Radrizzani1, V Lo Cicero1, S Soncin1, S Bolis1, D Sürder2,1, T Torre3,
F Siclari3, T Moccetti2, L Turchetto1
1
Cell Therapy Unit, Cardiocentro Ticino, Lugano, Ticino,
Switzerland, 2Cardiology Service , Cardiocentro Ticino, Lugano, Ticino,
Switzerland, 3Cardiac Surgery Service, Cardiocentro Ticino, Lugano, Ticino,
Switzerland
The Cardiocentro Ticino Cell Therapy Unit is authorized for the production of
experimental advanced therapy medicinal products (ATMP). The ATMP for the
upcoming CIRCULATE study (“Bone Marrow Derived Cell Therapy in Peripheral Arterial Disease”), currently under evaluation by Swiss regulatory authorities, is named VASASTIM and consists of fresh mononuclear cells isolated
from autologous bone marrow through density gradient centrifugation on low
density Ficoll-PaqueTM; cells are formulated in saline solution containing 5%
human serum albumin. The process has been successfully validated according to
Good Manufacturing Practices, for safety (sterility, endotoxin), identity (CD45/
CD34/CD133), purity (contaminant granulocytes and platelets) and potency
aspects (viability, CFC, CFU-F and invasion assays). The aim of the present
work was to further characterize the product and evaluate its stability, according
to specific requests formulated by regulatory authorities. Fourteen pre-clinical
VASASTIM batches were manufactured and extensively tested. All of them
comply with viability and purity specifications (viability 70%, granulocytes
55%, platelets/WBC10) and contain CD34+ (2.671.86%) and CD133+
(0.330.64%) cells; a consistent fraction of the cells express CXCR4 (3213%),
a marker known to correlate with functional activity of bone marrow-derived
mononuclear cells. The product contains hematopoietic precursors (CFC assay:
66984363 colonies/1E6 cells), mesenchymal precursors (CFU-F assay: 5640
colonies/1E6 cells) as well as cells with invasion capacity (Fluorimetric invasion
assay: Invasion Index 4718%) and angiogenic potential (CFU-EC assay: 3119
colonies/1E6 cells). Nine batches entered a stability program: they were stored at
10 C, previously defined as the optimal temperature for this product, and tested
for several parameters at 0-6-20-24 hours. Overall, the results indicate that
VASASTIM is stable for at least 20 hours at 10 C.
118
ENDOTHELIAL PROGENITOR CELLS THERAPY CONCOMITANT
WITH DERMATAN SULFATE INJECTION AFTER CAROTID
ARTERIAL INJURY ATTENUATES INFLAMMATORY RESPONSE
IN MICE
JA Godoy1, CC Werneck2, CP Vicente1
1
Department of Functional and Structural Biology, State University of
Campinas - UNICAMP, Campinas, Sao Paulo, Brazil, 2Department of
Biochemistry, State University of Campinas - UNICAMP, Campinas, Sao
Paulo, Brazil
Vascular interventions can damage endothelium, promote thrombosis affecting
endothelium regeneration. Cellular therapy using endothelial progenitor cells
(EPC) originated from mononuclear (MNC) bone marrow cells primed in
culture can promote re-endothelization. Dermatan sulfate (DS) is an antithrombotic glycosaminoglycan that can also be anti-inflammatory and inhibit
neointima formation. The objective of this work is to verify if the treatment with
DS alone or with EPC can inhibit the initial inflammatory response promoted by
arterial lesion. For this, we induced carotid artery lesion using a wire probe and
intravenously administered DS [20 mg/kg] alone or with freshly purified MNC
or EPCs primed in culture obtained from C57BL6 mice bone marrow. We
analyzed the molecules involved in adhesion (ICAM-1), leukocyte homing (Pselectin), vascular tone (eNOS) by western blotting and apoptosis (TNF-alpha)
and extracellular matrix metabolism (TGF-beta) by ELISA 1 and 3 days after
lesion. The treatment with DS or EPC alone decreased TNF-alpha in both 1
and 3 days. Injection of DS increases TGF-beta on day 1 and primed EPC or
MNC decreased TGF-beta on day 3. ICAM-1 decreased in all groups on day 1
and this decrease was maintained after 3 days. P-selectin decreased in animals
treated with DS or DS with EPC after 1 and 3 days. eNOS expression was
decreased on day 1 by DS or EPC alone and this decrease was maintained only in
DS + EPC treated animals on day 3. We can conclude that treatment with DS
was capable in avoiding apoptosis and leukocyte homing but negatively influenced vascular tone and extracellular matrix metabolism; EPC alone or with DS
showed the same results as DS alone, demonstrating the importance of EPC in
inflammatory response; MNC alone or with DS, promoted a higher leukocyte
adhesion and extracellular matrix degradation increasing inflammation in the
injury site. Financial Support: Fapesp 2012/23640-2 and Fapesp 2010/01119-3.
119
HUMAN UMBILICAL CORD ARTERIES AS POTENTIAL ARTERIAL
GRAFTS: A PROTEOMIC VALIDATION OF DECELLULARIZATION
PROTOCOLS
P Mallis1, I Gontika1, T Poulogianopoulos1, J Zoidakis2, A Vlahou2,
E Michalopoulos1, TK Chatzistamatiou1, AC Papassavas1,
C Stavropoulos-Giokas1
1
Hellenic Cord Blood Bank, Biomedical Research Foundation, Academy of
Athens, Athens, Greece, 2Biotechnology Division, Biomedical Research
Foundation, Academy of Athens, Athens, Greece
Introduction: Major achievements in creating decellularized whole tissue
scaffolds have drawn considerable attention to decellularization as a promising
approach for tissue engineering. In this study histological and proteomic analysis
was performed to evaluate the efficiency of the two decellularization protocol.
Methods and Results: In decellularization protocol A, umbilical arteries (n¼
20) were incubated in CHAPS and sodium dodecyl sulfate followed by incubation
in a-MEM with foetal bovine serum. In decellularization protocol B the umbilical
arteries (n¼20), were incubated in Hypotonic Tris and SDS followed by incubation in Nuclease solution. Decellularized umbilical arteries were completely
devoid of cellular and nuclear material while extracellular matrix remained intact
Native and decellularized arteries were digested using Proteinase K and DNA
content was measured. For the proteomic analysis the umbilical arteries were
snap frozen in liquid nitrogen and homogenized. The protein content was
measured using Bradford assay and analyzed by 2D Electrophoresis. Protein
spots were excised manually and tryptic digestion and Peptide Mass Fingerprinting was performed. Decellularized patches (3x3 mm) of umbilical artery
were seeded with 10,000 MSCs-RFP+ per patch, incubated for 2 weeks at 37o C
and 5% CO2 and no cytotoxic effect was observed.
Conclusion: Both decellularization protocols effectively removed the cellular
material while the ECM remained intact and supported MSCs seeding.Future
studies are warranted to elucidate the specific effects of altered structuree
function relationships on the overall fate of decellularized umbilical arteries.
120
DIRECT REPROGRAMMING OF HUMAN FIBROBLASTS TO A
CARDIAC FATE USING REPROGRAMMING PROTEINS
Z Ghazizadeh1, H Rassouli2, H Fonoudi1, M Alikhani2, GH Salekdeh2,
N Aghdami1, H Baharvand1,3
1
Department of Stem Cells and Developmental Biology at the Cell Science
Research Center, Royan Institute for Stem Cell Biology and Technology,
Tehran, Iran, Islamic Republic of, 2Department of Developmental Biology,
University of Science and Culture, Tehran, Iran, Islamic Republic of,
3
Department of Molecular Systems Biology at the Cell Science Research
Center, Royan Institute for Stem Cell Biology and Technology, Tehran, Iran,
Islamic Republic of
Heart disease remains one of the main causes of death worldwide. Direct safe
cardiomyocyte reprogramming of patients’ own fibroblasts potentially offers a
new therapeutic approach to cardiovascular regenerative medicine. In the present
study, we show that human fibroblasts converted into cardiomyocytes by directly
delivering of reprogramming recombinant cell-permeant form of OCT4, SOX2,
KLF4, and c-MYC (rOSKM) proteins followed by bone morphogenetic protein4 treatment and glycogen synthase kinase inhibition. The early reprogrammed
cells were be directly reprogrammed toward protein-induced cardiomyocyte-like
cells (p-iCLCs) over a period of three weeks without first undergoing reprogramming into or through a pluripotent intermediate. The transplantation of the
p-iCLCs also improved rat model of myocardial infarction. Our study provides a
transient and plastic developmental state in human fibroblasts which shortcut
reprograming toward patient cardiogmyocytes in a safe manner.
121
LONG TERM FOLLOW-UP OF CORONARY ARTERY BYPASS
GRAFTING WITH AUTOLOGOUS BONE MARROW CELL
THERAPY
AN Patel1, S Mittal3, RF Vina2, F Benetti2, N Trehan3
1
United States, 2Cardiac Surgery, Benetti Foundation, Rosario, Argentina,
3
Medanta Heart Institute, Medanta - The Medicity, Delhi, India
Background: Autologous bone marrow cell (BMC) therapy as an adjunct to
coronary revascularization has been performed for many years. A randomized
study was conducted using epicardial delivery to inject the BMC into the
myocardium as an adjuvant to coronary revascularization (CABG) therapy in
patients with decreased myocardial function.
Methods: Autologous bone marrow cells was performed in patients with an
ejection fraction of less than 35% undergoing CABG. The patients underwent
echocardiography, nuclear imaging, and cardiac catheterization to identify
ischemic regions of the heart and to guide in the selection of BMC injection
sites. The patients were prospectively randomized before the operative therapy
was performed. Patient follow-up was yearly after the initial year.
Results: There were 50 patients enrolled in the study: 20 in the pilot phase and
30 in the second phase after safety was evaluated and verified. Twenty-five patients had successful BMC injection into the myocardium + CABG. The other 25
patients had CABG only. There were no complications attributed to the BMC.
The patients have been followed for 10 years. There is a improvement in cardiac
function over the 10 years compared to the control group (see table). There is also
a survival advantage in the BMC group (22 pts) vs control (15 pts)* (*p<0.05).
Conclusions: Long term follow-up demonstrates that autologous BMC led
to significant improvement in myocardial function with survival advantage.
Larger studies will be required to further validate the findings of this study.
S39
Long term ventricular function
Ejection Fraction %
1 yr
5 yr
10 yr
CABG
CABG +BMC
36.8
34.0
29.8
46.0
43.1*
37.2*
*p<0.05.
122
RETROGRADE DELIVERY OF AUTOLOGOUS CONCENTRATED
BONE MARROW CELL THERAPY FOR PATIENTS WITH
CONGESTIVE HEART FAILURE - REVIVE-1 - A PROSPECTIVE
RANDOMIZED STUDY
AN Patel1, H Ince2, S Mittal3, G Turan2, N Trehan3
1
United States, 2Cardiology, University of Rostock, Rostock, Germany,
3
Medanta Heart Institute, Medanta - The Medicity, Delhi, India
Background: End stage hart failure have limited options due to few donor organs.
Cell therapy has shown promise but is limited by dosing, retention and cardiac wall
thickness. Our goal was to evaluate retrograde bone marrow cell delivery in patients with either ischemic (IHF) or non-ischemic heart failure(NIHF).
Methods: This was a prospective randomized, multi-center, open label
study of the safety and feasibility of retrograde infusion of nucleated cells
through the coronary sinus using the Harvest SmartPRePÒ Bone Marrow
REVIVE-1
NIHF
n¼30
BNP baseline (pg/ml)
BNP 1 yr (pg/ml)
EF baseline (%)
EF 1 yr (%)
IHF
n¼30
Ctrl
n¼6
BMAC
n¼24
Ctrl
n¼6
BMAC
n¼24
648
508
24.1
22.5
403
181*
25.1
30.6*
652
226
25.0
33.1
336
326
26.3
28.3
*p<0.05.
Aspirate Concentrate System (BMAC). Sixty (60) randomized subjects were
stratified by IHF and NIHF to receive either treatment or control (standard
of care) in a 4:1 ratio. Accordingly, 24 subjects were randomized to the
ischemic treatment group and 6 to the open label ischemic control group.
Similarly, 24 subjects were randomized to the non-ischemic treatment group
and 6 to the open label non-ischemic control group. Clinical and blood based
endpoints were collected.
Results: All 60 patients were successfully enrolled in the study. The
treatment groups had BMAC infusion without complication. The LVEFs and
BNPs are in the table below.
Conclusion: Retrograde BMAC delivery was safe. The NIHF had significant reduction in BNP while improving other clinical endpoints. This study
forms the basis for a larger clinical in patients with NIHF.
123
FATE OF HUMAN CRARDIAC PRECURSOE CELLS FOLLOWING
INJECTION IN THE SHEEP MYOCARDIUM USING A NOGA
CELL DELIVERY SYSTEM
SMP Fluri1,2, T Pedrazzini2, P Ruchat3, E Pruvot2, C Gonzales2, I Plaisance2,
D Locca2
1
Cardiologie, Hôpital de Sion, Sion, Switzerland, 2Medecine, CHUV,
Lausanne, Switzerland, 3Chirurgie, CHUV, Lausanne, Switzerland
Cell replacement therapy for heart failure in human has been shown to have
beneficial effects on remodeling, the mechanism of action remains unknown.
This study aim is to investigate the destiny of human cardiac precursor cells
(CPCs) up to 4 hours after injection into sheep myocardium and mechanical
S40
Poster Abstracts
heart diseases. Regardless of cell type and route of administration, injected cells
appear to vanish from the injection area, in which they should exert their
regenerative effect. One plausible way of increasing cell retention and effect is
injecting the cells in an in-situ cross-linking alginate hydrogel (AH). Before
initiating these trials, the characteristics and immunological properties of the
cells cultured in AH must be investigated.
Methods: Adipose-derived stromal cells (ASCs) were isolated from 3 healthy
donors, cultured in standard culture conditions and seeded in 1% AH with or
without RGD attachment motifs. After one week of culture, cell viability was
assessed using live/dead staining and ASC phenotype was characterized by flow
cytometry and triple differentiation. For test of immunomodulatory properties,
CD14+ cells were isolated from buffy-coats and differentiated to dendritic cells
(DCs). DCs were co-cultured for 24 hours with ASC in AH. DC maturation
markers were measured by flow cytometry.
Results: The ASCs behaved similarly in the two types of AH, except for a more
branched appearance with RGD incorporation. After one week of culture, the cell
viability was 95%, and the ASCs characteristics closely resembled the ISCT
criteria. CD90 was down regulated, but the levels increased to ISCT standards
when the cells were separated or sedimented from the AH. AH alone induced a low
level increase in DC maturation markers, while the AH containing ASCs did not.
ASCs alone significantly decreased the expression of DC maturation markers.
Conclusion: The ASCs retained most ASC specific markers while in the AH,
and quickly regained CD90 upon leaving the AH. The ability of the ASCs to
suppress an immunological response when seeded in the AH, is evidence of
sustained paracrine function of the ASCs.
125
EXOSOMES SECRETED BY ADULT HUMAN CARDIAC
PROGENITOR CELLS INHIBIT CARDIOMYOCYTE APOPTOSIS,
STIMULATE ANGIOGENESIS, AND IMPROVE CARDIAC
FUNCTION AFTER MYOCARDIAL INFARCTION
L Barile1, V Lionetti2, M Matteucci2, E Cervio1, T Torre1, F Siclari1,
M Gherghiceanu3, L Popescu3, T Moccetti1, G Vassalli1
1
Cardiocentro Ticino, Lugano, Switzerland, 2Scuola Superiore Sant’Anna, Pisa,
Italy, 3“Victor Babes’’ National Institute of Pathology, Bucharest, Romania
damages mediated by intramyocardial injections. In 2 sheep, CPCs isolated from
human right atrial appendage were injected into the left ventricular myocardium
with the NOGA (Biosense Webster, USA) catheter. Both animals underwent
cell injections 4 and 1 hours before euthanasia, whereas an injection of the same
volume of saline was performed 4 hours before sacrifice in one animal to assess
mechanical damage related to injections. Myocardial cubes around the injection
sites were harvested and embedded in OCT (Optimal Cutting Temperature)
compound. Analysis of cryosections showed large amounts of human HLA-1
positive cells intramyocardially (Figure 1) at both time points. Cells were present
in clusters and located in elongated lacunae surrounded by intact sheep cardiomycoytes. The same lacunae were identified in tissue samples after saline
injection, suggesting that myocardial fibers are pushed apart by the mechanical
pressure imposed by the injection. H&E staining (Figure 2) confirmed these
results. The size of cell clusters was larger after 1 hour that after 4 hours, indicating a decresasing cell retention over time. Reasons for this are multiple, but a
large lymphocytic infiltrate has been identified after 4 hours.
124
PRESERVATION OF PHENOTYPE AND IMMUNOMODULATORY
PROPERTIES
OF
ADIPOSE-DERIVED
STROMAL
CELLS
CULTURED IN ALGINATE HYDROGEL
B Follin1,2, M Juhl1, J Tratwal1, M Haack-Sørensen1, M Gad2, J Kastrup1,
S Cohen3, A Ekblond1
1
Cardiology Stem Cell Center, The Heart Center, Rigshospitalet, University
Hospital Copenhagen, Copenhagen, Denmark, 2Cell Technology Group,
Bioneer A/S, Hørsholm, Denmark, 3Avram and Stella Goldstein-Goren
Department of Biotechnology Engineering, Ben-Gurion University of the
Negev, Beer Sheva, Israel
Background: In recent years, multiple clinical trials have documented a
beneficial effect of treatment with various mesenchymal cell types for several
Background: Cardiac progenitor cell (CPC) transplantation improves cardiac
function after myocardial infarction (MI). This effect is largely mediated by
secreted factors. Exosomes (Exo) are secreted nano-sized membrane vesicles that
act as intercellular carriers of proteins and nucleic acids including RNA. Here,
we investigated the role of Exo in the paracrine secretion by human CPCs.
Methods and Results: Atrial appendage specimens were obtained from
patients who underwent heart valve surgery. CPCs were derived from the
cellular outgrowth of atrial explants using the primary ex vivo culture technique. CPC conditioned medium (CMCPC) stimulated tube formation by
endothelial cells (HUVECs) while also inhibiting mouse HL-1 cardiomyocyte
apoptosis after serum deprivation in vitro. CM-CPC depleted of Exo lacked
proangiogenic and antiapoptotic activities. These activities were restored by
adding back the purified Exo fraction to Exo-depleted CM. Exo-CPC, but not
Exo from normal human dermal fibroblasts (Exo-F), inhibited cardiomyocyte
apoptosis in a dose-dependent manner. miRNA transcriptional profiling
identified several miRNAs as differentially expressed in Exo-CPC vs. Exo-F. In
a rat model of MI, Exo-CPC-injected hearts, but not those injected with ExoF, showed significantly less cardiomyocyte apoptosis and scar, more blood
vessels, and improved changes in left ventricular ejection fraction 7 days after
MI compared to PBS-injected hearts (0.8+6.8% vs. -21.3+4.5%; p<0.05).
Conclusion: Exo accounts for proangiogenic and antiapoptotic activities of
human CPCs. Exo-CPC injection into infarcted hearts attenuates functional
impairment early after MI. As a cell-free product, Exo-CPC has a potential for
circumventing many of the limitations of cell transplantation for cardiac repair.
126
APPLICATION OF STEM CELL THERAPY FOR DILATED
CARDIOMYOPATHY: A SYSTEMATIC REVIEW AND METAANALYSIS OF RANDOMIZED CONTROLLED TRIALS
H Jeong1, H Yim1, H Park2, S Jeong3, H Kim4
1
Department of Preventive Medicine, The Catholic University if Korea, Seoul,
Korea, Republic of, 2Division of Cardiovascular Medicine, Seoul St. Mary’s
Hospital, Seoul, Korea, Republic of, 3Medical Library, Seoul St. Mary’s
Hospital, Seoul, Korea, Republic of, 4Clinical Research Coordinating Center,
Catholic Medical Center, Seoul, Korea, Republic of
Background and objectives: Dilated cardiomyopathy is the most common form
of non-ischemic cardiomyopathy and can lead to sudden cardiac death or heart
failure. Stem cell therapy has been studied in preclinical and clinical models of
ischemic heart disease, showing potential benefit. Few studies have addressed the
use of stem cells to treat patients with dilated cardiomyopathy and the results of
current studies were discrepant. The objective of this meta-analysis was to evaluate
the efficacy of stem cell therapy for dilated cardiomyopathy. To our knowledge,
there is still none evaluating stem cell therapy for dilated cardiomyopathy.
Methods: A systematic search was conducted through Pubmed, Embase, and
Cochrane data base from inception to March, 2013. The weighted mean difference (WMD) was calculated using the fixed effect model for net changes in left
ventricular ejection fraction (LVEF), left ventricular end-diastolic dimension
(LVEDD), 6-minute walk, and plasma NT-proBNP level. Also, odd ratio (OR) of
death was measured using the fixed effect model.
Results: Five randomized controlled trials with a total of 268 patients were
included in the analysis. Compared with the controls, patients who received stem
cell therapy had a significant improvement in LVEF by 6.45% (95%CI: 4.83-8.07,
I2¼29%), a reduction of LVEDD by -33mm (95%CI: -58 to -7, I2¼24%), an
enhancement of 6-minute walk by 142m (95% CI: 108-176, I2¼0%), and a
decrease in plasma NP-proBNP level by -1947pg/mL (95%CI: -2343 to -1552,
I2¼0%). In addition, stem cell therapy was related to lower risk of death, with the
OR being 0.43 (95%CI: 0.23-0.81, I2¼25%).
Conclusions: Stem cell therapy appears to be effective in treatment of patients
with dilated cardiomyopathy and may be valuable treatment option, particularly
for those challenging patients with limited therapeutic options.
127
A 3 MONTH TOXICOLOGY STUDY OF BONE MARROW
DERIVED MESENCHYMAL STROMAL CELLS MANUFACTURED UNDER GMP AND ADMINISTERED ONCE BY
INTRAMUSCULAR INJECTION
M Creane2, MM Elroy3, CS Dawood1,2, A Duffy1,2, T O’Brien2,1
1
Centre for Cell Manufacturing Ireland (CCMI), National University of
Ireland Galway, Galway, Ireland, 2Regenerative Medicine Institute
(REMEDI), National University of Ireland Galway, Galway, Ireland, 3Charles
River Preclinical Services, Tranent, Edinburgh, United Kingdom
S41
Endothelial progenitor cells (EPCs) described by Asahara et al. (1997) are
progenitors cells able to differentiate into adult endothelial cells and play role in
the repair of injured endothelium. Increasing the knowledge of EPC biology
represents an advance in the understanding the mechanism involving vascular
repair. The objective of this study was to understand the role of EPCs in
resolving arterial thrombosis induced by FeCl3 lesion. EPCs were obtained
from mice bone marrow mononuclear cells (MNCs), cultured and expanded for
5 passages in EPC medium and characterized by FACS and immunofluorescence using cellular markers as CD133, CD34, CD31, VEGFR2 and CD45,
uptake of Dil-acLDL.and tube formation in Matrigel. For the arterial injury
C57Bl6 male mice were anesthetized and the common carotid artery isolated
and injured with FeCl3. Animals were injected or not with 2x106 freshly isolated MNCs or cultured EPCs or HUVECs. Cells were labeled with Rhodamine G and injected via tail vein 10 minutes before injury. Thrombus formation
and migration of these cells were monitored by intravital microscopy and the
thrombosis time determined after cell injection and lesion using an ultrasound
flow probe. As results we observed that our EPCs were CD45low, CD34+/
VEGFR2+/CD31+, incorporated Dil-acLDL and formed tube-like structures.
We followed thrombus formation for 0, 3 and 10 min after injury and observed
that the injection of MNC or EPC decreased significantly thrombus formation
time from 4 to 8 min (p<0.05). Injection of HUVEC was unable to alter
thrombosis time. We also observed using intravital microscopy that EPC injection promotes thrombus embolization, what may be responsible to the
prolonged occlusion time. We conclude that EPCs injection alters thrombus
formation probably by affecting thrombus matrix remodeling altering its ability
to became stabilized. Further studies are necessary to determine the complete
role of these cells in thrombus formation and stabilization.
129
INCREASED HUMAN ADIPOSE DERIVED MESENCHYMAL
STEM CELLS AND PRO-B-TYPE NATRIURETIC PEPTIDE IN
PATIENTS WITH CRITICAL LIMB ISCHEMIA
H Farres1, CP Sutton1, A Hakaim1, A Zubair2
1
Vascular, Mayo Clinic, Jacksonville, Florida, United States, 2Cell Therapy,
Mayo Clinic, Jacksonville, Florida, United States
Atherosclerotic peripheral arterial disease (PAD) is a condition associated with
substantial morbidity and mortality and frequently results in lower limb amputation. The increased prevalence of PAD, in the absence of adequate therapeutic
options, suggests that limb amputation and death within these patients will
continue to rise. Mesenchymal stromal cells (MSCs) with their immunosuppressive and angiogenic properties may provide an exciting treatment strategy for
patients with PAD. However prior to a first in man study, preclinical toxicology
studies using a product manufactured under good manufacturing practices (GMP)
conditions are deemed a necessary regulatory requirement prior to the trial initiation. Toxicology studies for cell therapy should use a product manufactured under
GMP in the manner in which it will be used for the human study. The cell product
should be administered by the same route to be used in the human study.
Furthermore, as these cells are derived from humans, immunocompromised animals will be required. We studied the safety of human bone marrow derived MSCs
manufactured under GMP conditions following intramuscular administration in
nude mice. The administration of 300,000 MSCs was not associated with any
changes in body weight or food consumption. Furthermore no significant clinical
signs, hematological or major pathological changes were found in MSC treated
animals in comparison to vehicle controls. However in a number of mice, although
not significant, plasma creatine phosphokinase (CPK) was increased 2 fold in the
MSC compared to the vehicle treated groups. Nonetheless in the absence of any
histopathological findings the elevated CPK was deemed as an incidental finding
that was not related to the MSC administration. In view of these data we conclude
that the intramuscular administration of hMSCs was well tolerated and not associated with any adverse effects in nude mice.
Mesenchymal stem cells (MSCs) have been shown to improve regeneration of
injured tissues in vivo, however the mechanisms remain unclear. Several in vitro
studies and animal models have demonstrated improvement in MSCs paracrine
effects (trophic, immunomodulatory and angiogenic) under hypoxic conditions.
Moreover, several studies suggested that the B-type natriuretic peptide (BNP)
could be involved in the stimulation of postischemic vascular regeneration. This
study aims to investigate the effect of critical limb ischemia, in a human model, on
in-situ adipose derived mesenchymal stem cells (ADMSCs) and to determine
whether serum levels of N-terminal (NT) pro-B-type natriuretic peptide (proBNP) correlate with ADMSCs counts and associated paracrine effects. Lipoaspirate samples of greater than or equal to 10mL were collected from ischemic
limbs (ischemic group) and compared to a control group (without ischemia).
MSCs were characterized by frequency (MSC/ml), viability, differentiation potential, cytokines expression, and cell surface markers. Serum pro-BNP was
measured as well. MSCs counts were 9-to-10-fold higher in patients with ischemic
limbs (mean 7952 MSC/mL +/- 542) than in patients in the control group (mean
790 MSC/mL +/- 65). NT pro-BNP levels (1878-4757 pg/mL) were approximately 8-to-26-fold higher than in age- and sex-matched controls. Furthermore,
there were positive correlations between pro-BNP levels and MSCs counts in the
ischemic group. Patients with critical limb ischemia have higher serum levels of
NT pro-BNP and MSCs counts than controls. Increased serum levels of NT proBNP and MSCs counts can be considered, respectively, humoral and cellular
surrogates of ischemia and hypoxia in patients with critical limb ischemia. This
supports several recent studies that suggest increased production of peripheral
NT pro-BNP may be a stem cells-mediated response to stimulate angiogenesis in
the ischemic skeletal muscles of patients with critical limb ischemia.
128
EXPANSION OF ENDOTHELIAL PROGENITOR CELLS FROM
BONE MARROW IN VITRO AND YOUR ROLE IN ARTERIAL
THROMBOSIS FORMATION INDUCED BY FECL3
GD Carneiro1, JA Godoy1, CC Werneck2, CP Vicente1
1
Structural and Functional Biology, State University of Campinas, Campinas,
Sao Paulo, Brazil, 2Biochemistry, State University of Campinas, Campinas, Sao
Paulo, Brazil
130
PRELIMINARY STEPS FOR THE CREATION OF SMALL
DIAMETER VASCULAR GRAFTS
SP Lopez1, MN Rego1, MÁ Viejo1, MP Basterrechea1, JC Anadon2,
SA Cervantes2, JO Hernandez1
1
Laboratorio de Trasplante y Terapia Celular, Hospital Universitario Central
de Asturias, Oviedo, Asturias, Spain, 2Instituto Murciano de Investigación y
Desarrollo Agrario y Alimentario (IMIDA), Murcia, Spain
S42
Poster Abstracts
Coronary bypass surgery has become one of the most executed procedures in
the treatment of occluded coronary arteries. Actually, the ‘gold standard’ is
the autologous grafting, where saphenous vein or internal mammary artery is
used to bypass the diseased vessel(1). However, in patients who have had a
previous bypass surgeries or vascular disease, a second surgical intervention is
not recommended(2) due to risk of infection or swelling. For all these reasons,
there is a real need to develop alternatives to autologous grafting. In this
context, new biomaterials as silk-fibroin or decellularized supports are an
interesting option to generate small vascular grafts. To obtain raw silk fibroin
(SF), silkworm cocoons were boiled in Na2CO3 aqueous solution and rinsed
with water. Then, it was dried at room T and dissolved in Ajisawa’s reagent.
The final step was a dialysis against distilled water for 3 days at 4C in cellulose
tubular membranes. To decellularize the umbilical artery different kinds of
detergents at several concentrations were used. Umbilical cords were used in
order to isolate smooth muscle and endothelial cells. Samples were stored at
4C for a maximum of 24 hours in sterile HBSS containing antibiotics. In
order to isolate smooth muscle cells, arteries were chopped on rectangle
pieces using scalpel blades and were uniformly distributed on cell culture
dishes coated with collagen and cultured in DMEM, 10% FBS. HUVECS
were isolated from the umbilical vein using 2% Collagenase I and cultured in
M199 medium, 20% FBS. Smooth muscle cells were characterized by the
expression of a-actin. Moreover, Endothelial cells were characterized by
CD31 expression, both markers were determined by immunofluorescence.
Once the cells were properly characterized, 12x106 smooth muscle cells were
seeded for 21 days in the scaffolds using a bioreactor. After this time period,
the scaffold was fixed to obtain images by emission scanning electron microscope (FE-SEM).
131
FIVE YEAR FOLLOW-UP OF CORONARY SINUS DELIVERY
OF BONE MARROW CELLS FOR CONGESTIVE HEART
FAILURE
J Tuma2, A Carrasco2, RF Vina2, S Chirinos2, C Cruz2, AN Patel1
1
United States, 2Regenerative Medicine, Maison de Sante, Lima, Peru
Background: Randomized studies suggest that intracoronary infusion of
autologous cells improve heart function for patients with heart failure (HF).
However, results have been mixed due to high cell loss and poor delivery.
Coronary sinus delivery (CSD) of bone marrow cells holds promise for patients
with HF.
Methods: 77 patients were enrolled and completed 5 years follow up. Patients underwent SPECT evaluation; all had LVEF 25%, 40 patients with
ischemic HF (IHF) and 37 patients with non-ischemic HF (nIHF). For IHF
and nIHF at baseline: NYHA III/IV were 31/9 and 29/8 respectively; median
LVEF were 18.7% and 24.7% respectively; median EDV were 281 ml and 242
ml respectively and median ESV were 217 ml and 189 ml respectively. Median
numbers of MNC & CD34+ cells were 12*108 & 23*106 respectively. Cells
were delivered in 90 cc via a coronary sinus approach using a balloon occlusion
for 15 minutes.
Results: There were no adverse events related to the cell delivery. The
results related to EF, NYHA, EDV, and ESV are shown below in the table.
Conclusions: Infusion of autologous bone marrow cells into the coronary
sinus is safe and feasible. It is associated with significant improvement in
symptoms and functional capacity benefit. Further randomized studies are
warranted.
Follow-up results
1 Year
5 Years
IHF n¼40 NIHF n¼37 IHF n¼32 NIHF n¼30
DNYHA Class
DLVEF %
DEDV ml
DESV ml
*p<0.05.
1*
9.0*
40.0*
35.5*
2*
12.0*
29.5*
42.0*
1*
9.0*
16.0
31.0*
2*
19.0*
20.0
41.0*
132
EXPERIENCE ON CELLULAR SELECTION OF CD 133D OF BONE
MARROW ASPIRATES AND THE COLLECTION OF PERIPHERAL
BLOOD HEMATOPOIETIC PROGENITORS FOR CARDIAC
AUTOLOGOUS CELL THERAPY
WM Ceron, B Cordova, E Carrillo, I Perez
Bank of Blood, Rebagliati Martins Hospital, Lima, Lima, Peru
Introduction: The application of CD133+ stem cells in ischemic heart disease
has shown to have beneficial results in this kind of patients. The sources from
which we obtained the CD133+ cells have been commonly bone marrow and
mobilized peripheral blood, existing between the two of them, differences
relating to the amount obtained as a final product of cellular selection. AIM
Compare the efficiency, performance and purity of the cellular selection procedures for cardiac CD133+ cellular therapy.
Materials and Methods: We had 12 patients with bone marrow aspiration
(BMA) to obtain CD133+ cells were collected in volumes between 100ml and
400ml. CD133+ cells from peripheral blood were obtained in 3 patients by
mobilization with G-CSF (5 mg/kg/12hours) during 4 days, dividing the
mononuclear fraction by apheresis. In both cases a immunomagnetic positive
selection was performed by the CliniMACS system , according to the volume
and cell number, 1 or 2 vials of anti-CD133 antibodies for staining. In 4 bone
marrow procedures a red blood cell depletion was performed by centrifugation,
prior to staining with anti- CD133 in order to use a single vial of antibodies
rather than two.
Results: Quantity and purity of procedures with BMA this 4.5 - 67 million
cells and 32% - 94% respectively. In processes with mobilized peripheral blood
apheresis the quantity and purity is between 68-215 million and 70% -90%
respectively. Furthermore, the depletion of red blood cells in 4 procedures
(BMA: betwen 181ml and 267ml). About 40 mL of red blood cells was reduced
and calculate the percentage of loss of leukocytes evidencing 8% average.
Conclusions: The origin of the product for the cellular selection showed
marked differences. The mobilized peripheral blood is a safe and efficient
source for the isolation of CD133+ stem cells. The red blood cell depletion was
shown to be an alternative to optimize the use of monoclonal antibodies
allowing us to process a greater amount of BMA.
133
INTRAMUSCULAR TRANSPLANTATION OF PIG AMNIOTIC
FLUID DERIVED PROGENITOR CELLS HAS THERAPEUTIC
POTENTIAL IN A MOUSE MODEL OF MYOCARDIAL
INFARCTION
S Shaw1,2, S Peng3, S Chih3
1
Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taoyuan,
Taiwan, 2Institute for Women, University College London, London, United
Kingdom, 3Institute of Biotechnology, National Taiwan University, Taipei,
Taiwan
Acute myocardial infarction (MI) is a fatal event causing a large number
of deaths world-wide. MI results in pathological remodeling and decreased
cardiac function which could lead to heart failure and fatal arrhythmia.
Cell therapy is a potential strategy to repair the damage through enhanced
S43
relevance to therapeutic activity.
Materials & Methods: The proteome of MSC exosome was profiled by
LC-MS/MS and the predicted biochemical activities of the proteome were
validated by enzymatic and cellular assays.
Results: Mass spectrometry revealed a large and varied protein cargo with
the potential to regulate a wide array of biochemical and cellular processes.
These include enhancing glycolysis to increase cellular ATP production and
glycolytic intermediates for anabolic activities, inducing adenosine-mediated
activation of survival kinases (e.g. ERK and AKT) via CD73, reducing complement activation through CD59 and reducing denatured protein through 20S
proteasome.
Conclusions: As these processes are fundamental, non-tissue specific processes in ameliorating tissue injury and promoting tissue repair, MSC exosomes
could potentially underpinned the therapeutic efficacy of MSC in diverse disease indications. This would provide a rationale to transform present MSCbased therapies into MSC exosome-based therapies.
135
136
ASIA PACIFIC CELL BASED CLINICAL TRIAL SURVEYe 2013
STATUS REPORT
LK Chelluri
Stem Cell Lab, Global Hospitals, Hyderabad, Andhra Pradesh, India
angiogenesis or by modulation of the inflammatory process via paracrine
signaling. Amniotic fluid-derived progenitor cells (AFPCs) have been reported
to differentiate to several lineages and can be used without ethical concerns or
risk of teratoma formation. Since pigs are anatomically, physiologically and
genetically similar to humans and pregnant pigs can be an abundant source of
AFCs, we used porcine AFPCs (pAFPCs) as our target cells. Intra-myocardial
injection of AFPCs has been shown to cure MI in animal models. However,
intramuscular transplantation of cells has not been extensively investigated.
In this study, we investigated the therapeutic potential of intramuscular injection
of pAFPCs on acute MI. MI mice were divided into 1) PBS control, 2) medium
cell dose (1 x 106 cells per leg; cell-M), and 3) high cell dose (4 x 106 cells per leg;
cell-H) groups. Cells or PBS were directly injected into the hamstring muscle
20 minutes after MI surgery. Four weeks after MI surgery, the cell-M and cell-H
groups exhibited significantly better ejection fraction, significantly greater wall
thickness, smaller infarct scar sizes and LV expansion index compared to the
PBS group. Using in vivo imaging, we showed that the hamstring muscles from
animals in the cell-M and cell-H groups had RFP positive signals. In summary,
intramuscular injection of porcine AFPCs reduced scar size, reduced pathological remodeling, and preserved heart function after MI.
134
BIOCHEMICAL POTENTIAL OF MSC EXOSOME
R Lai1, R Yeo1, S Tan1, B Zhang1, Y Yin1, N Sze2, A Choo3, S Lim1
1
Exosome and Secreted Nano-vesicle, Institute of Medical Biology, Singapore,
Singapore, 2School of BIological Sciences, Nanyang Technological University,
Singapore, Singapore, 3Bioprocessing Technology Institute, Singapore,
Singapore
Introduction: Of the different stem cells in clinical trials, mesenchymal
stem cells (MSCs) are presently the stem cells in the most advanced stage of
clinical testing and for the widest range of indications. The ease of isolation
from adult tissues, large ex vivo expansion capacity and apparent therapeutic
efficacy in a wide range of disease indications have made MSCs the stem
cell of choice for regenerative medicine. Clinical and animal studies have
demonstrated that secreted trophic factors, and not stem cell differentiation, likely mediated much of the therapeutic efficacy of MSCs. This
paradigm shift in the therapeutic mechanism of MSCs has started to
transform MSC therapy from a cell- to biologic-based therapy. Our group
has identified the exosome, a secreted membrane vesicle, as an active
therapeutic factor in MSC secretion. In this study, we profiled the proteome of MSC to elucidate the biochemical potential of MSC and its
Background: Asia Pacific region has no survey program or tangible information on the cell based clinical trials, though a large number of them are
being executed. Aim: The clinical trial database created in this survey aims to
gather information on various aspects of the cell therapy implementation
w.r.t regulatory clearances, disease indications, cell type and source, route of
administration, cell dose, potency testing, number of patients under trial,
bio-distribution and monitoring of adverse events, safety, functional
evaluation.
Materials & Methods: It is a simple 20 point questionnaire with an estimated minimum 10 minute requirement for the completion through a software
generated program for better outreach. The target responses were from cell
therapy industry, members of various cell therapy related associations, through
social media.
Results: Of the targeted 10-15 responses for the first year, 11 responses
have been registered in the survey. Predominantly, the responses are from the
cell therapy industry originating from India, Malaysia and Thailand. Autologous cell therapy was being carried out by the 7 trials as against 4 allogeneic.
Interestingly, there are 6 responses to tissue engineered products for the topical
and IV route of administration. There are more cell therapy applications in
cardiovascular disease indication followed by the epithelial, musculoskeletal
and endocrine systems. Most of the clinical trials are under PhaseI/II applying
fixed dose and few responded to variable dose. Majority of the trials have
explored most optimum route of cell administration for direct homing. Except
for 2, none of the clinical trials performed either potency testing or carried out
bio-distribution study.
Conclusion: There is an increased need to carry out the survey through
professional societies for larger participation.
137
COMMON CITATIONS DURING FACT INSPECTIONS COMPARISON OF THIRD AND FOURTH EDITIONS OF THE
CELLULAR THERAPY STANDARDS
J Schwartz1, C Keever-Taylor3, D Gastineau2, P Warkentin4,5, K Wacker5
1
Pathology & Cell Biology, Columbia University Medical Center, New York,
New York, United States, 2Pathology & Laboratory Medicine, Mayo Clinic,
Rochester, Minnesota, United States, 3Medicine, Medical College of
Wisconsin, Milwaukee, Wisconsin, United States, 4Pediatrics & Pathology,
University of Nebraska Medical Center, Omaha, Nebraska, United
States, 5Foundation for the Accreditation of Cellular Therapy, Omaha,
Nebraska, United States
Background: The FACT-JACIE Cellular Therapy (CT) Standards are
designed to improve the quality of CT. Analysis of commonly cited standards
S44
Poster Abstracts
helps to identify areas that accredited facilities need to improve and that
FACT-JACIE needs to target for educational activities.
Methods: Post inspection citations reviewed and approved by the FACT
CT Accreditation Committee under the 3rd and 4th edition Standards were
compiled for analysis. Drill-down analysis of the 4th edition data was performed and then compared to the 3rd edition.
Results: A total of 114 inspection reports with 2505 citations were
analyzed for the 3rd Edition and compared with 180 reports with 2434 citations for the 4th Edition. Under the 4th edition, the average number of
citations in a single program was: 5 clinical, 2 marrow, 6 apheresis & 7
processing. The Quality management (QM) section was the most commonly
cited under both editions. However, the percentage of QM citations dropped
significantly from the 3rd to 4th edition within all parts of the program.
Audits were the most common QM citation (17%) while qualification &
validation, accounted for only 3&6% respectively. A significant increase in
the number of citations between the 3rd and 4th editions was noted in the
donor section (mostly related to informed consent & donor suitability), facility management and process control.
Conclusions: QM programs of FACT-accredited facilities improved
significantly between the 3rd and 4th editions of the FACT-JACIE Cellular
Therapy Standards, possibly due to correction of deficiencies between inspection cycles and heavy emphasis of QM during FACT educational sessions.
However, increased issues were noted for process control, in particular relating
to donor evaluation and management. Assessment of commonly cited standards
can guide future efforts to improve cellular therapy, such as educational sessions and in the development and organization of future editions of the
Standards and their associated guidance.
138
THE APPLICATION OF LEAN MANUFACTURING PRINCIPLES
TO A NATIONAL NETWORK OF CELLULAR THERAPY
LABORATORIES
D Hollyman1, J Thompson1, M Guttridge4, C Wiggins5, A Lubenko6,
V Day7, E Austin8, C Ash2, S Hurren3, J Smythe9
1
Cellular & Molecular Therapies, NHS Blood & Transplant, Birmingham,
United Kingdom, 2Specialist Services, NHS Blood & Transplant, Filton,
United Kingdom, 3SimplerÒ Consulting, Cambridge, United Kingdom,
4
Cellular & Molecular Therapies, NHS Blood & Transplant, Filton, United
Kingdom, 5Cellular & Molecular Therapies, NHS Blood & Transplant,
Southampton, United Kingdom, 6Cellular & Molecular Therapies, NHS
Blood & Transplant, Leeds, United Kingdom, 7Cellular & Molecular
Therapies, NHS Blood & Transplant, Sheffield, United Kingdom, 8Cellular &
Molecular Therapies, NHS Blood & Transplant, Liverpool, United
Kingdom, 9Cellular & Molecular Therapies, NHS Blood & Transplant,
Oxford, United Kingdom
NHS Blood and Transplant, through its Cellular & Molecular Therapies (CMT)
function, currently undertakes the testing, processing, storage and distribution of
cells for human application via HTA licensed and JACIE accredited facilities, and
supports approximately 50% of the current UK bone marrow transplantation
activity. Regenerative medicine, which among other areas encompasses cellular
therapies and tissue engineered products, is seen as a key future industry for the
UK. Central to NHSBT’s strategic role in regenerative medicine is the expansion
of CMT service activity and licensing by the MHRA, allowing NHSBT to support
the development and manufacture of novel cell therapies. We have an MHRA
license for IMPs and Specials in an Advanced Therapy Unit, established at Liverpool and are now expanding the sites to initially include Oxford, Birmingham and
Bristol followed by other geopraphically placed centres later. To create capacity
within the 7 existing laboratories an Operational Improvement Program (OIP) has
been undertaken based on the application of LEAN manufacturing principles. The
OIP program aims to deliver increased efficiency and reduced costs, with opportunities for improvement identified using Value Stream Analysis (VSA) and delivery of a future state through Rapid Improvement Events (RIEs). We will present
data from CMT LEAN events including workflow and takt time analysis, realised
savings and the identification and removal of waste. The design and set up of
LEAN work cells for different processes including the receipt of cellular therapy
products (449 hours/year capacity released), reporting of results (518 hours/year
capacity released) and cell therapy product issue at several sites with differing facility designs and layouts will be presented. Challenges to delivering an operational
improvement program within frontline clinical departments while maintaining
regulatory compliance and business-as-usual operations will also be discussed.
139
HCT/P
TRANSLATIONAL
PRODUCT
DEVELOPMENT:
REGULATORY AND QUALITY IMPACT
K St. Martin
National Marrow Donor Program, Minneapolis, Minnesota, United States
The precedent set by FDA for pharmaceutical and medical device industry
regulation indicates that it may be reasonable to expect ever-increasing promulgation of requirements for cellular therapy (CT) products. Specifically, regulation
surrounding HCT/P collection, storage, and processing will mature as efficacious
products and their practical clinical application continue to develop. The FDA’s
dissemination of Hematopoietic Progenitor Cells, Cord Blood (HPC(CB))
guidance documents for Investigational New Drug (IND) and Biologic License
Application (BLA) in recent years and the subsequent licensure of multiple Cord
Blood Banks (CBB), has affected industry by requiring rigorous manufacturing
quality controls. The National Marrow Donor Program (NMDP) holds an IND
to support the access and distribution of unlicensed minimally manipulated
allogeneic HPC(CB) that are made available for transplant when medically
necessary. In this capacity, both GTP and GMP regulations were applied to
existing operations in order to meet the intent of the FDA guidance. Retrospective analysis of this experience indicates that as licensed HCT/P biologic
manufacturers increase in number, the supporting infrastructure of laboratories,
suppliers and vendors may also be affected by the quality control requirements of
these regulated products. CBBs associated with the NMDP IND were individually qualified as manufactures in addition to satisfying NMDP Standards and
Participation Criteria. The IDM testing program is another example of transitioning from a ‘research use only’ mindset to a manufacturing environment.
Whenever possible, IDMs associated with the NMDP IND are routed through
CLIA certified laboratories necessitating FDA cleared screening test kits. As the
CT industry becomes familiar with manufacturing regulations, entities that
collect and process product under license or IND are obliged to demonstrate that
their processes meet current regulations and product specifications.
140
141
142
SELECTION OF BIOLOGICAL RAW MATERIALS FOR USE IN
THE MANUFACTURE OF ADVANCED THERAPY MEDICINAL
PRODUCTS
P Ginty1, B Sheridan2, J Barry1, N Mount1
1
Cell Therapy Catapult, London, United Kingdom, 2BSI Group, London,
United Kingdom
The manufacture of Advanced Therapy Medicinal Products (ATMPs) for
clinical application frequently requires the use of raw materials of biological
origin during key process steps. The use of such biological raw materials can
pose a risk to product quality / safety if they themselves are not manufactured
to quality and traceability standards that make them suitable for human use. In
the April 2013 meeting with the European Directorate for the Quality of
Medicines and Healthcare (EDQM) in Strasbourg, the European Medicines
Agency (EMA) revealed that the insufficient assessment of raw materials impact
on final product quality was one of the most common quality objections at the
Market Authorisation stage. The vast majority of biological materials used in
ATMP manufacture are non-compendial and if only research grade materials
are available to developers, demonstrating the appropriate level of quality at the
licencing stage remains challenging. The EDQM and the EMA clearly understand the need for intervention, as they have set up a task force with the aim
of harmonising the quality standards for raw materials via the European
Pharmacopeia. However, during the interim period, there is value in a practical
guidance document that will aid developers in assessing raw materials and the
potential impact they will have on product quality. Therefore, the Cell
Therapy Catapult and the British Standards Institution have brought together
a team of experts in the field of cell and gene therapy manufacture and regulation to compile and publish a publically available specification (PAS) aimed
primarily at early stage developers, to both fill the current void and complement the longer-term objectives of the EDQM and EMA.
143
CELL THERAPY CATAPULT PROPOSE A CELL HISTORY FILE TO
SUPPORT DEVELOPERS OF CELLULAR THERAPY PRODUCTS
J Barry, P Ginty, N Mount
Cell Therapy Catapult, London, United Kingdom
The starting material for many cellular therapies has the potential to be
incorporated into intermediate and final products with post-trial and marketing
commitments years or decades into the future. However, the precise record of
the history of such Tissue/Cells is often unavailable in a complete, accessible
and ordered format. Cell Therapy Catapult would like to propose a Cell History
File (CHF) for recording the processing and testing of the early stages of a
cellular therapy clinical product. In the EU there are currently a number of
regulatory ‘compendium’ documents which capture some of the downstream
processing of human Tissues and Cells; the Investigational Medicinal Product
Dossier and Product Specification Files for clinical trial products or Marketing
Authorisation Application for fully licensed product and EUSTITE have recommended a similar dossier for the upstream processing of the human Cells and
Tissues for human application. These documents provide an overview of the
production processes and testing to which the cells/tissues may have been
exposed. They do not, however, capture Lot/Batch specific information and as
such they do not preserve the record of the history of Tissue/Cells. The CHF
will capture this crucial information. The Cell Therapy Catapult believe the
CHF will be useful to academic groups as well as international sponsors and
manufacturers developing Cellular Therapy products. In a user friendly format,
it will enable the capture and preservation of the necessary traceability and batch
specific processing data crucial for both regulatory compliance for future submissions and due diligence exercises for products subject to future out-licensing.
144
HUMAN ADIPOSE-TISSUE DERIVED STROMAL CELLS IN
CLINICAL TRIALS: SETTING UP OF GMP PRESERVATION
PROTOCOLS
J Peyrafitte, S Casse, A Varin, S Fleury-Cappellesso, L Sensebe, P Bourin
STROMALab, CNRS 5273, Inserm U1031, EFS-Pyrenees-Mediterranee,
Universite de Toulouse, Toulouse, France
Introduction: Human adipose tissue-derived stromal cells (ASC) are used in
various clinical trials. Transport or storage of cell products may be required,
our aim was to define good-manufacturing practice (GMP) preservation protocols. This study focused on the characterization of fresh or cryopreserved
cells during cold storage in liquid solution.
Methods: The stromal-vascular fraction was obtained from lipoaspirates
after collagenase digestion and centrifugation. The adherent fraction was
expanded in vitro during 14 days (P1 ASC, 2 passages) in a GMP culture
medium. ASC were cryopreserved and/or stored at 4 C in liquid solution
during up to 48 hours. The evolution of cell characteristics and functions were
investigated at different time point.
Results: Freshly harvested ASC displayed good viability up to 28 hours in albumin alone. Supplementation with glucose improved viability up to 48 hours.
After 28 hours in storage medium, we noticed a decrease of proliferation and
colony-forming unit fibroblasts potential associated with an increase of apoptosis.
No significant changes in surface immunophenotype or differentiation potentials
toward adipogenic, osteogenic and chondrogenic lineages were observed. Finally,
liquid preservation resulted in a progressive decrease of immunosuppressive
properties. Interestingly, 28-hours liquid-preserved cells recovered same properties as fresh cells after one passage in culture. On the other hand, cell cryopreservation induced a strong inhibition of immunosuppressive capacity of the ASC.
Conclusion: We developed a GMP preservation protocol using human albumin supplemented with glucose allowing storage and shipment of cell therapy
products from production centers to therapeutic units. Nevertheless, regarding the
decrease of immunosuppressive potential during preservation, transport delays
must be reduced as much as possible. Moreover we determine that cryopreservation is not an appropriate technique to store ASC for cell therapy purposes.
145
A SINGLE-TUBE REAL-TIME PCR ASSAY FOR MYCOPLASMA
DETECTION AS A ROUTINE QUALITY CONTROL OF CELL
THERAPEUTICS
K Janetzko, G Rink, A Hecker, K Bieback, H Klueter, P Bugert
Institute of Transfusion Medicine and Immunology, Mannheim, Europe, Germany
S45
GMP-compliant manufacturing of cell-based products requires testing for absence
of Mollicutes species including Mycoplasma, Ureaplasma, Acholeplasma and Spiroplasma according to the European Pharmacopeia (EP). We developed a singletube real-time PCR assay including an internal control (IC) for rapid and sensitive
detection of all Mollicutes species stipulated by the EP. Primers and a TaqMan
probe (FAM labeled) were deduced from 16S rDNA sequence alignment of 18
Mollicutes species. A synthetic IC-DNA molecule with binding sites for the Mollicutes primers and an IC-specific TaqMan probe (VIC labeled) was designed. The
IC-DNA was added to cell culture samples prior DNA extraction. The analytical
sensitivity (genomes/ml) of the assay was determined on dilution series of DNA
from 12 Mollicutes strains followed by Probit analysis. Specificity was proven by the
use of DNA from other bacteria. The required 10 CFU/ml detection limit of the
PCR assay was validated using 9 Mollicutes species spiked into cultured BM-MSC
as a matrix. Analytical sensitivities of the PCR assay were in the range of 405 to 2,431
genomes/ml for 11 of the 12 tested mollicute DNA samples. The lowest sensitivity
was found for U. urealyticum (19,239 genomes/ml). The detection limit of 10 CFU/
ml could be demonstrated for all 9 Mollicutes species: A. laidlawii, M. fermentans,
M. hyorhinis, M. orale, M. pneumoniae, M. gallisepticum, M.synoviae, M. arginini,
S. citri. Mollicutes-specificity of the PCR assay was proven by negative results for
DNA samples from three different ubiquitous bacteria. Simultaneous IC detection
enabled monitoring of the entire process including DNA extraction and PCR
amplification. Our single-tube real-time PCR assay enables sensitive and specific
detection of Mollicutes contaminants. The assay can be performed directly on cell
culture samples by adding the IC-DNA followed by DNA extraction and real-time
PCR. The PCR is suitable for routine quality control of cell therapeutics.
146
VALIDATION OF A NUCLEIC ACID AMPLIFICATION
TECHNIQUE FOR THE DETECTION OF MYCOPLASMA IN
CELL-BASED MEDICINAL PRODUCTS (CBMPS)
N Theys, F Vandermeers
Strategy &Innovation, Quality Assistance, Donstiennes, Belgium
Introduction: The contamination of cell cultures by mycoplasma is a widespread and serious problem in biopharmaceutical production. The European
Pharmacopoeia (Eur.Ph.2.6.7) prescribes test methods for determination of
mycoplasma biosafety. These methods are time-consuming which is not relevant for CBMPs. The use of nucleic acid amplification technique (NAT) as an
alternative was developed and validated.
Material and methods: Amplification of 16S rRNA gene by quantitative
PCR was performed on 10 mycoplasma species (M.arginini, M.hyorhinis,
M.fermentans, M.synoviae, M.orale, M.pneumoniae, M.hominis, M.genitalium, M.salivarium, M.gallisepticum), Spiroplasma citri and Acholeplasma
laidlawii. Each reference strain was spiked in a cellular product at 10 GC/reaction. Specificity against close phylogenetic bacterial species, yeast and human
DNA was tested up to 105 GC/reaction. Moreover, cell cultures were spiked
with CFU-quantified mycoplasma mid-log phase culture preparations in order
to validate this method at 10 CFU/ml and estimate the GC/CFU ratio. Finally,
discriminatory positive control was also spiked in these samples to detect potential PCR inhibition or DNA loss during extraction.
Results and conclusions: All spiked samples were positive for the presence
of mycoplasma DNA at 10 GC/reaction and at 10 CFU/ml. In these analyses, no
PCR inhibition or DNA loss during the extraction was observed. Specificity study
showed that the assay did not detect DNA from human, yeast or gram-positive
bacteria with close phylogenetic relation to mycoplasma, such as Clostridium
acetobutylicum, Lactobacillus acidophilus and Streptococcus pneumoniae.
Additional assays on mycoplasma culture with low GC/CFU ratios are ongoing
for method comparability assessment. In conclusion, data from PCR-based mycoplasma test demonstrate that this assay meets compendia requirements for limit
of detection and specificity and constitutes an alternative of choice for rapid
biosafety testing of CBMPs.
147
VALIDATION AND GMP PRODUCTION OF MESENCHYMAL
STROMAL CELLS BATCHES FOR CLINICAL APPLICATION IN
ORTHOPEDICS AND RHEUMATOLOGY
M Codinach1, M Blanco1, C Torrico2, J Vives1, A Casamayor-Genesca1,
J Lopez1, R Coll1, J Garcia1, A Pla1
1
Divisió de Teràpies Avançades (Xcelia), Banc de Sang i Teixits, Barcelona,
Catalonia, Spain, 2Divisió de Teixits, Banc de Sang i Teixits, Barcelona,
Catalonia, Spain
S46
Poster Abstracts
Mesenchymal Stromal Cells (MSCs) have achieved a great potential in
Regenerative Medicine, although limited information exists about bioprocess
and production on Good Manufacture Practices (GMP)-grade, standardization
and quality controls. The present work describes our experience on the validation and GMP production of 25 consecutive bone marrow (BM)-derived
MSCs batches for clinical use. BM was obtained from posterior iliac crests of
patients. Manufacturing included isolation by density-gradient centrifugation
and plating, trypsinization and secondary expansion cultures and, harvesting
and formulation of a suspension containing 4010E6 viable MSCs. Quality
controls included cell counting and viability assessment; immunophenotyping;
Sterility Test, Mycoplasma detection and Endotoxin Tests conducted according to European Pharmacopeia (Eur. Ph.) and, finally, Gram Staining. All
products were shipped according to current EU Regulations. After validation,
25 MSCs batches for clinical application were manufactured. BM harvested
volumes ranged from 98 to 163 mL (median 132,1mL) containing 8,38E8 to
4,29E9 viable Nucleated Cells (NCs) (median 2,53E9). Cell concentrations
ranged from 5.74E6 to 3.56E7 NCs/mL (median 2.34E7). Final product volume was 10,0 mL (4-10,5 mL) containing a median of 4.02E7 (3.21E7-4.87E7)
viable MSCs. Antigen expression was consistent with MSCs phenotype as
described by ISCT. In a minority of the batches produced, we observed an
increase of HLA-DR expression compatible with the activation on the MSCs.
Sterility, Mycoplasma, Gram Staining and Endotoxin tests of the finished
products were always negative. We have established a GMP protocol, using
human sera as supplement, confirming its safety and feasibility. It is consistent
and reproducible and the bioprocess developed demonstrated to be robust, safe
and reliable. This work was cofounded by MEDCEL and FACTOCEL projects from the Spanish government (Ministerio de Ciencia e Innovación).
148
MULTICOLOUR FLOW CYTOMETRY QUANTIFICATION
OF IMMUNOAFINITY SELECTED CMV ANTIGEN SPECIFIC
T-CELL IN CLINICAL SCALE PREPARATIONS WITH TWO
DIFFERENT DEVICES
S Kloess1, R Esser1, M Marburger1, S Tischer2, C Priesner1, K Aleksandrova1,
B Maecker-Kolhoff3, H Heuft2, L Goudeva2, R Blasczyk2, L Arseniev1,
B Eiz-Vesper2, U Köhl1
1
Institute for Celltherapeutics, Medizinische Hochschule Hannover,
Hannover, Lowee Saxony, Germany, 2Institute for Transfusion Medicine,
Medizinische Hochschule Hannover, Hannover, Lowee Saxony,
Germany, 3Clinic for Paediatric Haematology and Oncology, Medizinische
Hochschule Hannover, Hannover, Lowee Saxony, Germany
Virus-specific T-cells (CTL) can be selected via the CliniMACS CCS using
either the CliniMACSPlus (Plus) or Prodigy (Miltenyi Biotec GmbH, Germany). Because of the low frequency of the target CTL a precise quality
control (QC) of the final product is a challenge, even after re-stimulation for
IFNg secretion. After proving donors’ CMV positivity and obtaining their
written inform consent 3 lymphaphereses were conducted. Re-stimulation with
MACS GMP PepTivator HCMVpp65 and selections of CMV-CTL were
carried out with the Prodigy and Plus simultaneously starting with 0.5-1x109
leucocytes. Single platform flow cytometry (FCM) with a panel of up to 9
colours and a successive gating strategy was applied for QC: CD3, CD4, CD8,
CD45, IFNg (positive) and CD14, CD19, CD56 (negative). 7AAD was used
for viable staining and Fluorospheres for counting. The selections were less
time consuming (<7 h hands-on time) with the Prodigy as compared to the
Plus (>12 h). Both devices provided similar results for the target fractions: 0.29.4 vs. 2.4-10.7x106 CD45+/7AAD- cells in 7-9.6 ml and 40-43 ml, respectively. The overall 7AAD negativity (19-63%) and the cell concentration (0.160.65 x106 cells/ml) were relatively low. The corresponding proportions of
CD3+IFNg+ cells ranged between 38.7-59.8% and 8.7-52.2%. Within 2 selection pairs predominantly CD8+/CD3+/IFNg+ cells were found (CD4:CD8
ratio 1:4.8). The last preparation contained more CD4+/CD3+/IFNg+
lymphocytes (CD4:CD8 ratio 4.8:1). The contaminating IFNg- T-cells were
2.9-11.3% and 5.1-36.7%, respectively. Although more than 1 x104 CTL were
collected with both machines the cell concentration was <1x106 cell/ml. In
combination with the relatively small volume extended analyses are not
possible. Thus a multicolour single platform FCM is the method of choice for
the accurate QC. Beside some initial technical difficulties with the CliniMACS
Prodigy both machines seem to target cell fractions with similar characteristics.
149
DESIGNING A CMC STRATEGY FOR CELL AND GENE
THERAPY
V Pimpaneau
Voisin Consulting Life Sciences, Rennes, France
Defining a CMC strategy is a key element to successful product development,
regardless of product type. In recent years, the implementation of the ATMP
regulation and guidelines together with the new quality paradigms developed in
ICH guidelines (Q8-10), and the refinement of GMP requirements (Annex 2),
all encourage science and risk based approach to become an integrated part of
product development. Identifying the CMC regulatory requirements at any
given development stage provides the opportunity to reflect on the product, its
specific characteristics and the potential technical challenges or limitation that
one may face during the course of development. This set of information will be
critical in defining the needs in terms of product characterization tools, production scales, comparability plans, risk mitigation etc. and will help drive
priorities and efforts throughout development. This presentation will also
discuss how anticipating the CTD content of Module 3 early on can help
identify potential gaps and define a regulatory frame as it relate to CMC requirements which, combined with a risk based approach, become the foundation of the CMC strategy that one can build upon as development evolves.
The presentation will also highlight the importance of the characterization
tools to help feed an efficient development cycle build on process and product
monitoring and on comparability. Learning objective: Understand the key
elements of CMC differences between ATMP and other medicinal products
and how they influence product development. Discuss the possible approaches
to overcome technical and regulatory challenges and how the knowledge of
product specificities can allow the integration of a risk based approach. Learn
how the development of the appropriate characterization tools and early
planning of comparability strategies are essential to successful product development and to the preparation of the Module 3.
150
SPECTRA OPTIAÔ AND ELUTRAÔ FOR THE PRODUCTION
OF MONOCYTE-DERIVED DENDRITIC CELL VACCINES
GS Andreassen1, L Skoge1, M Lundby1, RJ Smith2, G Kvalheim1, D Josefsen1
1
Dep of Cellular Therapy, Oslo University Hospital, Oslo, Norway, 2Biotech
and Cell processing EMEA, Terumo BCT Inc, Zaventem, Belgium
We investigated the Spectra Optia as a platform for collecting mononuclear
cells (MNC) from unmobilised patients and the Elutra performance to
further process these products to a monocyte enriched fraction for the
generation of dendritic cells. A total of 14 patients were enrolled: Ovarian
cancer (2), Prostate cancer (6), Glioblastoma (2), Lymphoma (2) and malignant melanoma (2). A single monocytapheresis procedure was completed
for each patient, using standard settings. Samples were collected prior to
apheresis, and from the harvested product. Cells were stored overnight
before elutriation for further enrichment of monocytes and production of
dendritic cell (DC) vaccines. Data are presented as Median (Range). During
apheresis 2.10 (1.03 e 2.80) total blood volumes were processed in 209 (110
e 330) minutes. A monocyte yield of 2.53 x 109 (1.35 e 4.56) total monocytes was collected in a final volume of 60mL (40 e 100). Monocyte
collection efficiency (CE1%) was also high at 62.9% (29.7% - 77.7%). We
also observed a moderate correlation (r2 ¼ 0.61) between pre-apheresis
monocyte count and monocyte yield per total blood volume processed. Nontarget cell residuals were extremely low, thus making ideal products for
further monocyte enrichment by elutriation. A median of 2.10 x 109 (0.89 e
3.18) total monocytes was subsequently elutriated. After processing, monocyte purity increased from 30% (16% - 67%) before elutriation to 88.6% (
71.0% - 97.0%) with an overall monocyte recovery of 64% (42% - 100%).
We conclude that Spectra Optia is able to isolate and enrich a highly purified
MNC fraction from patients in steady-state haematopoiesis. Moreover,
MNC products collected on Optia are ideally suited for subsequent elutriation to obtain highly enriched monocytes. Importantly, dendritic cell vaccines made from the enriched monocytes show no change in phenotype and
clinical efficacy when compared to DC vaccines made from COBE Spectra
and elutriation on Elutra.
151
A WEB BASED FORUM FOR EDUCATION & COMMUNICATION
IN A CHALLENGING WORKPLACE ENVIRONMENT
K Stasko, MPH, H Hansen
Cell Manipulation Core Facility, Dana Farber Cancer Institute, Boston,
Massachusetts, United States
Background: The Dana Farber Cancer Institute’s Cell Manipulation Core
Facility (CMCF) is approved to provide clinical stem cell transplant services to
all Harvard affiliated hospitals. Providing in-service education to clinical team
members is particularly complex given the everyday challenges of communicating technical details, allocating time for busy staff, and providing access to
restricted GMP lab space. In addition to educating the clinical team, it is
paramount to disseminate information as well as train internal department
members within the CMCF.
Methods: In order to meet these challenges, we have developed a website
which provides in-service training specifically centered on video and pictorial
methods of communication. The website features a facility tour, as well as an
overview of product types, all major processing steps, and standards for release.
Additionally, links to the site are present on CMCF cell processing standard
operating procedures.
Results: Prior to the implementation of the in-service training website, 8
staff members in the stem cell transplant laboratory had current working
knowledge of cell processing procedures. Post implementation, all 40 internal
departmental staff members, as well as the entire bone marrow transplant
program, have access to the most up to date cell processing techniques and
standards.
Conclusion: The in-service training website proved efficacious as an efficient method to expand the knowledge base of new technologists, non-technical staff members within the CMCF, and external bone marrow transplant
team members. By utilizing this simplified strategy, we have bridged the gap
that exists in our current training and education process.
152
ENSURING SUPPLY CHAIN OF CELL THERAPY PRODUCTS
DURING COMMERCIALIZATION: EASY RECONSTITUTION
AT THE BEDSIDE
S Snykers, P Willemsen, C Gumy, Y Greiling, B De Vos, C Dedry, E Sokal,
E Halioua
R&D, Promethera Biosciences, Mont-Saint-Guibert, Belgium
Promethera HepaStem is the company’s therapy product to treat serious
metabolic liver disorders. An European phase I/II clinical trial is ongoing to
treat Crigler-Najjar and Urea Cycle Disorders in a pediatric setting. As of
today, 20 patients have been treated with HepaStem. Currently, Promethera
is preparing its next clinical phases in US and Europe. The challenge is to
provide a drug product easy-to-reconstitute at clinical sites. In order to
guarantee a flexible, highly qualitative, and economically sustainable supply
chain during commercialization, Promethera has developed a ready-to-use
off-the-shelf product for direct reconstitution at the hospitals. The product
preparation is simply carried out like conventional reconstitution of freezedried sterile medicinal products (e.g. vaccine). The challenge was to find an
appropriate final container (i) compliant to liquid nitrogen storage, (ii)
allowing automated in-line filling and (iii) reconstitution without changing the
product quality (safety/identity/purity/potency). The Aseptic Technologies
closed vials perfectly fit within this concept. This approach does not only
simplify the preparation procedure and operation time, it also reduced the
storage footprint and improved the product’s quality in terms of viability,
yield, content uniformity and batch-to-batch consistency. Shelf-life, the keybottleneck of most cell therapeutic products, is substantially less critical. The
idea for the upcoming clinical phases is to provide an all-in-one kit, including
the cryopreserved cell therapeutic product HepaStem and all material needed
for reconstitution (syringe/adaptor/disinfectant/prefilled vial with reconstitution media). In conclusion, Promethera has developed a ready-to-use
product, allowing worldwide availability and easy reconstitution at the
bedside. This technology guarantees a sustainable supply chain during
commercialization and offers opportunities in the field of cell-based products
with limited shelf-life.
153
RED CELL DEPLETION OF BONE MARROW USING THE
THERMOGENESIS AUTOEXPRESS SYSTEM
S47
A Strano2, R Destro2, D Bovo2, M Gazzola2,1
1
Azienda Ospedaliera di Padova, Padova, Italy, 2Paediatric HaematologyOncology, Az.Ospedaliera-Università di Padova, Padova, Italy
The Thermogenesis AutoXpress (AXP) system is an automated closed device
commonly used to harvest the buffy coat from cord blood. We validated its use
with the bone marrow since it was available in our laboratory, which includes
also the Padova Cord Blood Bank. 16 bone marrow samples were obtained
from a second buffy coat collection during the processing of ABO incompatible
products with the COBE 2991. Each sample contained 3.8+/-2 x10E9 nucleated cells (range 0.8-8.9 x10E9) and 47+/-13 ml of red cells. The BM samples
were concentrated to 127+/-21 ml, HES was added at 20% to facilitate the
concentration and separation of the fractions: plasma, buffy coat and red cells.
After the 2 steps centrifugation, at 1400g for 20 minutes and at 80 g for 10
minutes, the buffy coat was concentrated into a 20 ml volume in the freezing
bag of the disposable set. The recovery of total nucleated cells and of CD34+
cells was 90+/-8.7% and 103,+/-9.7% respectively. Cell viability (7AAD) pre
and post AXP centrifugation was not significantly different (91.3+/- 4.1% vs
91.8+/- 4.2%). A consistent volume of red cell was measured in the final
product: 5+/-0.5 ml. These results are comparable with those obtained with
cord blood by our laboratory. In our hands the AXP system designed for cord
blood concentration showed an excellent performance also with bone marrow.
AXP system alone or in combination with COBE2991 can represent a reliable
system for the depletion of red cells especially in paediatric ABO incompatible
transplants.
154
BONE MARROW PROCESSING OF ABO INCOMPATIBLE
PRODUCTS BY THE COMBINED USE OF THE COBE 2991 AND
THE THERMOGENESIS AUTOXPRESS SYSTEM
A Strano2, R Destro2, D Bovo2, M Gazzola2,1
1
Azienda Ospedaliera di Padova, Padova, Italy, 2Paediatric HaematologyOncology, Azienda Ospedaliera-Università di Padova, Padova, Italy
We developed a new processing approach based on the combined use of
COBE 2991 and the Thermogenesis AXP system for depletion of red blood
cells in ABO incompatible bone marrow transplantation. Here we present the
results of 16 procedures. Bone marrow buffy coat were collected in two
consecutive steps using the COBE 2991 cell processor, and transferred in two
bags labelled buffy coat 1 and buffy coat 2. The mean volume of buffy coat 1
was 90+/-31 ml and it contained most of the total nucleated cells (TNC) and a
relatively low volume of red cells (14+/-9 ml). The mean volume of buffy coat
2 was 127+/-21 ml and it contained less TNC (3.8+/-2 10E9) and a higher
volume of red cells (47+/- 13 ml). Buffy coat 2 underwent a further concentration using the AXP system after the addition of HES at 20%. The
combined use of COBE 2991 and AXP system resulted in 91% reduction of
red blood cells (range 83 to 98%). The mean recovery of TNC was 88 +/13%. The initial mean volume of the bone marrow aspirates was 695+/- 322
ml and the final mean volume was 109+/- 38 ml. The TNC and CD34+ cells
infused were 3.6x10E8+/-7/kg and 3.4+/-2x10E6/kg respectively. The mean
volume of red cell infused was 0.48.+/- 0,17 ml/ kg. Engraftment occurred in
all the patients and no adverse reactions have been observed. This is a
convenient and reliable method for processing ABO incompatible products
with a reduced risk of contamination since processing is performed in closed
systems.
155
GMP LABORATORY FOR ACADEMIC CELLULAR THERAPY;
TISSUE ENGINEERING AND GENE THERAPY DEVELOPMENTS
AND
CLINICAL
TRIALS
SUPPLY
AT
MAIMONIDES
UNIVERSITY-ARGENTINA
GA Moviglia
CIITT, Universidad Maimonides, Buenos Aires, Argentina
To accomplish the legal national and international requirements for the
elaboration of elements for advanced therapies, the Maimonides University
of Buenos Aires, Argentina built a GMP laboratory of 400 square meters
divided in three different areas distinguished one from other through their
level of Biosafety: Level 2 (BIO 2) dedicated to quality control and quality
assurance test as well as animal studies. Level 3 (BIO3) dedicated to the
process of non contaminated human cells to be used in cell therapy and / or
tissue engineering, cell freezing and banking and Tissue Culture Media
S48
Poster Abstracts
elaboration. Level 4 (BIO4) is dedicated to the process of cells for gene
engineering or contaminated cells for cellular therapy. The door access to
each area and laboratory is controlled by personal magnetic card that allows
the use of each room according to the level of authorization of each user.
Bio 2 counts with a reception desk were each sample is registered, assigned
to a specific process and labeled with bar code etiquette. There is a laboratory with molecular biology equipment and patch clamp testing, a second
laboratory with FACS equipment and fluorescent microscopy, two laboratories with cell incubators, micromanipulator to nucleolus transfer, animal
cell processes and culture. Finally is a washing area with two double doors
autoclaves. The access to bio 3 is through a pressured concourse where there
is necessary to change clots. There are six equal culture rooms with all the
necessary equipment to do not need to go to a second room to complete a
cell process, avoiding cross contamination. A seventh laboratory to frozen
and storage cells as well as to prepare tissue culture media. The equipment
of BIO 4 is under construction for Baker Company. Liquid wastes are
decontaminated in a treatment plant and the water supplied came from an
own invert osmosis plant.
156
157
CYTOCOMPATIBILITY EVALUATION OF CELL DELIVERY
DEVICES USING CLINICALLY RELEVANT CELLS UNDER
EXAGGERATED USE CONDITIONS
S Charlebois1, MC Hiles2, N Ramachandran2, W McRoy1, M Poderycki1,
J Niehaus3
1
Product Discovery, MED Institute, West Lafayette, Indiana, United
States, 2Research, Cook Biotech, West Lafayette, Indiana, United
States, 3Laboratory Services, Cook General BioTechnology, Indianapolis,
Indiana, United States
Blood and blood products are used in front-line treatments for patients with
anemia, acute blood loss, inherited metabolic disorders, and post-operatively
following myeloablative therapies. Specific treatments using umbilical cord
blood and bone marrow-derived concentrates, both of which are comprised of
a mixture of hematopoietic stem cells, neutrophils, platelets, and red blood
cells, are being evaluated in early clinical studies. Therefore, we determined
that cord blood or bone marrow concentrate would be relevant cellular
models to evaluate medical device cytocompatibility. This study evaluated the
cytocompatibility of various cell delivery devices by characterizing cell recovery, cell viability, and cellular proliferative activity of human umbilical cord
blood-derived cells and bone marrow-derived cells following direct contact
with devices. Direct contact of the cells with the device was carried out for a
period of time far exceeding that expected for clinical use. This study also
assessed the limits (i.e., time restrictions) in the use of the blood or blood
product with cell delivery devices. Human umbilical cord blood or bone
marrow cells were characterized with and without exaggerated exposure with
devices by evaluating CD34+ cell concentrations, cell viability (by flow
cytometry), and proliferative capability based on the ability to form colonies
(colony forming units).
158
DESIGN VERIFICATION AND ENHANCED RISK MITIGATION
TESTS FOR CYTOCOMPATIBILITY EVALUATION OF CELL
DELIVERY DEVICES
S Charlebois1, N Ramachandran2, MC Hiles2, W McRoy1, M Poderycki1,
H Steidinger1, J Kuske2
1
Product Discovery, MED Institute, West Lafayette, Indiana, United
States, 2Research, Cook Biotech, West Lafayette, Indiana, United States
The purpose of this study was to evaluate the cytocompatibility of various cell
delivery devices using a battery of tests under exaggerated conditions. These
tests were carried out using a human embryonic cell line transfected with
Toll-like receptors (TLR) and a human monocytic cell line. The TLRtransfected cell line was used to evaluate the potential for cell surface receptor
modulation and consequent changes in downstream signaling pathways that
could occur when passing cells through a device. TLRs are well characterized
and respond to a range of relevant ligands; for these reasons, TLRs were used
as representative receptors to indicate modulation of cell surface receptors.
The human monocytic cell line was used to characterize the viability and
metabolic activity of cells following incubation with media exposed to the test
article for a set time. This assay evaluated the potential toxicity of substances
that might be released from the medical device on the cellular product being
delivered. Human monocytes were selected for use because they are wellcharacterized in the literature and familiar to academic and industrial labs.
For each assay, all devices were incubated with complete cell culture media
for a period of time far exceeding that expected for clinical use. Assays were
performed after a set exposure period to the delivery device exposed media.
Taken together, data generated from these tests may be representative of
alterations that could arise in therapeutic cells when processed through
medical devices and provide a basis for cytocompatibility evaluation of delivery devices.
159
A ROADMAP FOR IMPLEMENTING HCT/P DATA MANAGEMENT
SYSTEMS
M Lujan, L Sylvestri
stemTrak LLC, Cambridge, Massachusetts, United States
As the field of Cellular Therapy, which encompasses hematopoietic stem cell
transplantation and regenerative medicine, advances, the need for equally
advanced and sophisticated data management systems moves in lockstep.
Good clinical practice (GCP), third-party insurers and evolving regulatory
requirements for data management demand the need for secure and reproducible data management systems to handle the ever-increasing data loads.
Having a logical “systems” approach to the implementation of a comprehensive data management system would be an enormous advantage to the
different stakeholders in the field, especially for cell therapy and stem cell
transplant programs that have limited resources and personnel to plan, design
and execute such labor-intensive systems. Establishing a programmatic data
management system is not intuitive, it involves many different areas and
different expertise, and it requires considerable planning and input from the
final stakeholders to be successful. Based on nearly 20 years of experience in
the design, development, implementation, and validations of data management systems in both non-regulated and regulated laboratories, stemTrak has
established a functional “roadmap” for implementing data management systems that we believe will fulfill these broad and diverse needs. Drawing on
expertise and knowledge experts in database management systems we’ve
developed a helpful template that is flexible, expandable, captures the significant drivers of the system and will permit a user friendly approach to
establishing a program’s need for database management. This roadmap will
be presented and discussed.
160
DESIGN AND CONSTRUCT A GOOD MANUFACTURING
PRACTICE FACILITY FOR CELLULAR THERAPY PRODUCTS
K Thiagarajah1, G Ooi1, K Then1, C Wong2, K Then1, S Cheong1,3
1
CryoCord Sdn Bhd, Cyberjaya, Malaysia, 2Cytopeutics, Cyberjaya, Selangor,
Malaysia, 3University Tunku Abdul Rahman, Kajang, Selangor, Malaysia
Cellular therapy industry is a fast growing industry worldwide and more
companies are planning to build their own facility to manufacture cellular
therapy products (CTPs). To ensure the quality of CTPs, these facilities must
conform to relevant regulatory requirements. In Malaysia, a Good
Manufacturing Practice (GMP) facility is a pre-requisite for clinical trials
involving the use of CTPs. Therefore, CryoCord Sdn. Bhd. had decided to
forge ahead with the construction of such facility. In this report, we would
like to share some of the considerations in designing and constructing a GMP
laboratory for CTPs. In line with current and proposed future expansion of
the laboratory and services, a 10000 square feet facility was designed and
constructed according to PIC/S and ISO 14644 guidelines. This facility was
equipped with eight Grade B cleanrooms which will be used for processing
CTPs. Working flow system, operational cost of the laboratory, backup power system, heating validation air condensing system, cold water system and
fire fighting system were taken into account during the designing and constructing phases. Prior to construction, a comprehensive Site Master File and
User Requirement Specification (URS) were established to ensure that the
facility complies with PIC/S and ISO 14644 guidelines. At the same time, the
Validation Master Plan (VMP), which consists of a complete set of validation
protocol, forms and test reports which will be required for audit, must be
completed. Lastly, a proper communication between the contractor and the
project manager is crucial to ensure that the facility was built according to the
URS. In conclusion, a thorough thinking process, including an understanding
of related regulatory requirements, working flow system and working environment, is vital to designing and constructing a GMP facility for CTPs.
161
RELOCATION OF CRYOPRESERVED UMBILICAL CORD BLOOD
SAMPLES IN HIGH CAPACITY LIQUID NITROGEN FREEZERS
TO A NEW LABORATORY: CRYOCORD, A CORD BLOOD
BANKING EXPERIENCE
G Ooi1, K Thiagarajah1, C Wong2, VV Vijayan1, K Then1, K Yong1,
S Cheong1,3
1
Cryocord Sdn Bhd, Cyberjaya, Selangor, Malaysia, 2Cytopeutics, Cyberjaya,
Selangor, Malaysia, 3University Tunku Abdul Rahman, Kajang, Selangor,
Malaysia
Umbilical cord bloods (UCB) have been used to treat a variety of hematologic
disorders and other diseases. To maintain the quality and viability of UCB,
processed UCB have to be stored in liquid nitrogen freezers (LNF) at
cryogenic temperature until the samples are retrieved, thawed and transplanted. However, when a large number of cryopreserved UCB have to be
relocated to a new laboratory at cryogenic temperature, a challenge is presented. In this report, we would like to share our experience on relocating
more than ten thousand cryopreserved UCB in twelve LNF into our new
laboratory. Procedures including relocating cryopreserved UCB, transporting
dry shipper and validating the quality of UCB after relocation were presented
in this report. For quality control purpose, two weeks before LNF relocation,
donated UCB were processed, cryopreserved and stored at the each LNF. On
the day of relocation, half of the donated UCB were retrieved and analyzed
for TNC counts, CD34 count and cells viability. These results served as
control for later comparison. During UCB relocation, UCB were transferred
into high capacity dry shipper before transport to new laboratory. Upon
arrival at the new laboratory, LNF were serviced before transferring UCB
samples back into original LNF from dry shipper. For quality control, the
remaining donated UCB were retrieved and analyzed for the same tests
mentioned above. We found that there were no significant differences in all
three tests when comparing the UCB samples before and after relocation. In
conclusion, all UCB were successfully relocated into new laboratory while
maintaining the quality of UCB in terms of TNC counts, CD34 count and
cell viability.
162
VALIDATION OF CONDITIONS REQUIRED TO CONTROL THE
RATE OF FREEZING OF CELLULAR THERAPY PRODUCTS
CRYOPRESERVED BY OVERNIGHT STORAGE AT -80 C
C Keever-Taylor1, SM Heidtke1, S Konings1, DA Margolis2
1
Hematology & Oncology, Medical College of Wisconsin, Milwaukee,
Wisconsin, United States, 2Pediatrics, Medical College of Wisconsin,
Milwaukee, Wisconsin, United States
HPC products collected for later use require cryopreservation to maintain
viability. Optimal cell preservation requires both cryoprotectant and a slow
cooling rate (<2 degrees/min) from 4C to -45C. Thereafter, products may
be cooled more rapidly (5-10 degrees/min) to -80 C or less prior to LN2
storage. We use a programmable LN2 freezer to control the cooling rate
and document the desired conditions were met. However, in event of
equipment malfunction or for products not intended to restore hematopoiesis we typically store products overnight in a -80 C freezer using a
sandwich of Styrofoam blocks to insulate products to achieve a slow cooling
rate. Here we report a validation study to optimize this process for the
various bag sizes (50, 250 & 500 mL), sources (Miltenyi & Charter Medical)
and volumes (20-125 ml) at which we freeze products. We varied the dimensions and thicknesses of Styrofoam blocks and tested polyurethane foam
padding for the 50 mL bag. A probe inserted into the bag and connected to
S49
a data logger was used to record temperature every minute. Results
confirmed that products cooled too fast without insulation, averaging 5 C/
min to the heat of fusion and >2 C/min thereafter. 0.5” Styrofoam blocks
slightly smaller than the cassette were also ineffective. The cooling rate of
250 mL bags containing 60-70 mL and 500 mL bags containing 90-100 mL
was consistently <1 C/min when sandwiched in 1” thick blocks that
completely covered the cassette. 50 mL bags cooled too rapidly using Styrofoam but could be frozen at the desired rate using 4” thick polyurethane
foam cut so as to encase the smaller cassette. We also confirmed that a slow
cooling rate was maintained when up to 4 bags, two each sandwiched in two
1” blocks and stacked, were frozen in the same freezer chamber. We
conclude that slow cooling of larger bags can be achieved using a -80 C
mechanical freezer so long as insulating material is sufficiently thick and
covers the cassette, while smaller bags may require more insulation.
163
DEPLETION OF ERYTHROCYTES FROM BONE MARROW
GRAFTS
BY
BUFFY
COAT
FORMATION
USING
A
COMMERCIALLY AVAILABLE, EFFECTIVELY CLOSED SYSTEM
DEVICE
AJ Ribickas1, R Smilee1, WE Janssen1,2
1
Cell Therapies Facility, Moffitt Cancer Center, Tampa, Florida, United
States, 2Blood and Marrow Transplant, Moffitt Cancer Center, Tampa,
Florida, United States
Recent clinical data, including a large scale multi-center study, suggest that
there will be a continued role for harvested bone marrow as a source of hematopoietic progenitor cells (HPC) for allogeneic transplantation. As there is
a significant erythrocyte content in harvested bone marrow, major ABO
mismatched transplants generally require some form of erythrocyte depletion
from the graft to prevent potential morbidity or mortality from hemolytic
reactions at infusion. We have previously reported adaptation of the Haemonetics CellSaverÔ for laboratory use for Ficoll separation of MNC. We
have now added simple buffy coat formation for the purpose of erythrocyte
depletion to our repertoire of functions for this instrument which is primarily
marketed for intra-operative blood salvage. Unprocessed marrow is separated
in a bell-shaped bowl that is spinning at 4800rpm. Marrow is pumped into the
bowl at 100 mL/min until the bowl is approximately 3/4 full at which point
pump speed is reduced to 20 mL/min. A buffy coat layer is observed moving
to the top of the packed RBC content of the bowl. As the buffy coat layer
reaches the top of the bowl, the waste bag clamps are closed, and the
collection bag clamps are opened to effect collection. Once the buffy coat
layer has been collected, the pump and bowl centrifuge are stopped. The
clamps are then closed on the collection bag, and reopened on the waste bag.
The bowl is then emptied of the buffy coat depleted marrow. The red blood
cells from the depleted marrow are saved in a satellite bag to be used in
further processing of the marrow when additional volume is needed to fill the
bowl during a sequence. The sequence above is repeated until the entire
volume of marrow has been processed. Depending on the initial volume of
marrow to be processed, 1-4 sequences are performed. We have performed
this procedure with two bowl sizes, namely 125 mL capacity (n¼7) and 225
mL capacity (n¼5). The extent of RBC depletion, and recovery of both TNC
and CD34+ cells is reported in the table.
Bowl Size
125 mL
225 mL
Mean RBC
depletion %
Mean TNC
recovery %
Mean CD34
recovery %
97.7
79.1
55.3
52.7
65.1
63.3
164
A PRELIMINARY SURVEY FOR RESEARCH STUDIES OF THE
NATIONAL POLICIES ON HUMAN GENETIC MODIFICATION
IN MEXICO
S50
Poster Abstracts
IO Osadolor1,2, GR Tecpanecatl1
1
Bioethics, Instituto de Ciencias Juridicas de Puebla, A.C., Puebla, Puebla,
Mexico, 2Law and Bioethics, Instituto de Estudios Universitarios., Puebla,
Puebla, Mexico
1
Bioethics, Instituto de Ciencias Juridicas A.C., Puebla, Puebla, Mexico, 2Law
and Bioethics, Instituto de Estudios Universitarios, Puebla, Puebla,
Mexico, 3Facultad de ciencias de la Salud. Licenciatura en Medico Cirujano.,
Universidad Autonoma de Tlaxcala., Tlaxcala, Tlaxcala, Mexico
Genetic modification, often referred to as gene therapy, is a procedure
whereby the genetic content (DNA sequence) of a cell, many cells or a whole
organism is modified. Most often, non-functional or misfunctioning genes
are replaced, manipulated or supplemented with healthy genes. In humans,
there are two categories of genetic modification: somatic and germline.
Somatic gene therapy consists of introducing a gene or gene segment into
specific tissues or organs (excluding germline cells or reproductive cells) in a
human subject with the aim of treating or curing an existing condition.
Unlike germline genetic modification, somatic gene therapy does not alter
the genetic make-up of future generations because the altered gene does not
exist in reproductive eggs or sperm. Germline gene therapy, on the other
hand, is a more controversial technique because the introduction of a gene
into germline cells will result in heritable changes that affect future
offspring. Germline gene therapy is not currently scientifically possible in
humans (Isasi et al, 2007). In Mexico, humanity is facing a policy deficit at
national level concerning social oversight and control of the new technologies of human genetic modification. In the fifteen years since the birth of
Dolly the sheep alerted the world to the prospect of human cloning and a
new, high-tech eugenics, Mexico has not adopted the policies needed to
bring human genetic technology under responsible societal governance. This
is a briefed analysis of national policies on genetic modification techniques
focused on the legal and ethical standards that have been adopted and the
regulatory systems that exist to control and govern genetic modification in
Mexico. According to The General Health Law of May 7, 1997: Regulation
to the General Health Law on Scientific Health Research (1985), twelve
articles were established.
Embryonic stem cell research and therapeutic cloning are permitted, but
reproductive cloning is banned (the laws were just amended in 2004). Mexico
has a thriving stem cell industry but has not yet implemented formal regulations on stem cell research. Indeed, some Mexican doctors are already using
stem cells to treat chronically ill foreigners, including Americans, who suffer
from conditions such as cerebral palsy, autism and paralysis. These unregulated therapies have been criticized by some in the international medical
community. During the 2003 legislative year, the Chamber of Deputies in
Mexico’s Parliament debated legislation about human cloning. In January
2003, the chair of the Chamber’s Health Commission, announced that there
would be a propose legislation to ban human cloning in Mexico. One month
later, deputies from one of the major parties announced their intention to
exclude research cloning from any ban on human cloning. The issue was
debated by the Deputies in April 2003, and at year’s end, on 3 December, a
bill banning both reproductive and research cloning was approved by the
Chamber of Deputies. The President of the Mexican Academy of Sciences
and researchers at the National Autonomous University of Mexico (UNAM)
responded immediately by vigorously criticizing the Chamber’s action
(Macedo and Barba, 2003).
165
A REGULATORY POSITION OF THE ETHICS OF GENETIC
ENGINEERING IN MEXICO
IO Osadolor1,2, GR Tecpanecatl1
1
and Bioethics, Instituto de Estudios Universitarios, Puebla, Puebla, Mexico
Bioengineering has the potential to transform our lives in many positive
ways. Rejection of this new technology on the ground that it is unnatural or
inherently immoral is unwarranted and seems to be based on little more
than an instinctive adverse reaction. Biotechnology is an extension of already
accepted and well-established techniques, such as directed breeding, but
with the distinct advantage of producing more predictable and more rapid
results. There are risks involved with this new technology, but provided that
it is appropriately regulated, its potential benefits outweigh its harms.
Legislators and other responsible decision-makers should not implement
regulations that unduly restrict implementation of genetic engineering. In
particular, existing mechanisms that ensure the safety of testing protocols
should be sufficient for somatic genetic therapies for humans. With respect
to germline enhancements for plants and animals, we recommend a better
coordinated effort of the Mexican regulatory agency to ensure there are no
gaps in the regulatory framework. Enhanced organisms should be rigorously
evaluated and tested in isolated conditions prior to their release in the wild.
Germline alterations for humans should not be prohibited outright, certainly
not in advance of their availability. However, given the special risks posed by
human germline alterations, each proposed alteration needs to be carefully
evaluated, not just with respect to immediate benefits and harms, but also
with respect to the effects that the proposed alteration may have on our
social structure and the distribution of social goods. Some have compared
genetic engineering to a Pandora’s box. If mythological analogies are
appropriate, the Center for Inquiry believes a better one would be a comparison to the gift of fire from Prometheus: genetic engineering can provide
immense benefits provided it is used prudently and carefully regulated and
controlled.
166
THE MEXICAN NATIONAL LEGISLATION CONCERNING
HUMAN REPRODUCTIVE AND THERAPEUTIC CLONING
IO Osadolor1,2,3, GR Tecpanecatl1, MS Rodriguez3
167
BIOETHICAL ANALYSIS OF THE NEUROREGENERATIVE
THERAPIES BASED ON THE USE OF STEM CELLS IN MEXICO
1
Mexico, 3Facultad de Ciencias de la Salud. Licenciatura en Medico Cirujano,
Universidad Autonoma de Tlaxcala, Tlaxcala, Tlaxcala, Mexico
One of the main goals of regenerative medicine is to make use of stem cells in
the treatment of this type of anomalies. However, the multiple possibilities of
obtaining stem cells raise the need for a prior analysis of the suitability use of
bioethics. The first step of bioethical analysis of neuroregeneratives therapies
requires a proper biological understanding of stem cells. In addition to the
classic types of stem cells, cell induction in recent studies have shown that the
microclover and potentiality, characteristics of this type of cells, may be
induced in any cell through the action of certain molecular signals. The second
step of bioethical analysis of neuroregeneratives therapies is the study of the
anthropological consequences derived from the donation and transplant of
cells. The review of different anthropological models allows to conclude that
data provided by the developmental biology are those of greatest relevance at
the time of making an assessment of this type. The third and final step of
bioethical analysis of neuroregenerativas therapies corresponds to the ethical
assessment. Analysis of the two main existing models, the cognitivist and noncognitivist, it is concluded that such valuation from a cognitive model allows a
more objective and therefore greater accurate analysis. In the legal field, the
analysis of the international and national regulations specifically devoted to this
topic highlights the danger posed by the use of a model based on consensus and
regulatory minimalism, non-solid anthropological status reference. As a whole,
bioethical analysis of neuroregeneratives therapies based on the use of stem
cells is concluded that adult stem cells, those derived from the umbilical cord
and certain types of induced cells (RiPS) offer more real and safe therapeutic
application possibilities.
168
THE EUROPEAN ATMP ‘HOSPITAL EXEMPTION’: A MODEL
FOR NORTH AMERICA?
B von Tigerstrom
The need to create a regulatory framework for regenerative medicine that
rigorously protects patient safety, but is flexible enough to allow innovation in
response to unmet needs, is the subject of ongoing discussion in many countries. In Europe, the 2007 Advanced Therapy Medicinal Products (ATMP)
regulation includes the “hospital exemption,” applying to an ATMP “which is
prepared on a non-routine basis according to specific quality standards, and
used within the same Member State in a hospital under the exclusive professional responsibility of an individual medical practitioner, in order to comply
with an individual medical prescription for a custom-made product for an individual patient.” An ATMP falling within this exemption does not require
authorization at the European level but is regulated by individual member
states. Designed as a specific exemption allowing for small-scale medical
innovation, this provision could provide a model for national laws in other
countries. This paper examines the potential for the hospital exemption to be
adapted and used outside Europe, focusing on the United States and Canada. It
considers the potential for a national law to include an exemption along similar
lines, allowing for state or provincial regulation within the scope of the
exemption. This includes consideration of the distinct jurisdictional issues in
those countries (i.e., the powers of the federal and state or provincial governments) and the structures that exist or could be developed to provide appropriate regulatory oversight at the state, provincial, or local level. It will also take
into account the questions and criticisms that have been raised regarding the
hospital exemption and its implementation in Europe, to see how some of those
concerns might be addressed in adapting the model. Conclusions will be drawn
about whether and how this model could be used appropriately in other parts of
the world.
169
THE LEGAL STATUS OF ENGINEERED TISSUE AND ITS
IMPLICATIONS
B von Tigerstrom
As a complex and rapidly developing technology, tissue engineering raises a
range of legal, ethical, and social issues that have not yet been fully examined.
Tissue-engineered products do not fit neatly into existing categories, either for
regulatory purposes or in the legal, policy, and ethical frameworks that govern
uses of human tissue. This paper explores the legal status of engineered tissue
and the practical and ethical implications that flow from it. Tissue engineering
sits at the intersection of two legally significant sets of categories. First, human
tissues and organs that are intended to be used for transplantation are generally
subject to legislation that sets out distinct consent requirements and typically
places strict limits on donor compensation and the sale of tissues and organs.
The details of these laws vary among jurisdictions, and it is not always clear
whether they extend to tissues and organs that are not directly transplanted but
are used to produce engineered tissues. Underlying this uncertainty is an
ethical question: to what extent are the concerns that justify prohibitions on
compensation and sale relevant to tissue engineering? What should the implications be for how the creation and implantation of engineered tissues and
organs are governed? Should they be treated more like implantable medical
devices, which can be bought and sold, or more like human organs, which
cannot? Second, the creation of engineered tissues and organs, especially those
custom-made for individual patients, seems at times to more closely resemble a
form of surgical technique or medical practice than the manufacture of a
product; in other cases, a manufacturing process, even a small-scale one, yields
a product that can be marketed. This distinction between practice and product
has legal and ethical significance in several dimensions, including liability, intellectual property, and appropriate regulation, as well as for the question of
sale or compensation.
170
THE PRESENT AND FUTURE OF CELLULAR THERAPY IN
MEXICO
1
Mexico, 3Facultad de Ciencias de la Salud. Licenciatura en Medico Cirujano.,
Universidad Autonoma de Tlaxcala., Tlaxcala, Tlaxcala, Mexico
In Mexico, cell therapy is not highly recognized by a great percentage of the
population. Though some medical practitioners do apply it in some of the
private and public clinics, the majority of the Mexican population still questions
about its origin, clinical applications and benefits. It is well known that stem cell
therapy offers limitless possibilities, enables the creation of new cells and tissues
and treats diseases of the body by replacing cells and tissues thereby aiding in
S51
overcoming illnesses and extending life. Naturally, stem cell therapy is often
regarded as a gift, it is of vital importance to establish and fully discuss its
ethical, legal and social implications since the therapies it offers can greatly
change the biological concept of the patient. A great number of patients had
suffered some adverse reactions and subsequent death in receiving cell
therapy, especially pulmonary embolism and lymphoma in most of the clinics
in Mexico, the causal relationship with stem cell therapy is unclear. According
to some experts, however, ‘it is already known among scholars through preclinical trials using animals that side effects such as pulmonary embolism and
lymphoma can occur’. The death of some patients receiving stem cell therapy
in Mexico had not received widespread domestic coverage. In a statement, the
Mexican legislations have urged the citizens not to participate in “unapproved” regenerative cell therapy, advised patients and their families to avoid
such procedures and asked the government to construct a new medical services framework that would include legal revisions. Cell therapy can be
performed under rigorous conditions in exceptional cases, even if clinical
trials or research studies have not been formally approved. If the appropriateness of stem cell therapy cannot simply be judged on the basis of approval
or disapproval by law or administrative guidelines, then where does the
problem actually reside in Mexico?
171
ODD MAN OUT: CELL AND GENE THERAPY PRODUCTS IN A
SMALL MOLECULE WORLD - HOW FDA GUIDANCE CAN HELP
B Griley
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston,
Texas, United States
Cell and gene therapy (CGT) product development moved out of the laboratory and into clinical research approximately 25 years ago. At that time there
weren’t similar products being regulated by the FDA so the review process was
designed to mimic the processes used to develop small molecules. As the field
has matured and more products have been developed, it has become increasingly obvious that the small molecule model of clinical trial design isn’t ideal for
use with CGT products. In response to this concern, in July 2013 the FDA
released a draft guidance entitled: “Considerations for the Design of EarlyPhase Clinical Trials of Cellular and Gene Therapy Products”. The draft
guidance presents features that are unique to CGT products that may influence
the design of clinical trials. Those features include the different nature of
clinical safety issues specific to the product characteristics; the complicated
logistics and feasibility of manufacturing CGT products; and the limited
available pre-clinical information for CGT products The draft guidance goes
on to provide a comprehensive description of the elements of early phase CGT
clinical trial design that may be impacted by the unique features of the products
and provides recommendations on how these elements can be addressed. The
elements include: 1. the trial objectives; 2. the study population; 3. the need for
a control group and/or study blinding; 4. the initial dose selection; 5. the
treatment plan; and 6. the monitoring and follow-up plan. The guidance
provides useful information for sponsors and investigators developing clinical
trials designed to evaluate CGT products. Equally important, it provides
valuable insight into current FDA thoughts on these products and the related
clinical trials.
172
GENE THERAPY CLINICAL TRIALS: THE AUSTRALIAN PATH
TO THE END OF SUFFERING
J Macpherson, J Rasko
Cell & Molecular Therapies (CMT), Royal Prince Alfred Hospital, Sydney,
New South Wales, Australia
The Therapeutic Goods Administration has authority over conduct of clinical
trials. Medicines from genetically modified organisms (GMO) may be regulated as prescription medicines or biologicals. The Office of the Gene Technology Regulator (OGTR) has authority over dealings involving genetically
modified organisms (GMO). CMT plays a pivotal role in the conduct of cell
and gene therapy clinical trials at our site and has held a licence for AAV vector
clinical trials. We reviewed the regulatory requirements and determined the
local approval path for several gene therapy scenarios. Conduct of GMO
dealings is first managed via an appropriately constituted Institutional Biosafety
Committee (IBC). When a licence is required the Sponsor must submit an
S52
Poster Abstracts
application for OGTR approval. The clinical introduction of viral vector requires a Licence for a Dealing Not involving Intentional Release. The
manufacture of human primary cells modified by viral vector is classified as a
Notifiable Low Risk Dealing (NLRD). Once administered to a subject the
GMO is outside the scope of the regulations. Clinical trials are conducted
under exemption from marketing approval, via the Clinical Trial Exemption
(CTX) or Clinical Trial Notification (CTN) schemes. CTX is required for
gene therapy studies unless the use of the biological is supported by evidence
from previous clinical use; or has received clinical trial approval for an equivalent indication from a national regulatory body with comparable regulatory
requirements. Approval to conduct gene therapy clinical trials has evolved
substantially. Currently primary approval resides with the TGA with advice
from the local IBC. Gene therapy clinical trials have a strong track record of
regulation in Australia. Declaration: JEJ Rasko, chairs both the TGA Advisory
Committee on Biologicals and the OGTR Gene Technology Technical
Advisory Committee. This work has not been endorsed by government
agencies and is not intended to represent their positions.
173
IMPLEMENTATION ROUTES OF ADVANCED THERAPY
MEDICINAL PRODUCTS DEVELOPED IN ACADEMIA IN
CLINICAL PRACTICE IN THE NETHERLANDS
P Meij1, M Hoozemans-Strik2, J Veenman3, LA Veltrop-Duits1
1
Clinical Pharmacy and Toxicology, Leiden University Medical Center,
Leiden, Netherlands, 2Research, Prevention and Patient Support, Dutch
Cancer Society, Amsterdam, Netherlands, 3Team Science and Innovation,
ZonMw - The Netherlands Organisation for Health Research and
Development, The Hague, Netherlands
The regulatory framework in The Netherlands allows clinical use of
Advanced Therapy Medicinal Products (ATMPs) (1) within a clinical study
protocol, (2) within the Hospital Exemption and (3) as registered drug.
Although a lot of ATMPs were tested in clinical trials in the Netherlands, in
the period of 2009 e 2012 about 56 ATMPs have been tested in clinical
studies, up till now in whole Europe only few ATMP are registered as medicinal product. Most ATMPs developed within the academia in The
Netherlands, which were shown to be safe and effective seem to remain in
developmental phase and are thereby not available for standard clinical care.
For some ATMPs other ways have been found for clinical use of the product.
This project aims to identify how effective and safe ATMPs, which have been
developed within academia in The Netherlands, can be efficiently become
available for clinical care. Industry can be involved in the development, but is
not leading. An inventory on the different development and implementation
routes for ATMPs in academia will be made, focussing on the clinical
development route and the implementation of the product in the clinic.
Different research groups and other stakeholders in the field will be interviewed. At least two implementation routes within the oncology field will be
analyzed in detail, where hurdles and successes will be identified. Differences
in the development and implementation between an ATMP and a regular
medicinal product will be characterized. The ultimate aim is to come to a
clear structure for the implementation routes in the clinic for successful
ATMPs developed within academia leading to availability of the treatments
for patient care. The project is supported by The Dutch Cancer Society
(KWF Kankerbestrijding) and The Netherlands Research Organisation for
Health Care and Development (ZonMw).
174
THE EU HOSPITAL EXEMPTION SCHEME FOR ADVANCED
THERAPIES: A VALUABLE TOOL TO SUPPORT INNOVATION
OR A REGULATORY PATH LEADING TO A FRAGMENTED
MARKET? EXAMPLES OF NATIONAL IMPLEMENTATION IN
FRANCE AND UK
AD Poiseau1, N Thomas2
1
Voisin Consulting Life Sciences, Rennes, France, 2Voisin Consulting,
London, United Kingdom
Article 28 of Regulation EC/1394/2007 on Advanced Therapy Medicinal
Products allows for a hospital exemption as an alternative for mandatory
centralized marketing authorization for “products prepared on a nonroutine
basis according to specific quality standards and used within the same Member
State in a hospital under the exclusive professional responsibility of a medical
practitioner, in order to comply with an individual medical prescription for a
custom-made product for an individual patient”. As highlighted during the
public consultation on the ATMP Regulation organized by the European
Commission during 2013, the national implementation of this Article 28 varies
across Member States. The presentation will illustrate this fact by showing how
differently France and UK have interpreted the concepts of “non-routine basis,” “specific quality standards” and “custom-made products.” This presentation will show how the legal provisions adopted nationally result in the nonalignment of the hospital exemption schemes in these two Member States. It
will explain the ‘conservative approach’ adopted in France, where the hospital
exemption framework is considered as an exception to the rule and where
clinical investigation on those products prepared on non routine basis is still
required (unless otherwise justified). In comparison, the presentation will show
how the UK has used the “Specials” framework (named patient use) to
implement a simpler system, allowing the use of products in patients under the
direct responsibility of the prescribing physician as long as the product
manufacture has obtained successful GMP inspection. The presentation will
further discuss the level of GMP requirements required by this hospital
exemption regulatory route in both countries and the obstacles encountered by
the hospitals to fulfill these requirements considering their endeavor in
developing innovative products for unmet medical need and promoting early
phases clinical studies.
175
OVERCOMING MARKET ACCESS HURDLES FOR ATMPS EARLY
IN DEVELOPMENT; PAST, PRESENT AND FUTURE
R Mouchotte1, M Deans2, AD Poiseau3
1
Voisin Consulting Life Sciences, Lausanne, Switzerland, 2Voisin Consulting
Life Sciences, London, United Kingdom, 3Voisin Consulting Life Sciences,
Rennes, France
A proportion of new medicines authorized by the European Commission,
based on the CHMPs benefit-risk opinion fail to be reimbursed or to penetrate
the pharmaceutical market as expected because they fail to meet the different
needs and expectations of Health Technology Agencies (HTA) and Pricing and
Reimbursement (P&R) bodies. This situation has been seen in particular for
recently approved Advanced Therapies. For example, ChondroSelect is reimbursed nationally in Belgium, the Netherlands, and Spain, and through private
payers in the UK whereas it was denied for reimbursement in France. Provenge
faced difficulties in persuading doctors to adopt its prostate cancer vaccine
Provenge. Oncologists and urologists considered that reimbursement issues
contributed to the slow uptake of the treatment. The fact that ATMPs are
generally developed for “niche” or rare disease indications, have complex
mechanism of action that might be difficult to clarify completely and have high
cost of goods represent the main barriers to reimbursement. This presentation
will analyse the difficulties ATMP developers currently encounter to obtain
reimbursement in Europe and the recent actions taken by regulatory authorities, HTA and industry bodies to address such difficulties, in particular the
European Early Dialogue programs. The presentation will propose strategies
that sponsors can use to incorporate Health Technology Agencies (HTA) and
Pricing and Reimbursement (P&R) bodies data expectations into product
development plans.
176
177
178
SOP TO MASTER PROCESS RECORD (MPR) TO BATCH
PROCESS RECORD (BPR): A PARADIGM FOR OPTIMIZING
CELL PROCESSING DOCUMENTATION
M Hackett1, I Prawoko1, WE Janssen1,2
1
Cell Therapies Facility, Moffitt Cancer Center, Tampa, Florida, United
States, 2Blood and Marrow Transplant, Moffitt Cancer Center, Tampa,
Florida, United States
Accreditation organizations, such as FACT or JACIE, and regulatory bodies,
such as FDA or EMA, require production documentation for any therapeutic
agent, including cellular therapeutics. At minimum, documentation must
reflect all steps of production, persons responsible for conduct of each step,
sampling of product for quality and safety testing, and the results of such
testing. Optimal documentation, generally referred to as a ‘batch process record’ (BPR), will have greater than minimum detail, and in addition to the
forementioned qualities should be sufficient to re-create how a specific product
was made and to verify full compliance with the associated SOP(s). We have
created a paradigm that forms a linkage between procedural SOPs, a ‘master
process record’ (MPR) that serves as a template, and the final BPR. This
paradigm has been codified in the form of both policy and procedure, and
establishes a rule set that defines: (1) the format for processing steps in an SOP
that will facilitate generation of the MPR; (2) the structure of the MPR; and (3)
requirements for completion, review and both individual and cumulative
analysis of BPRs. In practice, the implementation of this paradigm, which has
only recently been put into place, has brought about SOPs wherein each step
describes only a single discrete task and defines necessary measurements to
determine if the task has been successfully completed. These new SOPs afford
greater clarity than was observed prior to implementation, and facilitate
monitoring and audits to assess SOP compliance of production activities.
Similarly, MPRs created within a commercially available record maintenance
software package provide for superior capture of processing detail than was
obtained formerly. Our next steps with this paradigm will be to establish direct
cumulative assessment of the final BPRs and to determine the overall benefit to
our quality management efforts with respect to extent of detail captured, efficiency of monitoring by the quality team, and degree of documentation
compliance by the production staff.
179
INCREASED HEMATOPOIETIC PROGENITORS CELLS (HPC)
MOBILIZATION
FROM
BONE
MARROW
ASSOCIATED
WITH MORTALITY FOLLOWING TRAUMA HEMORRHAGIC
SHOCK
MK Sharma, S Mohanty, A Selvi, DN Rao, SK Bhoi
Emergency Medicine, Lab Medicine, Stem Cell Facility, Biochemistry, All
India Institute of Mediical sciences, New Delhi, New Delhi, India
Background: Trauma remains a significant public health issue and is the
leading cause of death in persons younger than 40 years. Up to 50% of early
deaths are due to massive hemorrhage. Late death following these injuries is
usually associated with infection, sepsis, and the development of the multiple
organ dysfunction syndrome. Hematopoietic failure has been observed in
experimental animals and human following shock and injury. Bone marrow
failure is one facet of the multiple organ dysfunction syndrome and is
commonly seen in patients recovering from severe trauma and hemorrhagic
shock.
Methodology: Peripheral blood sample from 39 patients with Trauma
hemorrhagic shock, those who have admitted within 8h post injury. HPC were
analyzed by Sysmex XE-2100 hematology. Than assessed with clinical details.
Results: Peripheral blood stem cell (PBSC)/ HPC was significantly
(p<0.05) increased in a nonsurvival patients as compare to survival patients
with T/HS.
Conclusions: Our data indicate that in human, Trauma hemorrhagic shock
lead to increased peripheral blood hematopoietic progenitor cells (HPC) that
display marker of mortality. But still unclear the exact molecular mechanism
Non survivors Vs Survivors Trauma hemorrhagic shock
Variable
Non survivors
(n¼14)
Survivors
(n¼25)
Hematopoeitic
0.40 (0.01,2.73) 0.03 (0.0,0.85)
progenitor cells (%)
GCS
4.50 (3,15)
15 (3,15)
Shock Index (SI)
1.35 (0,2.14)
1.25 (0,2.26)
p value
0.001
0.005
0.578
*Data are expressed in Median (Min, max) **Mann- Whitney or
Wilkinson Rank sum test ***Non survivors (n¼14) Survivors
(n¼25).
S53
and signaling pathway involved in change the behavior of bone marrow
microenvironment. Keywords: Trauma hemorrhagic shock, Pro-inflammatory
cytokine, HPC, SI, GCS.
180
POST THAW VIABILITY OF CRYOPRESERVED HPC WITH
INCREASED NUCLEATED CELL CONCENTRATION
C Wright1, K Zarkos1, R Brown1, S Larsen1, Z Anwar1, E Newman2,
J Trotman2, J Gibson1
1
Institute of Haematology, Royal Prince Alfred Hospital, Camperdown,
Sydney, New South Wales, Australia, 2Haematology, Concord Repatriation
General Hospital, Sydney, New South Wales, Australia
Aim/Background: All autologous HPC products require cryopreservation to
enable the products to be stored for a prolonged period between collection and
infusion. The routine cryopreservation procedure requires addition of 10%
DMSO as a cryoprotectant. HPC products can be volume reduced prior to
cryopreservation for the following reasons: To reduce the risk of DMSOassociated adverse effects on the recipient. To reduce the amount of time
required to infuse the product at transplant. To reduce the amount of storage
space required in the cryostorage tank.
Methods: The HPC Apheresis products from the Spectra Optia (29
collections from 19 patients) were plasma reduced prior to cryopreservation
to a NCC not greater than 50 x 107/mL. All HPC products were cryopreserved with 10% DMSO and cryopreserved using a controlled rate
freezer. Viable CD34 enumeration was performed using single platform
methodology.
Results: An increase in the NCC used in the cryopreservation protocol to
50 x 107/mL showed a significant inverse correlation between NCC and the
recovery of post-thaw viable CD34+ cells (range 43.3-93.3%) of the cryopreserved product (r¼-0.57; p <0.05). By comparison, products stored using a
protocol of a NCC of not greater than 20 x 107/mL had a post thaw recovery of
approximately 80% while products stored with a NCC of 50 x 107/mL would
have a post thaw recovery of approximately 55%. Engraftment data for
transplant patients whose collections were harvested during the study period
were shown to be not significantly different (Neutrophils t ¼ 0.51; p ¼ 0.61 and
Platelets t ¼ 0.25; p ¼ 0.80) from the median of historical controls. This data is
supported by a review of published data.
Conclusion: Increasing NCC by volume reduction caused a loss of viability
of CD34 cells.
181
MASS PRODUCTION OF FUNCTION NEUTROPHILS FROM
HUMAN HEMATOPOIETIC STEM CELLS BY SERUM-FREE
CULTURE STRATEGY
C Yao, Hu, C Mu
Department of Chemical Engineering and Materials Science, Yuan Ze
University, Chung-Li city, Taiwan
In recent years, the number of people who receive chemotherapy increased
due to the rising trend of malignancy. Hematopoietic stem cells (HSCs) from
bone marrow, peripheral blood and cord blood are widely used in transplantation after cancer chemotherapy. Among them, there are several limitations of cord blood hematopoietic stem cell transplantation, such as slow
recoveries of neutrophils and platelets. The aim of this study is to develop a
neutrophil induction medium to increase the number of neutrophils in vitro,
to accelerate the recovery of autoimmune system of patients after cord blood
HSC transplantation and to overcome the limitation of clinical application. In
this study, HSCs were isolated from cord blood and were expand by a serumfree HSC expansion system (SF-HSC medium) that was developed previously
by our lab. Then we screened five of cytokines (SCF, G-CSF, GM-SCF, IL-3
and IL-1 beta) and optimized the concentration of cytokines that can effectively induce serum-free expanded HSCs into CD66+/MPO+ cells. In addition, we also checked CD66+/MPO+ cells by neutrophil-associated surface
antigens, such as CD11b, CD13, CD15, CD16, CD33, CD64, TLR-2 and
TLR-4, and neutrophil-specific functional assays, such as phagocytosis assay,
chemotaxis assay and cellular reactive oxygen species (ROS) production assay,
challenged with Pseudomonas aeruginosa after transplanted neutrophils in
NOG mices. Finally, we confirmed induced cells were functional neutrophils.
S54
Poster Abstracts
In conclusion, we have successfully established a neutrophil induction medium
that can effectively induce HSCs into mature and functional neutrophils in
vitro. We believe that combination of SF-HSC medium and neutrophil induction medium can generate a large amount of functional neutrophils and
provide a promising cell source for basic research and clinical application in
the future.
182
TO EVALUATE THE EFFECT OF GROWTH FACTORS
(EPO, IL3,GM-CSF) ON IN-VITRO HEMATOPOIETIC STEM
CELL GROWTH/DIFFERENTIATION FOLLOWING TRAUMA
HEMORRHAGIC SHOCK
MK Sharma, SK Bhoi, S Mohanty, DN Rao, A Selvi
Emergency Medicine, JPNATC, All India Institute of Medical Sciences, New
Delhi, New Delhi, India
Background: Trauma remains a significant public health issue and is
leading cause of death in persons younger than 40 years. Up to 50% of
early deaths are due to massive haemorrhage. Late death following these
injuries is usually associated with infection, sepsis, and the development of
the multiple organ dysfunction syndrome. Excessive pro-inflammatory
cytokine, hyper catecholamine that induced hematopoietic progenitor cell
apoptosis lead to the persistent anemia that means delay recovery of patients with trauma hemorrhagic shock. Therefore, our aim was of this study
to evaluate the effect of growth factors (rh-EPO, rhIL-3, rh-GM-CSF) on
in-vitro hematopoietic progenitor cells growth among trauma hemorrhagic
shock patients.
Methodology: Bone marrow aspirates were collected on day 3 (n¼8)
those patient who have survived. Bone marrow cells were cultured for he-
matopoietic progenitor’s cells growth (CFU-GM, BFU-E, and CFU-E
colonies) with or without additional growth factors (rh-EPO, rhIL-3, rhGM-CSF).
Results: Bone marrow CFU-E colony significantly increased (p<0.05) with
rhEPO, rh-GMCSF and rhIL-3 but BFU-E and CFU-GM colonies was not
significantly increased with growth factors (rhEPO, rh-IL3, rhGM-CSF) as
compare to baseline (without additional growth factor).
Conclusion: Our studies suggested bone marrow dysfunction in a T/HS.
Still need to evaluate the signalling pathway and its correlation with bone
marrow failure following trauma hemorrhage.
183
POST-THAW CHARACTERIZATION AT CORD BLOOD BANKS:
DOES IT CORRELATE WITH TRANSPLANT CENTER
PRODUCT CHARACTERIZATION AND ENGRAFTMENT?
EK Storch1,2, S Avecilla2, R Reich-Slotky2, S Mayer2, K van Besien2,
M Cushing2
1
New York Blood Center, New York, New York, United States, 2New York
Presbyterian Hospital, New York, New York, United States
Background: Cryopreserved umbilical cord blood transplants are
increasingly used as a source of hematopoietic stem cells, however quality
of cords vary significantly. Post-thaw testing at cord banks is not yet
routinely available or standardized, and cord blood bank (CBB) viability
and potency assessments may differ from that of receiving centers. We
attempt to correlate post-thaw testing at CBBs with that of our transplant
center.
Methods: All charts of umbilical cord units received from CBBs were
reviewed. Results of post-thaw segments from CBBs were compared to postthaw viabilities and colony-forming count assays (CFCs) obtained in our lab.
We also compared post-thaw CBB results to engraftment. Statistics were
performed with Pearson correlation.
Results: Post-thaw viability was available from 13 source CBBs for 19/85
charts. Other data was inconsistent including TNC, total CD34 and CFC.
Nine banks determined viability using 7-aminoactinomycin D (7-AAD), 3
using trypan blue, and 1 using acridine orange. Two banks reported CD45
viability. No centers provided statistics such as measures of central ten-
Post-thaw characterization
NYP*
CD34 7AAD
Trypan
blue
CFUs
CFU-E (DAY7)
CFU-E colony counted on day 7, BFU-E and CFU-GM counted on day 14
with or without growth factors.
Variable
CFU-E
BFU-E
CFUGM
Base line
(Without additional
growth factor)
(n¼8)
(1)
70.0* (0,424)
46 (0,185)
19.5 (0,84)
*p value <0.05, Friedman Test.
With EPO
(2U/ml)
(n¼8)
(2)
EPO
4U/ml
(n¼6)
(3)
82.42
(21.07)
95 (3.16)
10.11
(7.02)
Cord bank*
88.19
(10.94)
90.25
(4.35)
53.42
(77.63)
Correlation
coefficient
0.48
0.44
0.41
p-value
0.12
(NS)
0.56
(NS)
0.28
(NS)
* Mean (SD).
EPO
6U/ml
(n¼6)
(4)
IL-3
3U/ml
(n¼8)
(5)
GM-CSF
6U/ml
(n¼8)
(6)
119* (0,544) 177*(0,611) 180* (0,562) 93.5* (0,2056) 123.0* (0,1723)
80.5 (0,226) 81.5 (0,255) 100.5 (0,188) 87.5 (0,202)
63. (0,183)
21.5 (0,65) 40.0 (0,72) 51.0 (0,78) 22.5 (0,38)
36 (0,93)
COB.1
EPO 1U/ml, IL3 1.5U/ml,
GM-CSF -3U/ml
(n¼5)
(7)
389* (0,1377)
71.0 (0,235)
20.0 (0,84)
dency or variability to aid in the interpretation of results. For 10/19 units
viability method was not specified but obtained upon inquiry. The correlations between CD34 7-AAD, trypan blue (r ¼ 0.48, r ¼ 0.44) CFCs
(r ¼ 0.41) or CBB viability to recipient engraftment (r ¼ 0.06) were not
significant.
Conclusion: Post-thaw viabilities and CFCs showed significant variation
between reporting cord blood banks and our internal assessment, and had no
correlation with engraftment. This study highlights limitations of inter-facility
product comparisons, and suggests it is necessary to establish consistency in
characterization of cord blood units.
184
OPTIMIZATION
OF
POST-THAW
VIABLE
CD34
ENUMERATION FOR CRYOVIALS OF HEMATOPOIETIC
PROGENITOR CELL PRODUCTS
R Reich-Slotky2, J Kim2, EK Storch3, S Avecilla1, M Cushing1
1
Transfusion Medicine, Weill Cornell Medical Center, New York, New York,
United States, 2Transfusion Medicine, New York Presbiterian Hospital, New
York, New York, United States, 3New York Blood Center, New York, New
York, United States
Background: Testing CD34 viability on concurrent small aliquots of cryopreserved hematopoietic progenitor cell (HPC) products can help estimate
product viability prior to transplant. Cryopreserved HPC are washed before
issue in our institution, thus cryovials are also washed prior to testing. The
current standardized viable CD34 enumeration protocol is not optimized for
testing cryopreserved products. Since cryopreservation can compromise cell
membranes, washing of cells in a small volume may result in lower apparent
viability. In order to optimize CD34 enumeration conditions for this sample
type, we compared CD34 viability of washed HPCs and their concurrent
washed and unwashed cryovials, and examined the correlation with transplant
outcomes.
Methods: 11 autologous or allogeneic HPC products were analyzed. Plasmalyte/ACD-A solution was used for washing, and samples were kept on ice
and analyzed within one hour. Trypan blue (TB) was used for total nucleated
cell (TNC) viability and the ISHAGE protocol was used for viable CD34
enumeration. Results are reported as percentage for viable TNC, viable CD45+
cells and viable CD34+/CD45+ cells. Paired t-test and Pearson Correlation
were used to compare HPC and cryovial results. Regression analysis was used
to test CD34 viability as engraftment predictor. Statistical analyses were performed using SAS 9.0.
Results: There are significant differences for all viability measurements,
including CD34, for washed cryovials (Table). However, with the unwashed
cryovial sample, there is no significant difference for viable CD34
(p-value¼0.8215). There is a positive correlation between CD34 viability of
unwashed cryovials and the HPC products (r¼0.878). When controlling for
cell dose, regression analysis indicated that CD34 viability of unwashed
cryovials is a significant predictor of ANC engraftment time (p-value¼0.01).
Conclusions: Our results suggest that measuring CD34 viability of an
unwashed concurrent cryovial provides a reliable tool for assessing HPC
product viability and engraftment potential while eliminating unnecessary
processing steps.
Comparison between washed HPCs and washed and unwashed
concurrent cryovial.
HPC
Viability (Mean Test
SD
TB
CD45
CD34
Washed
Cryovial
(Mean SD)
92.1 3.4 41.6 19.9
59.1 15.4 35.9 14.9
54.4 24.8 26.9 18.3
Unwashed
Cryovial
t-Test
(Mean t-Test
(p-value)
SD)
(p-value)
<0.0001 51.6 21.4
<0.0001 52.7 16.6
0.0007 52.3. 20.1
0.0001
0.0787
0.8215
All results are in percentage (%). TB¼Trypan Blue, CD45¼Viable
CD45, CD34¼Viable CD34
S55
185
MULTIPLEXED FLOW CYTOMETRIC CHARACTERIZATION OF
CD34 AND CD3 CELL CONTENT IN HPC PRODUCTS
M Ancharski1, S Avecilla3, D Sutherland2, M Cushing3
1
Transfusion Medicine/Cellular Therapy, NewYork-Presbyterian Hospital,
New York, New York, United States, 2University of Toronto, Toronto,
Ontario, Canada, 3Weill Cornell Medical Center, New York, New York,
United States
Background: The characterization of stem cell products requires the use of
separate flow cytometry templates to quantify stem cells and T-lymphocytes.
The current methods are time consuming and costly. By multiplexing the
ISHAGE protocol template to include CD3, costs and turnaround time can be
reduced without change in specificity or sensitivity.
Study Design: A paired sample comparison of two flow cytometry protocols
was performed using peripheral blood, bone marrow, HPC-apheresis, and cord
blood as samples. The current method in use relies upon the BD Enumeration
kit and the ISHAGE protocol template for CD34 enumeration (2 Trucount
tubes). CD3 enumeration relies on a flow cytometry assay which includes the
use of CD3 and CD19 monoclonals and associated isotype controls (3 Trucount tubes). The proposed multiplexed method for combined CD34 and CD3
cell content is based on the BD Enumeration kit with the addition of a CD3APC monoclonal antibody. In the new protocol, CD34 enumeration is still
performed with ISHAGE gating, however additional subset analysis using
similar sequential gating logic was applied to perform CD3 enumeration from
the same sample and dataset (2 tubes). An acceptability criterion in the comparison was defined to be an allowable Total Error of 5% for single-platform
comparisons.
Results: 17 samples were run and analyzed in parallel and all met the
allowable error of 5% for the single-platform method. There was a 60%
reduction in the use of Trucount tubes and elimination of 3 monoclonal
antibodies.
Conclusion: The proposed multiplexed protocol was found to be clinically
equivalent using stringent acceptability criteria. The reduction in resource
utilization was found to be substantial in reagent usage alone, and further
analysis on the reduction of analysis, labor, and turn-around-time is planned.
While achieving efficiency strides, the new protocol also maintains both the
high sensitivity and specificity of the incumbent assay.
186
COMPARISON OF EX VIVO T CELL REMOVAL USING
DIFFERENT CLINIMACS PROCEDURES
T Soh1, P Law2, L Tan1
1
Laboratory Medicine, National Unversity Hospital, Singapore, Singapore,
Singapore, 2Orthopaedic Surgery, National University Hospital, Singapore,
Singapore, Singapore
T cells number in a stem cell graft is directly related to the risk of graft
versus host disease (GVHD) in patients undergoing haematopoietic stem
cell transplantation (HSCT). In this study, we compared our single
institution, non-randomized comparison of different CliniMACs T cells
depletion procedures from mobilized HPC, Apheresis, which include
CD34+ selection and CD3+, CD3+/CD19+ and T cell receptor (TCR)
ab+ depletion. Results are summarized in Tables 1. Statistical analysis showed
Table 1. Log of T cell depletion and % Recovery of CD34+
Cells
Cell Type
N
Depletion Log
% Recovery
Median
Median
Selection
CD34+
11 4.48 (3.74 Depletion
CD3+
12 3.81 (3.40 CD3+/CD19+ 3 4.37 (4.03 TCRab+
6 3.70 (3.33 -
5.97) 82.00 (72.23 - 93.19)
4.59) 92.98 (85.74 - 102.90)
4.61) 92.71 (86.73 - 97.77)
4.86) 95.29 (90.53 - 99.55)
S56
Poster Abstracts
that CD34+ selection achieved significantly better T cell depletion (p < 0.05)
than CD3+ depletion and TCRab+ depletion. However, recovery of CD34+
cells was statistically higher for the three depletion procedures than the CD34+
selection procedure. There are no differences amongst the depletion procedures in term of T cell log reduction and CD34+ cell recovery. Our results
suggested that if a purer CD34+ stem cell graft is desired, depletion procedures appear to be the preferred methodology than CD34+ selection. On the
other hand, if a lower T cell dose in the stem cell graft is desired, CD34+
selection should be the method of choice.
187
EXPRESSION PROFILE OF GALECTINS (1, 9, 11 AND 13) IN
HUMAN BONE MARROW DERIVED MESENCHYMAL STEM
CELLS IN DIFFERENT CULTURE MEDIUMS
F Jenhani
CNTS, cellular therapy cytometry and immunology, El manar II, Tunisia
In this study we analyzed MSCs cultured and expanded cells in three mediums: basic growth medium consisting of alpha-Minimum Essential Medium in which HPL replaced FBSin order to investigate their morphology,
plasticity, proliferation differentiation to osteoblasts,adipocytes, and vascular
smooth muscles and their capacity to express galectins in terms of the
mediums’ compositions. MSCs were characterized and defined according to
the ISCT minimal criteria. So first, we demonstrated that BM-MSC
adhered to plastic. Second as measured by flow cytometry all MSC derived
from BM-MSC were nearly 100% positive for the makers CD90 CD105,
and CD73 (95%), while they lacked expression of CD45 CD34, and
CD14 ( 2%). Third, BM-MSC differentiated to osteoblasts, adipocytes
and vascular smooth cells. To detect the expression of galectins 1, 9, 11 and
13, flow cytometry (FC) and RT-PCR were used. The expression of
galectins in MSCs cultivated in the three culture mediums was observed:
the standard medium: alpha-MEM+ FGF2, the medium (with 5% HPL),
the medium (with 10% HPL). The flow cytometry revealed that all
the galectins of interest (Gal-1, Gal-3, Gal-9, and Gal-13) are expressed in
BM-MSCs, in above mentioned culture mediums,. It thus becomes clear
thatexpression percents of galacetins revealed by the flow cytometry in
hBM-MCSs depend on the composition of the medium and the type of the
galectin. The best Galectins’ expression was visualized in the standard
medium (alpha-MEM /FGF2). Previous studies have shown that the supplementation of fibroblast growth factor 2 (FGF2) in vitro selects for the
survival of a large number of MSCs enriched in pluripotent mesenchymal
precursors. MSCs maintains their multilineage differentiation potential
during in vitro expansion and prolongs their life span. The data suggests
that the increase of galectins in the presence of FGF2 is due to the
improvement of the growth the proliferation and differentiation potential
of MSCs.
188
RECOVERY OF VIABLE CD34 FROM THAWED CORD BLOOD: A
REAL CONUNDRUM
J Kurtzberg1,2, T Gentry2, A Balber2
1
Pediatrics, Duke University Medical Center, Durham, North Carolina,
United States, 2Duke Translational Medicine Institute, Duke University
Medical Center, Durham, North Carolina, United States
Specifications for CD34 viability and recovery from cryopreserved and thawed
umbilical cord blood have yet to be defined. Various banks, transplant centers,
registries and regulatory bodies use terminology about CD34 viability or recovery of viable CD34 cells with precision. Comparability between methods or
assays used to measure or calculate viable CD34 cells have not been established.
Knowledge about the meaning of a viable CD34 or percent recovery of viable
CD34 cell result is poor amongst transplanters and registries utilizing this data.
In the US, the specifications set forth in the FDA Guidance for Cord Blood
Licensure is equally confusing and based on a error (pre 2005) when validated
assays for viable CD34 essentially did not exist. In those historical times, one
approach to quantitation of viable CD34 cells involved measurement of the
total nucleated cell count multiplied by the percent viability measured by
trypan blue multiplied by the percent of CD34 cells enumerated by flow
cytometry. Recent assays incorporate vital dyes, like 7-AAD, into flow
cytometry panels with CD34. A viable CD34 count/uL as well as viability of
CD34 cells are reported. These can be multiplied by the volume of product
recovered post thaw to quantitate a total viable CD34 count. This can be
divided by the pre cryopreservation viable CD34 cell content to calculate
percent recovery of viable CD34 cells. These approaches yield different absolute results. Also, these assays often have a preparation step using lysis of red
blood cells which also lyses some of the nucleated white blood cells in the
product, a step which, along with prolongation of timing of the assay run, may
reduce the measured amount of recovered viable CD34 cells. Other variations
will be discussed. The transplant and banking communities need to understand
what viable CD34 means and the scientific and banking communities need to a
new approach to measurement of and the language used to talk about viable
CD34 cells.
189
VALIDATION OF AN ALTERNATIVE TRANSPORT SYSTEM FOR
CRYOPRESERVED HAEMOPOIETIC PROGENITOR CELLS
PG Dyson, R Tocchetti, S Hiwase
Haematology, SA Pathology, Adelaide, South Australia, Australia
Introduction: To centralise processing of Haemopoietic Progenitor Cells
(HPC) collected at multiple sites it is critical to have a safe and effective means
of transporting cryopreserved cells to the transplant site. The transport system
must be safe for staff as well as product. Conventional metal liquid nitrogen dry
shippers used for transport of cryopreserved cells are bulky and heavy creating a
significant health and safety hazard for staff. The requirement for liquid nitrogen to maintain vessel charge can also be a logistic limitation. Dry ice (frozen
CO2) may be used to maintain product at <-80 C but is limited by the
requirement for a reliable and consistent supply of dry ice bricks. Aim. We
aimed to develop a method for transporting cryopreserved HPC that ensured
product and personnel safety as well as being easy to maintain and cost effective.
Method: Our system utilised a polycarbonate insulated transport shipper,
containing liquid nitrogen absorbent pillows within an insulated foil bubble
wrap bag. The shipper could be charged very quickly and safely prior to
transport.. The conditioned container packed with cryopreserved cells was
light enough to be easily carried by any staff member.
Results: Initial validation was performed utilising expired cryopreserved
HPC products. Data loggers were used to monitor product temperature during
“transport,” The system was validated to maintain temperature at <-80 C for a
median of 8.4 hours. Further validation was performed to demonstrate that the
shipper maintained temperature at <-80 C when transport included a challenge
of repeated openings to mimic the bedside infusion of 8 HPC bags. Further
verification was performed on line during repeat HPC transports and infusions.
Conclusion: We have a system for HPC transport validated to maintain
product at <-80 C for a median of 8.4 hours. The system is cost effective and
safe for product and staff.
190
DONOR COMBINED T AND B CELL DEPLETION OF
HAPLOIDENTICAL HEMATOPOIETIC CELL GRAFTS FOR
TRANSPLANTATION IN CHILDREN
B Wagner1, A Scharpf1, J Gossner1, B Lowigus1, R Henschler1, MH Albert2
1
Transfusion Medicine, Cell Therapy Products and Hemostaseology,
Klinikum der Universitaet Muenchen, Muenchen, Germany, 2Pediatric
Hematology/Oncology, Dr. von Haunersches Kinderspital der LudwigMaximilians-Universität, Muenchen, Germany
As a curative mode of treatment in various diseases the importance of
allogeneic hematopoietic cell transplantation (HCT) has been increasing.
CD3/19 depletion (CD3/19D) enabled the use of haploidentical (HI) donors as an option preserving CD34-ve populations e.g. NK cells, accessory
cells and CD34-ve HPCs. Our report on 10 CD3/19D procedures for 10
children undergoing HIHCT illustrates possible relations to graft quality.
Patients and Methods: 10 children with AML/MDS (5), neuroblastoma (2),
hepatoblastoma (1) and SCID (2) underwent HIHCT. Aliquots from
HPC(A)s from 10 HI parental donors were subjected to 10 CD3/19D
procedures. Loading time and number of stages and cell counts (PLT, NC,
CD34+ HPCs, lymphocyte subsets) including viability of the respective
HPC(A)s, intermediate and final products were analysed for variation and
correlation. Results: Like in HPC(A)s cell counts in the final graft varied
considerably: 21.513.8 (mean+SD)x 10e9 NCs,186.8107.8x 10e6
CD34+ HPCs, 58.957.3x 10e5 T cells, 18.816.3x 10e5 B cells and
49.434.4x10e7 NK cells. T and B cell depletion averaged 3.2logs0.5, and
3.3logs0.7, respectively. Despite several processing steps the platelet
depletion (94.210.1%) and the yields of NCs (54.75.7%) and CD34+
HPCs (80.110.3%) were consistent in contrast to that of NK cells
(53.321.2%). NCs in the final product were significantly correlated with
the amounts of B and NK, but not T cells. The residual amount of T, but
not B or NK cells was closely correlated with the loading time per stage, but
inversely associated with the numbers of stages performed, the former being
negatively related to the NC concentration applied. PLT counts in the graft
were significantly correlated with residual B, but not T or NK cells (R2
0.469-0.794; p<0.05). Conclusions: Our results demonstrate that within
CD3/19D procedure T and B cell depletions seem to adhere to different
rules as well as the preservation of NK cells and CD34+HPCs in the
engineered graft.
191
INCREASING STEM CELL DOSE PROMOTES THE LONGEVITY
OF THE GRAFT IN MICE TRANSPLANTED WITH HUMAN
CORD BLOOD STEM CELLS
A Dolnikov, N Xu, S Shen, T O’Brien
Cord and Marrow Transplant Laboratory, Sydney Children’s Hospital,
Randwick, New South Wales, Australia
Haematopoietic stem cell (HSC) transplantation is being increasingly
offered as a curative option for patients with hematologic malignancies.
Stem cell dose has been recognized to be important in allogeneic transplantation. HSCs shorten time for hematologic engraftment when given in
higher doses. Studies correlating stem cell dose with long-term outcomes
are limited. Although sub-optimal stem cell dose was associated with inferior overall and progression free survival and lower non-relapse mortality, it
is not clear whether this was associated with poor bone marrow function or
rather attributed to the most common causes of myelosuppression, viral
infections, relapse, rejection and GVHD. To compare long-term donor
haematopoiesis in the recipients of low and high doses of stem cells, we used
severely immunocompromised NOD/SCID-IL2Rgnull (NSG) mice transplanted with different doses of cord blood CD34+ stem cells to monitor
multilineage human chimerism within more than 30 weeks. We have
demonstrated that mice transplanted with low stem cell doses exhibited
premature decline in donor haematopoiesis in peripheral blood. In addition,
early skewing to myeloid cell lineage in expense of lymphoid lineage
characteristic of replicative stem cell aging was observed in mice received
low stem cell dose. The decline in human cell chimerism in periphery appears to be associated with stem cell exhaustion in the bone marrow. We
have also shown the excessive proliferation of transplanted stem cells in the
recipients of low stem cell dose that may impose an additional “replicative
stress” upon the graft and lead to pre-mature stem cell exhaustion. The
results of this study suggest that premature decline in stem cell regenerative
capacity may be the factor contributing to the inferior long-term survival of
the recipients transplanted with low stem cell dose and highlight the
importance of increasing stem cell numbers in transplants to maintain the
longevity of the graft.
192
STEADY STATE PERIPHERAL BLOOD STEM CELLS FATE
DEPENDS ON HIF EXPRESSION AND ANTIOXYDANTS
P Brunet de la Grange1,2,3, J Chevaleyre1,2,3, M Vlaski1,2,3, P Duchez1,2,3,
V Lapostolle2,3, F Mazurier3, A Villacreces2,3, V Praloran2,3, Z Ivanovic1,2,3
1
R&D Laboratory for Cell Ingineering, French Blood Institute, Bordeaux,
France, 2CIRID, CNRS UMR 5164, Bordeaux, France, 3University of
Bordeaux, Bordeaux, France
S57
The role of Hypoxia and its key regulators, HIF-1 and HIF-2, remains to
be defined for circulating Steady Sate Peripheral Blood (SSPB) Heamtopoietic Stem Cells HSC. In this work we tested the ability of Hypoxia
and antioxidants-complemented medium to maintain the HSC activity
inside SSPB CD34+ cell population. We first evidenced that Side Population (SP) activity of SSBP CD34+ cells is linked to very primitive cells
according to proliferation, CFC content and engraftment capacity in
NSG mice. By culturing SSBP CD34+ cells at atmospheric concentration
(20% O2, control) and low O2 concentrations (3% and 1% O2), we
showed that SP activity decreased by 10 fold at 20% and 3% O2 whereas
the proportion and number of SP cells were increased by 2.6 fold at 1%
O2. In this condition, the inhibition of hypoxia-induced stabilization of
HIF-1a and HIF-2a using specific shRNA led to a 2.3 and 6.3 fold
decrease of SP cells for shRNA/HIF-1a and shRNA/HIF-2a, respectively. Similarly, SSBP CD34+ transduced cells with either shRNA/
HIF-1a or shRNA/HIF-2a failed to give a sustained hematopoietic
reconstitution of NSG mice. By culturing SSBP CD34+ cells at 20%
O2 with antioxidants-supplemented medium and cytokines inducing the
stabilization of HIF-1a, we observed a 21 fold increase in SP cells
number. The phenotypic characterization of these SP cells using CD34,
CD38, CD90 and CD133 antibodies showed a 5.9 to 10 fold expansion
depending on the phenotype. This result was corroborated by the xenotransplantation assays in NSG mice that showed an 11 fold increase in
huCD45 chimerism after primary transplantation when cells were injected
after 7 days of culture compared to uncultured cells. Besides, these cells
displayed a secondary engraftment capacity. These data show that SSPB
HSC fate is dependent on hypoxic regulatory pathways and environmental capacity to decrease the ROS content. This work strengthens the
possibility to use SSBP as a source of HSC for research and cellular
therapy.
193
FRENCH NATIONAL PROFICIENCY TESTING STUDY FOR
THE STANDARDISATION OF THE CFU-GM PROGENITOR
ASSAY
V Affraix, B Panterne, C Sabatini, C Maurin, I Fabre, N Charlier-Bret,
F Cano
Laboratory Controls Division, ANSM, Saint-Denis, France
The Laboratory Controls Division of the French Health Product Safety
Agency (ANSM) had organized an annual external quality control (EQC)
of preparations of Hematopoietic Stem Cells (HSC) from 2000 to 2013.
From this experience, it appeared that it was necessary to prepare new
technical guidelines for the clonogenic assay. Indeed the present guidelines did not give sufficient tools of validation. The analysis of data
collected from the EQC allowed to elaborate recommendations which
have been placed in public inquiry for three months in 2013. In order to
evaluate these ones, a Profiency Testing Study was conducted with 29
control laboratories of HSC. To that end, samples of fresh cord blood,
culture medium and cell culture procedure were sent to participants.
Three conditions were proposed: seeding 200 cells CD34+ per dish
(compulsory requirement) with initial cord blood then according to laboratories practices, seeding at another cell concentration and/or after
deserythrocytation. For the reference condition, coefficients of variation
are inferior to 40%, while discrepancies vary much more for the two
other conditions (with a maximum value of 116.6%). In order to carry on
the assessment of deserythrocytation procedures initiated during this
study, we performed additional testing of sedimentation. They have
shown that this simple and fast method can be used with reliability but
only in intra-laboratory conditions. Alongside, comments received during
the public inquiry were studied. The answers were documented by
analyzing data from EQC that showed an improvement of reproducibility
of results obtained in 2011-2012. Eventually, the recommendations were
adjusted according to our studies and will be evaluated by the Committee
of biological products of the French Pharmacopoeia. All our results
showing that the clonogenic assay can be used reliably to evaluate the
quality of hematopoietic grafts, should allow to base their publication in
early 2014.
S58
Poster Abstracts
194
AUTOMATED WASHING OF AUTOLOGOUS HEMATOPOIETIC
STEM CELL GRAFTS AFTER THAWING DOES NOT IMPAIR
ENGRAFTMENT
B Calmels1,5,3, A Drezet3, C Huynh1, A Autret4, A Stoppa2, R Bouabdallah2,
D Coso2, C Malenfant1, C Lemarie1,5, C Chabannon1,5,3
1
Centre de Therapie Cellulaire, Institut Paoli-Calmettes, Marseille,
France, 2Departement d’Onco-Hematologie, Institut Paoli-Calmettes,
Marseille, France, 3Inserm UMR 1068, CNRS UMR 7258, AMU 105, Centre
de Recherche en Cancerologie de Marseille, Marseille, France, 4Unite
Biostatistiques, Departement de la Recherche Clinique et de l’Innovation,
Institut Paoli-Calmettes, Marseille, France, 5Inserm CBT-510, Centre
d’Investigations Cliniques en Biotherapie, Marseille, France
Autologous hematopoietic stem cell transplantation (AHSCT) is widely used
during treatment of hematologic diseases. Cryopreservation is mandatory,
allowing administration of high dose chemotherapy. Reinfusion of thawed
grafts without washing might induce side effects. However, washing remains
controversial since CD34+ cell loss is supposed to be higher. There are no
reports comparing these approaches for their relevant clinical endpoints, i.e.
hematopoietic reconstitution. To determine whether washing has a detrimental effect on engraftment, we retrospectively compared two cohorts,
receiving either washed or unwashed autologous grafts. We selected 218
AHSCT thawed at the bedside and that fall within four diagnosis groups
with homogeneous intensive chemotherapy regimens, and retained 65
AHSCT with CD34+ cell doses within 2.0-5.0x10e6/kg. We then selected
580 AHSCT among the same 4 intensive regimen for which grafts were
homogeneously processed (washing on CytoMate) and retained 260
AHSCT with CD34+ cell doses within 2.0-5.0x10e6 CD34+/kg. To
compare these two groups, we used a case-match design: each of the 65
unwashed AHSCT was matched with 2 of the 260 washed AHSCT, using 4
variables: sex, age, diagnosis and CD34+ cell dose. The case-match analysis
exhibits, as expected, no difference in terms of age, sex ratio, diagnosis and
CD34+ cell dose. Median time to reach 0.5x10e9/L neutrophils is compa-
rable, with 12.4 and 12.5 days in the unwashed versus washed groups,
respectively. Our approach allowed us to compare two matched cohorts
issued from 2,930 AHSCT performed over 10 years at a single institution.
This accurate matching leads us to conclude that washed autologous grafts
do not compromise engraftment as compared to bedside thawing. Moreover,
using devices such as CytoMate or Sepax allow for standardization,
improved stability of thawed cell products and determination of infused
CD34+ cells.
195
NEW POTENTIAL AGENTS FOR CRYOPRESERVATION OF
HEMATOPOIETIC AND ADIPOSE STROMAL CELLS
J Svalgaard1, E Haastrup1, A Brink1, C Clausen3, B Holst3,
K Theilgaard-Mönch2, K Reckzeh2, A Fischer-Nielsen1
1
Department of Clinical Immunology, Rigsospitalet, Copenhagen,
Denmark, 2Finsenlab, Copenhagen Biocenter, Copenhagen,
Denmark, 3Bioneer A/S, Hoersholm, Denmark
Introduction: Cryopreservation of cells and tissue is important in both
clinical settings and the field of biological research. The most commonly
used cryoprotective agents (CPAs) is dimethyl sulfoxide (DMSO). However, DMSO exerts temperature and concentration dependent cell toxicity
and, also, often causes side effects when administered to patients.
Therefore, there is a need for safer and non-toxic CPAs replacing DMSO.
We investigate whether Pentaisomaltose, a proprietary formulation of
isomaltooligosaccharides with molecular weight 1kDa and Pentaisomaltoside, the hydrogenated version of Pentaisomaltose, could serve as alternatives to DMSO. Both formulations are developed by Pharmacosmos
Denmark.
Materials and methods: Hematopoietic stem cells (HSCs) from mobilized
peripheral blood and adipose-derived stromal cells (ASCs) purified from
liposuction based adipose tissue were frozen in cryoprotective medium
containing either 10% DMSO, and different concentrations of Pentaisomaltose or Pentaisomaltoside, using a controlled rate freezer. Outcome
measures are 1) post thawing viability and 2) in vitro function as assessed by
colony forming unit for HSCs and proliferation and differentiation potential
for ACSs.
Results: Viability data for both HSCs and ASCs indicate a potent cryoprotective effect of both CPAs investigated as compared to DMSO. The experiments performed so far, suggest a viability of approximately 90-100% for
both cell types relative to DMSO. Also, in vitro function tests performed so far
indicate values comparable to DMSO.
Conclusion: The presented data, although generated from only a few
samples, suggests that both Pentaisomaltose and Pentaisomaltoside are potential alternatives to DMSO. Parameters related to the freezing protocol are
still being optimized and further in vitro and in vivo experiments need to be
performed to address whether the proliferative, differentiation and functional
properties are intact following cryopreservation.
196
A NOVEL PROCEDURE TO IMPROVE FUNCTIONAL
PRESERVATION
OF
HEMATOPOIETIC
STEM
AND
PROGENITOR CELLS IN CORD BLOOD STORED AT 4 C
BEFORE CRYOPRESERVATION
Z Ivanovic1,2, L Rodriguez1,3, P Duchez1,2, M Plainfosse3, B Dazey1,
V Lapostolle2,1, M Vlaski1,2, P Brunet de la Grange1,2, B Delorme3,
J Chevaleyre1,2
1
Aquitaine-Limousin, Etablissement Français du sang, Bordeaux,
France, 2UMR 5164, CNRS/University Bordeaux 2, Bordeaux, France, 3R&D
Biotherapy, Macopharma, Tourcoing, France
Functional preservation of the relatively small amount of cells of interest in
collected cord blood units (CBU) during the period of transportation to the
bank prior to its processing and cryopreservation, is very important for the
graft potency. In order to improve the storage efficiency, i.e. functional
preservation of stem and committed progenitor cells in cord blood units, we
conceived an approach based on two principles: first- providing a better
nutritive and biochemical environment to stem and progenitor cells and
second - preventing the hyperoxygenation of these cells transferred from a
low (1.1 to 4% O2 in the cord blood) to an atmospheric oxygen concentration
environment (20 to 21% O2). Our results clearly demonstrate that the
maintenance of hematopoietic stem cells and committed progenitors in the
cord blood could be ensured by combining the prevention of oxygenation/
CO2 leak and by optimizing the nutritive environment of the storage medium. These conclusions are based on the assessment of stem cell activity and
progenitors by the functional approaches: i) the capacity of hematopoietic
reconstitution of immunodeficient mice, ii) the capacity of ex vivo expansion
after the storage period and iii) progenitors’ capacity of in vitro colony formation. Applying this storage procedure it is possible to maintain the full
functional capacity of CBU graft for 72h with respect to Day-0 (D0), i.e. to
get a graft with better functional capacities in comparison to the method
currently used where CBUs are stored for 24h in gas-permeable bags and
without medium. In this study, we demonstrated not only the proof of
principle of our approach, but also developed a clinical-scale kit and performed a preclinical assay demonstrating the feasibility and efficiency of our
CBU preservation protocol.
197
CHARACTERIZATION OF BONE MARROW-DERIVED CD34D
CELLS WITH DIFFERENT MIGRATORY POTENTIAL BY
MICRORNA FINGERPRINTING AND ANTIBODY ARRAY
C LeBlon1, B Warbington1, D Weinsten1, D Mallison2, D Olijnyk2,
S Paterson2, D Dunbar2, S Ridha2, V O’Brien2, M Lin1, T Fong1, W Chan1
1
Progenitor Cell Therapy, LLC, Allendale, New Jersey, United
States, 2Sistemic, Ltd., Glasgow, Scotland, United Kingdom
Background: AMR-001, an autologous CD34+ cell product derived from
mini-marrow harvest, is currently undergoing Phase II trials to treat acute
myocardial infarction (AMI). At the time of AMR-001 infusion, it is believed
that the infarct-region stromal derived factor-1 (SDF-1) levels are peaked. It
was found that improvement in cardiac perfusion and infarct size correlated
with the mobility potential of CD34+ cells, as mediated by a SDF-1 gradient.
We have initiated a study to identify potential microRNAs (miRNAs) and
proteins that may be used as biomarkers that correlate to CD34+ cell migratory
potential.
Methods: In vitro transwell migratory assays were performed on purified
CD34+ cells derived from bone marrow of healthy donors. After 4 hours at
37 C, CD34+ cells that migrated into the lower chamber in the presence of
SDF-1, the non-mobilized cells in the upper chamber, and untreated cells were
harvested. The miRNA expression profile was analyzed (Sistemic, Ltd) using
microarray slides (Agilent). A biotin label-based antibody array (RayBiotech)
was used for the detection of 1000 proteins.
Results: Hierarchical clustering analysis of the miRNA data showed that
mobilized cells grouped separately from the non-mobilized/untreated cells.
Sixty-three miRNAs were upregulated in the mobilized samples compared to
non-mobilized/untreated samples, including two miRNAs which have a reported pro-angiogenic role in migratory cells. Twenty-six proteins had higher
expression in mobilized cells compared to non-mobilized cells. Several of the
identified proteins have a role in migration or angiogenesis.
Conclusion: Analysis of the miRNA and protein profiles of the CD34+
cells identified a number of miRNAs/proteins that represent possible
markers for a migratory phenotype. qPCR and ELISA assays will be performed to verify the specific miRNAs and proteins identified. This approach
will enable the development of a biomarker assay for migratory potential of
AMR-001.
198
EXPERIENCE IN A PUBLIC CORD BLOOD BANK USING A
SEGMENT-BASED ALDEHYDE DEHYDROGENASE ASSAY AS A
BIOMARKER FOR UMBILICAL CORD BLOOD POTENCY
K Shoulars1, JD Troy2, T Gentry1, P Noldner1, K Page1, A Balber1,
J Kurtzberg1
1
Duke University Medical Center, Durham, North Carolina, United
States, 2The EMMES Corporation, Rockville, Maryland, United States
Cryopreserved cord blood units (CBU) provide a source of hematopoietic
stem cells (HSCs) for transplantation for patients in need of a suitable
donor. Primary graft failures or delayed engraftment occur in 10-15% of
patients following cord blood (CB) transplant. This may be due to low
potency, defined as low levels of engrafting HSCs. Transplant centers select
CBUs based on human leukocyte antigen (HLA) match, pre-cryopreservation
total nucleated cell count, and, in some centers, viable CD34+ content.
These methods do not account for insults during cryopreservation and
thawing that may impact overall potency. Prior studies have shown that the
post-thaw hematopoietic colony forming units (CFU) content of transplanted CBUs predict engraftment and reflect potency. However, post-thaw
CFU assays require >14 days, so relevant data are usually not available at
the time of CBU selection or transplantation. We found that CFU content
of fresh CBUs correlates with the content of cells expressing high levels
of the enzyme aldehyde dehydrogenase (ALDHbr). Retrospective studies
indicated that ALDHbr content of segments associated with transplanted
CBUs correlated with engraftment. Therefore, we developed a rapid
ALDH-based potency assay using CBU-attached segments that could be
performed when HLA confirmatory typing was requested. From March
2010 and August 2013, segments from 2766 CBUs were analyzed. The
number of viable (7-AAD-), CD45+, ALDHbr, and CD34+ cells were
measured by flow cytometry using multiparameter gating and CFUs were
enumerated. The percentage of ALDHbr (r¼0.82) and [ALDHbr viable
CD34+] cells (r¼0.72) correlated well with CFUs measured (n¼2730, both
S59
p<0.0001). In contrast, CFU correlated less well with viable CD34+
(N¼2727, r¼0.28, p<0.0001). These correlations were valid regardless of
time in cryostorage. Approximately 800 assayed CBUs have been administered to patients. Thus, transplant outcomes data will be available to
compare with laboratory metrics.
199
LWPQ: AN IN SILICO DESIGNED MULTI-CORE SUPERAGONIST MOTIF-LIKE REGULATORY PEPTIDE FOR THE
ACTIVATION OF HUMAN STEM CELL TRANSCRIPTS USING
AN INTEGRATED COMPUTATIONAL APPROACH
N Grigoriadis, I Grigoriadis
Biogenea pharmaceuticals, Thessaloniki, Greece
The role of KIT ligand CXC chemokine receptor CXCR4, fibroblast
growth factors and their receptors (FGFRs), Notch or its ligand Jagged1
and retinoic acid (RA) signaling in determining the fate of these cells in the
regulation of normal hematopoietic stem cells is well-known An increasing
amount of evidence from experimental and computational bioinformatic
analysis suggests that there are many domains in DNA sequences that
remain evolutionarily conserved. In some cases, these conserved patterns in
a collection of unaligned DNA and protein sequences present the same
functional and regulatory properties and are significant for the molecular
role of these sequences. Discriminative motif finding algorithms aim to
increase the sensitivity and selectivity of conserved motif discovery by
utilizing a specific set of DNA and protein sequences, and searching for
binding sites and homolog repeats among the sets of the selected sequences.
In the present study we introduce a combined bioinformatic software-based
discriminative methodology to detect short, highly and most conserved
motifs between the DNA sequences within the FLT3, CXCR4, CKIT,
HOXB4, JAGGED1, FGF1 proteins and then, on finding out a multitarget motif conserved featured superagonist peptide about them including
their physical regulatory properties, as well as their function as an ex-vivo
positive modulator for the enhancement of human stem cell expansion
rates.
200
ROLE OF APOPTOSIS IN CRYOPRESERVED HCT/P
J Tassy, G Koehne, J Tonon, P Maslak, K Smith, M Pessin, RC Meagher
Cellular Laboratory Medicine, Memorial Sloan- Kettering Cancer Center,
New York, New York, United States
Human cells, tissues, or cellular tissue-based products (HCT/P) are
routinely cryopreserved and stored for varying periods of time in liquid
nitrogen vapor freezers (LN2) in preparation for patient bone marrow
transplantation. The duration of storage in the cryopreserved state may
cause a gradual decline in cell viability, and hence cell functionality. As a
quality measure, stability of storage studies are periodically performed on
these cryopreserved cells to assess their condition and how long term storage
may affect them. The role of apoptosis (programmed cell death) and its
possible deleterious effects on HCT/P viability and functionality during
long term storage are not well understood. In a preliminary study, we
examined the effects of apoptosis on thawed HPC, Apheresis, (HPC(A))
products in hopes of conveying more information on this mechanism of cell
damage post-thaw. 10 samples of cryopreserved HPC(A) products from
patients collected between 2004 and 2010 for bone marrow transplantation
were examined for various stability testing (pre/ post cryopreservation).
Routine HPC(A) product testing was performed including enumeration of
CD34+ cells, 7AAD viability, confirmation of sterility, and analysis of colony
forming units (CFU), in conjunction with direct measurement of apoptotic
cells using a flow cytometric methodology. Preliminary results using 10
samples for post-cryopreservation viability testing (65-89%), reveal a close
correlation between number of viable cells observed and length of time
HPC(A) products were stored in LN2 vapor. Results from this study indicate the need for further investigation on the role of apoptosis in cryopreserved HCT/P to assess whether change in cryopreservation methods
or improvements in cryoprotectants are needed to optimize cryopreservation
conditions for HCT/P.
S60
201
USE OF STERILIZED, LYOPHILIZED PLATELETS
MULTIPLE APPLICATIONS
N Ramachandran, MC Hiles
Cook Biotech, West Lafayette, Indiana, United States
Poster Abstracts
FOR
Clinical and non-clinical work within the last decade has changed the
conventional thinking about platelets. In addition to hemostasis, platelets
are now recognized as playing a major role in wound healing, immune
modulation and tissue regeneration. Autologous platelets are gaining
acceptance for various wound healing applications while allogeneic platelets are still reserved for infusion for hemostasis. The biggest disadvantage
is that these platelets have a shelf life of 5 days. Lyophilization is one
technique that has been evaluated to increase the shelf life of platelets and
almost all lyopilization techniques use some kind of preservative or sugars.
We have developed a process of lyophilization for allogenic platelets which
are past their useful life for infusion but are still quite well suited for
seeding provisional matrix within wounds. This process doesn’t use special
additives and optionally includes terminal sterilization to yield an efficient,
safe, off-the-shelf, freeze-dried product. This product was characterized by
residual moisture content and presence of key growth factors (PDGF,
TGF-b1, FGF and VEFG). All these growth factors are able to stimulate
cell proliferation, matrix remodeling and angiogenesis in wound healing.
An extensive analysis of these growth factors in the product postprocessing indicated that their values (per 106 platelets) were very close
to values obtained from fresh platelet rich plasma from healthy volunteers.
Comparisons of growth factors were conducted to detect differences
between methods of sterilization and different vial materials used in
the lyopilization process. Functional assays were conducted to verify the
activity of the freeze-dried product. All tests showed that the processed product maintains the growth factor levels and cell stimulation
activity appropriate for seeding the provisional matrix in a topical or
surgical wound.
202
203
STEM CELL ENGINEERING TOWARDS THE OPTIMIZATION
OF THE EX-VIVO EXPANSION OF HUMAN HEMATOPOIETIC
STEM/PROGENITOR CELLS FOR CELLULAR THERAPIES
CL da Silva1,2, PZ Andrade2,3, AM Soure2, FD Santos2,3, G Almeida-Porada4,
JS Cabral1,2
1
Department of Bioengineering, Instituto Superior Tecnico, University of Lisbon,
Lisbon, Portugal, 2Stem Cell Bioengineering and Regenerative Medicine
Laboratory, Instituto Superior Tecnico, University of Lisbon, Lisbon,
Portugal, 3Cell2B - Advanced Therapeutics, SA, Biocant Park, Cantanhede,
Portugal, 4Wake Forest Institute for Regenerative Medicine, Wake Forest Institute
for Regenerative Medicine, Winston-Salem, North Carolina, United States
Umbilical Cord Blood (UCB) transplantation has faced a significant increase
recently due to the unique features of UCB hematopoietic stem/progenitor
cells (HSPC) for the treatment of blood-related disorders. However, the low
cell numbers available per unit significantly impairs its widespread use for
transplantation of adult patients. We have been focused on using rational
stem cell engineering approaches targeting the maximization of UCB HSPC
expansion. Human UCB CD34+-enriched cells were co-cultured with human bone marrow (BM) mesenchymal stem/stromal cell (MSC)-derived
feeder layers using a cytokine-supplemented serum-free medium. Several
parameters were studied namely different initial stem/progenitor content,
cytokine/growth factor cocktails and the impact of low oxygen tension
values (2-10%) compared to normoxia (21% O2). Importantly, a dynamic
co-culture system using plastic microcarrier-immobilized human bone
marrow (BM) MSC was evaluated to support expansion of UCB CD34+enriched cells. A design of experiments strategy was applied to the optimization of cytokine cocktails supplementing serum-free culture medium,
resulting in an increased cell productivity with a reduction in culture costs
by 50-65% compared to previously established protocols. The impact of the
initial CD34+ cell content was studied showing that a high (>90% CD34+
cells) initial progenitor content was not mandatory to successfully expand
HSPC. The effect of physiological O2 levels (5-10%) were found to be
beneficial for an efficient expansion of UCB HSC. Concerning the expansion performed under dynamic conditions in spinner flasks, clinical meaningful CD34+ cell doses e 19 millions e were produced for a putative
transplantation strategy of an adult patient, in a short time period (10 days).
Overall, our results provide the basis for the establishment of efficient and
controlled scalable culture systems for the generation of clinically significant
cell numbers for cellular therapies.
204
NEUROPROTECTIVE ROLE OF 17B-ESTRADIOL ADMINISTRATION ON ALTERED AGE RELATED NEURONAL PARAMETERS
IN FEMALE RATS
P Kumar, RK Kale, NZ Baquer
School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Aging is a multifactorial process involving neurodegenerative changes in cell
morphology and biochemistry. During aging the brain experiences structural,
molecular, and functional alterations. Aging in females and males is considered
as the end of natural protection against age related diseases like osteoporosis,
coronary heart disease, diabetes, Alzheimer’s and Parkinson’s disease. These
changes increase during menopausal condition in females when the level of
estradiol (E2) is decreased. The objective of this study was to observe the
changes in activities of membrane linked ATPases (Na+K+ ATPase,
Ca2+ATPase), antioxidant enzymes (superoxide dismutase (SOD) glutathione
S-transferase (GST), intrasynaptosomal calcium levels, membrane fluidity and
neurolipofuscin accumulation occurring in brains of female rats of 3 months
(young), 12 months (adult) and 24 months (old) age groups, and to see whether
these changes are restored to normal levels after exogenous administration of
estradiol (0.1 mg/gm body weight for one month). The results obtained in the
present work revealed that normal aging was associated with significant
decrease in the activities of membrane linked ATPases, antioxidant enzymes
and an increased in neurolipofuscin, intrasynaptosomal calcium levels in brain
of aging female rats. The present study showed that E2 treatment reversal the
changes to near normal levels. E2 treatment appears to be beneficial in preventing some of the age related changes in the brain, an important anti-aging
effect of the hormone.
205
UNDIFFERENTIATED HUMAN FETAL STEM CELLS PRODUCE
STRUCTURAL AND FUNCTIONAL IMPROVEMENTS IN THE
SPASTIC HAN-WISTAR RAT MODEL OF ATAXIA
TL Uhlendorf1, R Nuryyev1, CS Malone1, AO Kopyov2, L Peltz2, J Prieto2,
T Keelinghamm2, RW Cohen1, O Kopyov2
1
Biology, California State University, Northridge, Northridge, California,
United States, 2Celavie Biosciences, LLC, Oxnard, California, United States
We assessed whether transplanted, brain-derived, fetal human stem cells
(hFSC) could improve functional deficits in the cerebellum and hippocampus
of male spastic Han-Wistar (sHW) rats. The mutant rat strain displays
neurodegeneration of Purkinje cells in the cerebellum and CA3 pyramidal
neurons in the hippocampus, resulting in forelimb tremor, hind leg rigidity,
uncoordinated movements, muscle wasting, and a shortened lifespan. To
ameliorate the observed neurodegeneration, we implanted Celavie’s nontumorigenic and hypoimmunogenic hFSCs displaying a normal karyotype
into mutant rats. At 30 days of age, mutants received cyclosporine to suppress
the rat’s immune system. At 40 days, mutants were injected bilaterally with
500,000 live stem cells (LhFSC), 500,000 dead stem cells (DhFSC), or
500,000 live human embryonic kidney cells (HEK 293T) into the cerebellar
nucleus or CA3 region of the hippocampus. Rats implanted with LhFSC in
the cerebellum showed statistically significant improvement of motor activity
scores after 60 days compared to DhFSC or HEK injected mutants. There
was no significant motor activity improvement for those rats with hippocampal LhFSC, DhFSC and HEK implants. At 65 days, rats with LhFSC
transplants maintained greater weight than rats with DhFSC and HEK cells
in the cerebellum, but not in the hippocampus group. The LhFSC cerebellum group also demonstrated significantly greater longevity (p<0.05) than
the DhFSC and HEK cerebellum groups. To examine the survivability of
transplanted LhFSC, all animals were fixed and prepared for morphological
evaluations. Our assays showed surviving LhFSC labeled with Q-dots in both
the cerebellum and hippocampus after 30 days post-transplant. Our results
demonstrate that implanted human, brain-derived fetal stem cells reduced
the ataxic symptoms and extended longevity in the sHW rat, suggesting
future clinical benefit for the utilization of these stem cells in treatments of
neurodegenerative diseases.
206
PROTECTION OF BRAIN CELLS IN ORGANOTYPIC SLICE
CULTURES
EXPOSED
TO
OXYGEN
AND
GLUCOSE
DEPRIVATION IS SPECIFICALLY MEDIATED BY CORD
BLOOD CD14+ CELLS
A Saha, S Buntz, J Kurtzberg, A Balber
Robertson Clinical & Translational Cell Therapy Program, Duke Tanslational
Medicine Institute, Durham, North Carolina, United States
We are developing clinical products derived from human cord blood [CB]
mononuclear cells [CBMC] to protect the brain from acute hypoxic injury. We
have standardized an organotypic mouse brain slice culture model to identify
CBMC subpopulations that protect brain cells from death following oxygenglucose deprivation [OGD]. To prepare CBMC, <2day old CB units were
centrifuged on FicollÒ, treated with NH4Cl, and washed in medium. Brain
slice [300mm] cultures established from P1 or P2 C57BL/6J mice were maintained for 8-10 days on membrane filters over serum free medium under
normoxic conditions, subjected to OGD [glucose free medium in <1 % O2; 1
hour], and then returned to normal conditions. CBMC were then added on top
of the slices. Co-cultures were maintained 72h. OGD induced death was
measured by propidium iodide staining. Adding CBMC reduced cell death in a
dose dependent manner; 25,000 CBMC reduced cell death 80 5% [mean SD, n¼5]. Peripheral blood [PB] mononuclear cells showed 3-fold less protection. Adding 125,000 CBMC to the medium below the membrane instead of
directly to slices reduced brain cell death 60 8 % [mean +/-SD; n¼3], suggesting that CBMC produce diffusible protective factors. To identify what
types of cells mediate protection, we immunomagnetically depleted specific cell
types and added depleted CBMC populations to OGD shocked cultures.
Depleting CD14+ cells reduced the protective activity of CBC 3.5-fold, but
depleting CD3+, CD19+, or CD34+ cells did not remove protective activity.
Positively selected CD14+ also protected brain cultures from OGD, but CD3+
and CD19+ enriched populations did not. CD14+ cells selected from PB were
4-fold less active than CD14+ cells isolated from CB. Thus, CD14+ cells from
CB are uniquely active in protecting brain cells from OGD induced death. We
are now exploring how CB CD14+ cells interact with brain glia and neurons in
these cultures and the molecular mechanisms by which CD14+ cells protect
brain cells.
207
HUMAN PLURIPOTENT STEM CELLS AMELIORATE NMDAINDUCED HIPPOCAMPAL DEGENERATION AND RELATED
FUNCTIONAL DEFICITS
SK Uppal1, J Saenz1, TL Uhlendorf1, AO Kopyov2, L Peltz2, J Prieto2,
RW Cohen1, O Kopyov2
1
Biology, California State University, Northridge, Northridge, California,
United States, 2Celavie Biosciences, LLC, Oxnard, California, United States
Seizures, trauma and many neurologic diseases induce damage to the CA3
region of hippocampus, resulting in extensive deficits in spatial navigation,
memory consolidation, and depressive-type behaviors. Current drug treatments have limited effectiveness in addressing these memory problems. To
attempt to use regenerative medicine to ameliorate these deficits, we used
Celavie’s human fetal, brain-derived, pluripotent, nontumorigenic, hypoimmunogenic stem cell line with a normal karyotype (hFSC). These stem
cells have previously shown an ability to migrate, differentiate and reduce
structural and functional deficits in other neurodegenerative models. We
determined if hFSCs injected into an NMDA-lesioned hippocampus survive
and possibly differentiate into mature functional neurons, thereby diminishing any behavioral and neuronal deficits. We induced hippocampal
degeneration by stereotactically lesioning the CA3 regions bilaterally with
the neurotoxin NMDA in 50 day old male Wistar rats (1 ml containing 7.5
mg/ml; -3.5 mm AP; 2.0 L and -2.5 V). At 54 days of age, live hFSC,
frozen-killed hFSC or HEK293T cells (500,000 cells of each type in 5 ml of
S61
cell suspension media), or cell suspension media (5 ml) were bilaterally
implanted directly into the NMDA damaged area. The rats’ spatial memory
was tested two weeks later (68 days) with the Morris water-maze task, and
novel and place-object tests. Our results confirmed that rats receiving live
hFSC implantation performed significantly (p<0.005) better in the water
maze task than any of the control groups. Novel and place object assays
showed no significant differences among all the treatment groups. Immunohistochemistry results confirmed the survival of implanted hFSCs up to
28 days post-implantation in the rat CA3 region. Our study has shown that
hFSC were able to survive in vivo and improve hippocampal functionality,
highlighting the potential promise for stem cell treatment of brain damage
in neurodegenerative diseases.
208
NEURONAL AND GLIAL CELL COMPOSITION IN A MOUSE
BRAIN SLICE CULTURE MODEL IS USEFUL IN DEVELOPING
HUMAN CORD BLOOD DERIVED CELLULAR THERAPIES FOR
NEONATAL HYPOXIC-ISCHEMIC BRAIN INJURY
S Patel, A Saha, S Buntz, J Kurtzberg, A Balber
Robertson Clinical and Translational Cell Therapy Program, Duke
Translational Medicine Institute, Duke University and Medical Center,
Durham, North Carolina, United States
Our laboratory is developing novel cord blood (CB)-derived cellular therapies for patients with neuronal damage resulting from hypoxia-ischemia. To
understand how CB cells mediate response to injury, we adapted and characterized the organotypic mouse brain slice culture model. The cellular
composition of these brain slice cultures can be used to better understand the
mechanisms underlying hypoxic injury and beneficial effects of cell therapies.
We used immunofluorescence and image analysis to enumerate glial and
neuronal cells in C57BL/6J mice-derived brain slices cultured on a semipermeable membrane for 21 days in serum free medium. We compared the
cellular composition of ex vivo brain slices to that of neonatal mouse brains.
Selected brain slice cultures or brain sections were fixed in 4% paraformaldyde on ex-vivo culture or postnatal days 1, 3, 6, 9, 12, 15 and 21.
Immunohistochemical staining differentiated astrocytes (GFAP), neurons
(NeuN), oligodendrocytes (olig2) and microglia (Iba1) in cultured brain
slices. Contiguous images of the periventricular regions were analyzed using
fluorescence confocal microscopy. In brain slice cultures, neurons comprised
approximately 40% (SD +/- 7%) of the total cell population, while glial cells
made up 60% (SD +/- 3.5%) throughout the 21 day culture period.
Conversely, in the age-matched neonatal brain sections, neurons maintained
an average of 60% (SD +/- 6%) of the cellular composition, and remaining
glial cells were 40% (SD +/- 4%). Both in vivo and in vitro, the relative
proportions of the three glial cell populations were the same. These results
establish concordance between the in vivo and ex vivo systems and validate
the ex vivo brain slice model for use to further investigate effects of hypoxic
injury. We have used this model system to investigate the protective effects of
cord blood mononuclear cells after acute hypoxic injury on different types of
brain cells.
209
210
DUOC-01, A CANDIDATE CELL THERAPY PRODUCT DERIVED
FROM BANKED CORD BLOOD, ACCELERATES BRAIN
REMYELINATION IN NSG MICE FOLLOWING CUPRIZONE
FEEDING
A Saha1, S Buntz1, S Patel1, GK Matsushima2, A Wollish1, J Kurtzberg1,
A Balber1
1
Robertson Clinical & Translational Cell Therapy Program, Duke
States, 2Department of Microbiology & Immunology, University of North
Carolina Scool of Medicine, Chapel Hill, North Carolina, United States
We are developing a candidate cell therapy product, DUOC-01, derived
from banked cord blood for use in the treatment of CNS demyelination.
We have adapted the cuprizone model of reversible brain demyelination to
S62
Poster Abstracts
determine whether DUOC-01 can accelerate remyelination of neurons in
immune-incompetent NOD/SCID-IL2Rgnull [NSG] mice. Male, 7-9 week
old NSG mice were adapted to a milled lab chow diet for one week and
then shifted to chow containing 0.2% (w/w) cuprizone. After 5 weeks,
brains were harvested from cuprizone-fed and from controls kept on
standard chow for subsequent assessment of the degree of demyelination
and gliosis induced by cuprizone. Remaining animals were returned to
normal lab chow to allow remyelination to begin. One day after the change
in diet, one group of mice was stereotactically injected in the corpus callosum [CC] region with 105 DUOC-01 cells. Cells were manufactured
using protocols suitable for clinical use. Control mice were injected with
excipient only. Brains were harvested from DUOC-01 treated and control
mice 1 week following injections, and cryosections were prepared. Myelination was assessed by Luxol-fast blue-periodic acid Schiff-staining [LFB]
and immunohistochemistry [MBP]. Organization of neurons [NHF] and
distribution of astrocytes [GFAP] and oligodendrocytes [Oligo2] were also
assessed by immunohistochemistry. The CC midline region and more
lateral regions of NSG mice were severely demyelinated with gliosis
following cuprizone feeding. LFB staining one week after cell treatment
showed that mice injected with DUOC-01 had significantly increased
myelination [p<0.0006] and decreased gliosis and cellular infiltration
[p<0.01] in the CC region compared to mice injected with excipient. No
abnormalities were noted in the brains of animals maintained on standard
chow throughout the protocol and injected with DUOC-01 or excipient.
These data demonstrate the potential activity of DUOC-01 in treating
demyelinating conditions.
211
ADIPOSE MESENCHYMAL STROMAL CELLS (AMSC) AS A BASE
OF A COMBINED CELL THERAPY FOR CHRONIC SPINAL
CORD
INJURY
(CSCI)
PATIENTS.
CLINICAL
AND
ELECTROPHYSIOLOGICAL RESPONSE AFTER SIX MONTH OF
TREATMENT
M Moviglia-Brandolino, GA Albanese, RF Vina, A Perusso, J Huerta,
SC Piccone, G Etchegaray, N Blasetti, GA Moviglia
CIITT, Maimónides University, Buenos Aires, CABA, Argentina
Introduction: To improve time and quality of clinical response of BEN
therapy, (Cytotherapy 2006;8:202-209 and Spinal Cord, 2009;47:499-503) BM
MSC have been replaced by aMSC. (MED therapy).
Methods: Under Compassionated Use and after local bio ethic comity
approval, 5 cSCI patients ASIA A (4) and B (1) received MED therapy. Adipose
tissue was obtained by lipectomy and dissociated with collagenase 4 in a GMP
facility. aMSC were isolated through attachment, cultured for a week. 1/2
aMSC harvested and implanted through intra lesion feeding artery infusion
(IA). After a week a cyto-aphaeresis (Ospira). Effecter Cells against CNS
proteins from the Buffy Coat were activated, expanded and negative selected
using Clinimacs. 1/2 EC were IV infused. Second EC half co cultured for 24 hr
with aMSC which differentiate into NPC. They were IA implanted. Patients
underwent to a program of physical therapy. Clinical outcome was evaluated
through physical examination and SEP and EMG tests.
Results: The five patients showed progressive improvements in their
clinical condition that correspond to function of muscles innervated by
motor nerves originated at 3 to 10 spinal cord levels under the lesion. For
this reason they recover complete absent functions as sustain the head,
improve the trunk tone, develop ability of trunk rotation flexion and
extension. Three of them recovered the ability to be standing up and give
(with two arms support and assistance) small steps. EMG and SEP registers
shown general wave shape morphology, intensity, amplitude and transmission speed values coincident with clinical findings predicting recovery
of function.
Conclusion: It is a proof of concept of MED therapy for cSCI. There also
shown that electrophysiology registers are valid and objective methods to
evaluate the response of these patients.
212
ADIPOSE STEM CELLS TRANSPLANTATION IN
TRAUMATIC BRAIN INJURY MODEL
A Giannakopoulou2, I Dori2, C Bekiari2, S Petrakis1, I Grivas2,
A Tsingotjidou2, G Koliakos1,3, G Papadopoulos2
A
RAT
Biohellenika Biotechnology Company, Thessaloniki, Greece, 2Faculty of
Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki,
Greece, 3Medical School, Aristotle University of Thessaloniki, Thessaloniki,
Greece
1
Pluripotent mesenchymal stem cells (MSCs) are thought to participate in
tissue repair through trophic support and immunomodulation when transplanted into damaged tissues. Adipose tissue is considered as an attractive
source of MSCs, generating the so called adipose stem cells (ASCs). The
present study examines host brain responses induced by ASCs intracerebroventricular (ICV) transplantation in an animal model of traumatic
brain injury (TBI). For this, 3-month old rats received succesively localized
TBI and ICV transplantation of Venus+ ASCs. Normal rats transplanted
with ASCs and TBI rats transplanted with medium, were used as controls.
Survival, spatial distribution and integration of ASCs in the host parenchyma were examined at one and six weeks post transplantation. Proliferative activity of both transplanted ASCs and endogenous reactive cells,
focusing on hippocampal radial glia like neural stem cells (NSCs), was
explored with antibodies for Ki67 and BrdU. Interactions of transplanted
ASCs with innate brain inflammatory cells were assessed with the markers
Iba-1 and GFAP, respectively. According to the results obtained, ASCs one
week after their transplantation exhibit proliferative activity, integrate into
brain parenchyma migrating mainly to the periventricular area, whereas
some of them acquire morphology of mature cells with ramified processes.
ASCs migration was more profound six weeks after their ICV transplantation, as they reach TBI site through white mater tracks and gather
around pre- existing or newly formed blood vessels. Both presence of ASCs
and TBI have a positive impact on hippocampal NSCs proliferation bilaterally. In addition, ASCs enhanced brain inflammatory responses with no
existing evidence of their phagocytocis or destruction. As inflammation can
participate in both beneficial and detrimental outcomes, the findings of this
study suggest that ASCs transplantation may contribute to the acceleration
of host brain repair mechanisms.
213
EFFICACY AND SAFETY OF STEM CELL-BASED THERPIES FOR
PATIENTS WITH STROKE: A SYSTEMATIC REVIEW AND
META-ANALYSIS OF SINGLE-ARM STUDIES
H Yim1, H Jeong1, Y Kim2, S Jeong3, H Kim4, S Jo1
1
Department of Preventive Medicine, The Catholic University of Korea,
Seoul, Korea, Republic of, 2Department of Neurology, Seoul St. Mary’s
Hospital, Seoul, Korea, Republic of, 3Medical Library, Seoul St. Mary’s
Hospital, Seoul, Korea, Republic of, 4Clinical Research Coordinating Center,
Catholic Medical Center, Seoul, Korea, Republic of
Background and objectives: Stroke is a major cause of mortality and
disability in adults worldwide. Nevertheless, currently, very few therapeutic
options are available. Cell-based therapy is a potential new approach in the
treatment of ischemic stroke. However, the effects of these treatments are
not yet fully understood and there is a lack of firm evidence on the efficacy
and safety of stem cell therapy for those patients due to the absence of
sufficiently powered randomized controlled trials. Therefore, we performed
a meta-analysis of available single-arm studies using stem cell-based therapy
in patients with stroke.
Methods: A systematic search and critical review of the literature published from its inception through January 2013 was performed. The articles
included in the search were restricted to the English language, studies with
at least 5patients, and those using cell-based therapies for treating stroke.
Data analyses were performed using Comprehensive Meta-analysis version
2.2. (Biostat Inc., Englewood, NJ) and the G3data graph analyzer version
1.5.3(GNU General Public License). We conducted random effects model
meta-analyses to assess efficacy and safety outcomes. The quality of studies
was assessed using the Newcastle-Ottawa Scale and Cochrane Risk of Bias
assessment.
Results: We included 10 single arm studies in the meta-analysis. The
pooled mean difference in NIHSS between baseline and follow-up points was
5.11 points (95%CI: 2.78-7.44) significantly increased. Pooled incidence rate to
achieve on mRS2 was 0.38 (95% CI: 0.23-0.55) at follow-up points. In
addition, pooled incidence rates of occurring infection, seizure, and death after
cell transplant were 0.15 (95% CI: 0.08-0.26), 0.14 (95% CI: 0.08-0.25), and
0.13 (95% CI: 0.08-0.23) respectively.
Conclusion: The present systematic review suggests that stem cell-based
therapies for patients with stroke are effective and safe. However, properly
designed randomized controlled trials are required.
214
ADIPOSE
MESENCHYMAL
STROMAL
CELLS
(AMSC)
DIFFERENTIATE INTO NEURAL PROGENITOR CELLS (NPC)
AFTER 24 HOURS OF CO CULTURE WITH EFFECTER CELLS
(EC) AGAINST CENTRAL NERVOUS SYSTEM (CNS) PROTEINS
N Blasetti, LA Ugartemendia, GA Moviglia
Introduction: In 2006 (Cytotherapy 2006, 8:196-201) was reported that BM
MSC co cultured with anti-CNS EC differentiate into NPC. To prove that
aMSC share the same property and this process may be conducted under GMP
rules we performed the following experiment.
Method: Adipose tissue obtained by lipectomy and dissociated with collagenase 4 in a GMP facility. aMSC were isolated by attachment and cultured for
a week. EC were obtained from peripheral blood mononuclear cells, concentrated, were activated and expanded culturing them in DEMEM + human
recombinant Insulin (Humalin), and 1% of Cerebrolysin . After 96 hours cells
were harvested, washed and marked with anti CD8, CD56 and CD25. and
negative selected with Clinimacs. EC and aMSC were co cultured for 24 hr to
120 hours. To test the aMSC differentiation into NPC cells were immunefluorescent stained with anti nestin, tubulin 3, neu66, GFAP and Sox2 and
MBP. and analyzed using confocal microscopy and FACS analysis.
Results: After 24 hours most of aMSC showed positive stain to nestin . At
48, 96 and 120 hours free cells and neurosphere structures showed positive
stain for the rest of cell markers. In the neurosphere was able to distinguish
cells positive for neu66, GFAP and sox2 proving multiple linage differentiation
of these structures. No microbial contamination, persistence of lymphocytes or
immune magnetic microbeads was detected in the harvested NPC cells.
Conclusions: aMSC may differentiate into NPC done from an adult individual
without use of any cytokine, neurotropin, gene transpher, in a GMP facility.
215
TISSUE DISTRIBUTION OF A CORD BLOOD-DERIVED CELL
PRODUCT FOLLOWING INTRATHECAL TRANSPLANTATION
R Storms1, C Liu2, T Gentry1, J Zhou1, A Ozamiz1, B Rusche1, A Balber1,
J Kurtzberg1
1
Robertson Clinical and Translational Cell Therapy Program, Duke
States, 2Department of Surgery, Duke University, Durham, North Carolina,
United States
We have developed an umbilical cord blood-derived cell product, DUOC-01, as
a potential adjunct therapy for patients with certain inherited leukodystrophies.
In clinical practice, DUOC-01 cells will be transplanted only after systemic cord
blood transplantation and engraftment. Importantly, the DUOC-01 cells will be
derived from the same cord blood unit that is to be used for the systemic transplant. To facilitate neural repair, the DUOC-01 cells will be delivered by
intrathecal injection. The goals of this study were to determine the tissue distribution of DUOC-01 cells following their intrathecal injection, and to determine how long they remain detectable in vivo. Five different DUOC-01 cell
preparations were cultured per GMP-compliant Standard Operating Procedures. For each transplant, 105 cells were delivered by intrathecal injection into
neonatal ( 2 days old) NOD/SCID-IL2Rgnull mice. After periods of up to 56
days post-transplantation, the mice were sacrificed to analyze six tissues (brain,
spinal cord, lungs, liver, spleen and bone marrow) for their content of human
cells, as determined using quantitative PCR to detect human Alu DNA sequences. Within the first 24 hours post-transplantation, the DUOC-01 cells were
detected within multiple tissues in 5 of 5 mice. These included the brain, spinal
cord, lungs or, to a lesser degree, the liver. Human cells remained detectable
within 11 of 20 mice that were analyzed between 7 and 56 days post-transplantation. However, at these later time points, the cells were detected only
within the brain and spinal cord. Furthermore, in 8 mice that had human cells
detectable within both the brain and spinal cord, the human DNA was more
prevalent in the brain. These studies indicate that immediately following intrathecal injection the DUOC-01 cells distribute to both neural and non-neural
tissues. However, in the long-term, the cells only remained detectable in the
neural spaces encompassed by the brain and spinal cord.
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216
ANGIOGENIC PERFORMANCE OF MAGNETIZED ENDOTHELIAL
PROGENITOR CELLS FOR STROKE THERAPIES
A Rosell1, E Carenza2, V Barceló1, A Morancho1, J Montaner1, A Roig2
1
Neurovascular Research Laboratory, Vall d’Hebron Research Institute,
Barcelona, Spain, 2Nanoaprticles and Nanocomposites Group, 2Institut de
Ciència de Materials de Barcelona (ICMAB-CSIC), Cerdanyola, Spain
IEndothelial Progenitor Cells (EPCs) are good candidates for cell-based therapies
to treat ischemic diseases by inducing angiogenesis. We propose that EPCs can be
magnetized with iron oxide superparamagnetic nanoparticles (SPIONs) and guide
them into the ischemic brain with an external magnetic device to enhance neurorepair. SPIONs were synthesized by the thermal decomposition. EPCs were coincubated for 24 hours and cell viability and cell function were assessed (MTT,
tubulogenesis, migration and growth factors secretion). Cell magnetization was
verified by SQUID magnetometry and transmission electron microscopy (TEM).
A Magnetic Resonance (MR) study was conducted to determine the relaxometric
properties of SPIONs and magnetized EPCs. In vivo, magnetized EPCs were
guided to specific cortical areas with an magnet device after intravenous administration and brain angiogenesis was measured in a mouse model of cerebral
ischemia. EPCs were successfully magnetized by SPIONs without affecting cell
viability. TEM showed SPIONs stored in cytoplasmatic endosomes/lysosomes
and EPCs were tracked in T2 weighted images by MRI. Magnetized EPCs were
fully functional and the secretion of important growth factors was enhanced
compared to non-magnetized EPCs. In this regard a proteome array showed a fold
change >2 in magnetized vs. control cells for FGF, PDGF-BB, PD-ECGF or
IGFBP-3, among others. MR images showed that brain tissue under the influence
of magnetic forces accumulated hypointense signals consistent with magnetized
EPCs engraftment after intravenous administration. Finally, in a mouse model of
ischemia magnetized EPCs were intravenously administered and vessel density was
found increased in specific cortical areas of animals with implanted magnetic devices. We show that EPCs magnetization with SPIONs might be a powerful tool
for precise cell guidance and to potentiate their angiogenic properties through
growth factors’ secretion in the context of ischemia.
217
NEURONAL
PROGRAMMING
OF
BONE
MARROW
MESENCHYMAL STEM CELLS
E Andr
e1,2,3, P Resnier1,2, L Sindji1,2, AP Lopez3, P Schiller4, C Passirani1,2,
B Seijo3, A Sanchez3, C Montero-Menei1,2
1
Micro et Nanomedecines Biomimetiques, inserm U1066, Angers, maine et
loire, France, 2PRES LUNAM , University of Angers, Angers, maine et loire,
France, 3Pharmacy and Pharmaceutical Technology, University of Pharmacy,
santiago de compostela, La coruna, Spain, 4Biochemistry & Molecular Biology,
Miami Miller School of Medicine, Miami, Florida, United States
Hungtington’s disease (HD) is an autosomal dominant genetic disease, associated
with the progressive loss of the GABAergic neurons in the striatum. Among the
strategies to cure this disease, is cellular transplantation to restore lost cells.
Clinical studies with foetal GABAergic precursors have shown promising results,
but problems with availability and ethical issues limit their use. Mesenchymal
stem cells (MSC) are an interesting source of cells for brain regenerative medicine
and more particularly the “Marrow-Isolated Adult Multilineage Inducible”
(MIAMI) cells a homogeneous subpopulation, which express several pluripotency markers (Oct4, Sox2, Nanog), may differentiate toward a neuronal
phenotype and secrete tissue repair factors. In order to enhance their neuronal
differentiation, we chose to use RNA interference (siRNA) against the neuronal
inhibitory factor REST to commit them toward a neural/neuronal phenotype(3).
Non-viral nanoparticle-based vectors, which have an efficient transfection level,
associated to low toxicity, have been developed for this neuronal programming
purpose. Two different novel systems have been formulated for neuronal programming: lipid nanocapsules (LNC) and nanoparticles based on sorbitan esters
(Span). Adapting the method by Heurtault et al, we optimized basic lipid nanocapsules (LNC) associating siRNA (size: 95nm and z potential : +10mV). On the
other hand, we developed nanoparticles based on sorbitan esters (Span) and
pullulan associating siRNA (size: 200 nm, z potential: - 43 mV). Both systems
provide an efficient siRNA association (around 50%), showing appropriate stability during storage and a good efficiency of transfection. Indeed, forty-eight
hours after the transfection, the siRNA seem to induce neuronal differentiation.
Therefore we can conclude that LNC and Span nanosystems seem to be two
promising systems with appropriate characteristics for siRNA transfection and
cell therapy.
S64
Poster Abstracts
218
ENDOVASCULAR RETINAL INFUSION OF BONE MARROW
HEMATOPOIETIC STEM CELLS FOR PIGMENTOUS RETINITIS
J Arturo1, C Perez1, O Segura2, OS Guerrero2, Y Bastidas1, L Larios1
1
Molecular and Regenerative Medicine, Inmugen Corporation, Bogota,
Colombia, 2Haemodinamia, Diagnosticos e Imagenes, Bogota, Colombia
There is more than 75 eye and constitutional disorders that may be associated with Pigmentous Retinitis (PR), a disease characterized by progressive
retinal degeneration and photoreceptors apoptosis , which affects 1 in 3,500
people. Symptoms include glare and photopsia, altered color perception,
night blindness, tunnel vision , and progressive reduction of central vision.
With an hereditary origin, in most cases may be involved a large number of
genes and patterns ( Autosomal dominant , Autosomal recessive and sexlinked ). Today the current treatments are palliative with oral vitamins and
macular vasodilators whereas gene therapies and surgical procedures are
under evaluation without significant results. Considering the clinical history ,
retinography and visual field diagnostic of PR , previous ethical comitee
aprobation, we performed a treatment in three patients , with implantation of
autologous bone marrow autologous hematopoietic stem cells by catheterism
through guide catheter platform No .6.0 F and guide endovascular navigation
system under maping road No. 1.5 F , with supra selective ophthalmic artery
infusion to to ensure the arrival of the cells to the retina During the first
month of monitoring improvement in symptoms of phosphenes, photopsia,
night blindness and color vision were persistently improved. Within 3
months after increase bilateral visual acuity, allowing patients to maintain a
capacity for self-care and perform activities of daily living so far (48 months
later). The amount of retinal lesions in comparative analysis was reduced in
the 3 patients. The procedure does not produce any complications. We
conclude after performing evaluation and follow treatment with autologous
bone marrow hematopoietic stem cells is a safe therapeutic option in patients
with pigmentous retinitis, with improve in life quality , which must be
evaluated in future clinical trials.
219
ANALYSES OF CD90 ROLE IN THE GROWTH, OSTEOGENIC
DIFFERENTIATION AND MORPHOLOGY, IMMUNOGENIC
PROPERTY OF HUMAN MESENCHYMAL STEM CELLS (MSC)
D Moraes1,2, O Toledo2, L Gamarra4, F Araújo3, T Sibov4, L Marti4,
R Azevedo1, D Oliveira1
1
Genetics and Morphology, University of Brasilia, Brasília, Distrito Federal,
Brazil, 2Ciencias da saúde, University of Brasília, Brasilia, Distrito Federal,
Brazil, 3Farmacia, University of Brasília, Brasilia, Distrito Federal,
Brazil, 4Instituto do Cerebro, Instituto Israelita Albert Eisntein, São Paulo, São
Paulo, Brazil
MSCs are isolated from several human tissues and expanded in vitro. These
cells promptly differentiate into multiple connective tissue lineages, such as
osteoblasts, chondrocytes and adipocytes. Studies showed that human MSC
have unique immunological properties: they are not immunogenic, they do
not simulate alloreactivity, they scape from T citotoxic and NK cells lysis
activity. The imunophenotypic characterization of MSC is positive for
expression of cell markers CD90, CD105, CD73, CD117, CD44, CD166,
CD29 and STRO-1. This work has as goal the analysis of CD90 glycoprotein
role in the morphology, growth, differentiation of dental pulp MSCs, and
immunological response of CD90 through the reduction of CD90 expression
using RNA interference mechanism. Lentivirus particles were used to create
stable MSCs clones expressing shRNA against CD90 and a scramble shRNA
control. Clones were isolated via puromycin selection. The reduction of
CD90 expression, confirmed in flow cytometry assays, was analysed using
FLOJO software. Morphology was analysed using phase contrast optical
microscopy and flow citometry. For proliferative rate analyses the number of
adherent cells was determined by hemocytometer. For differentiation analyses, cells were induced for osteogenic differentiation and their calcium
deposits were stained with Alisarin red. We investigated calcium concentration through Espectometry. We found that a significant decrease in CD90
levels does not affect MSCs immunogenic properties, proliferation, differentiation and morphology. The partial reduction of CD90 expression leads:
to a decrease in CD166 and CD44 expression and to an increase in calcium
concentration and mineralization when compared to our control cells. This
work supported by CNPQ.
220
A NOVEL ANIMAL COMPONENT-FREE CULTURE MEDIUM
FOR EFFICIENT DERIVATION AND EXPANSION OF HUMAN
MESENCHYMAL CELLS
R Wagey1, J Yau1, E Hadley1, M Wong1, C Duronio1, A Sampaio1, C Miller1,
T Thomas1, A Eaves1,2, SA Louis1
1
STEMCELL Technologies Inc., Vancouver, British Columbia,
Canada,, 2Terry Fox Laboratory, BC Cancer Agency, Vancouver, British
Columbia, Canada
MesenCultÔ-ACF, a novel animal component-free (ACF) culture medium, was
used to derive and expand mesenchymal progenitor cells (MPCs) from primary
human bone marrow mononuclear cells (BMMC) and adipose tissues (AD). MPCs
were isolated from primary BMMC by plating 10,000 - 50,000 cells/cm2 in MesenCultÔ-ACF or in a serum-containing control medium. Clonogenic growth was
evaluated using the Colony-Forming Unit-Fibroblast (CFU-F) assay. To evaluate
MPC expansion from primary BMMC, cells were plated at 3 e 5 x 104 cells/cm2 in
MesenCultÔ-ACF or 5 x 104 e 1.0 x 105 cells/cm2 in control medium. For subsequent subcultures, cells were plated at 1500 e 3000 cells/cm2. Expansion cultures
of MPCs from AD were initiated with 500 - 1250 cells/cm2 in either MesenCultÔACF or control medium. The proliferative potential of MPCs from either cell
source in each medium was determined by counting cell number at each passage (P)
up to P8 and paired t-test was used for statistical analysis. Total CFU-F derived per 1
x 106 BMMC was significantly higher in MesenCultÔ-ACF than in control medium (51 8 versus 29 5; mean SEM; n¼6; p<0.05). Average fold-expansion at
each subculture of MPCs from BMMC from P1 to P8 (31-46 days) in MesenCultÔ-ACF was significantly higher than in control medium (6.6 1.0 versus 4.0 0.8; mean SEM; n¼6; p<0.05). Similarly, the average fold-expansion of ADderived MPCs from P1 to P8 (25 to 39 days) was also significantly higher in
MesenCultÔ-ACF than in control medium (10.6 1.2 versus 2.1 0.2; mean SEM; n¼3; p<0.01). Cells cultured in MesenCultÔ-ACF differentiated robustly
under the appropriate differentiation cultures into adipocytes, osteogenic cells and
chondrocytes, as visualized by Oil Red O, Von Kossa, and Alcian Blue staining,
respectively. In summary, MesenCultÔ-ACF efficiently derives and expands MPCs
directly from primary tissues under animal component-free culture conditions.
221
PHENOTYPIC EXPRESSIONS OF REPROGRAMMED OSTEOSARCOMA CELL LINES
P Choong1,2, H Teh2, H Teoh1, H Ong2, K Choo2, S Cheong1,2, T Kamarul3
1
Stem Cell Transplantation, PPUKM-MAKNA Cancer Centre, Universiti
Kebangsaan Malaysia Medical Centre, Malaysia, Kuala Lumpur, Kuala
Lumpur, Malaysia, 2Faculty of Medicine and Health Sciences, University
Tunku Abdul Rahman (UTAR), Selangor, Selangor, Malaysia, 3Tissue
Engineering Group, National Orthopaedic Centre of Excellence for Research
and Learning, Department of Orthopaedic Surgery, Faculty of Medicine,
University of Malaya, Kuala Lumpur, Kuala Lumpur, Malaysia
Reprogramming of cancer cells have brought us closer to generate patient-specific
induced pluripotent stem cells (iPSC) with cancer properties for understanding of
cancer biology and drug screening for patient-specific cancer therapy. We used
Yamanaka factors, OCT4, SOX2, KLF4 and c-MYC, to transduce all osteosarcoma cells. Transduced cells were transferred to inactivated mouse embryonic
fibroblast (iMEF) on Day 3 post transduction. Colonies were manually picked on
Day 15 - Day 20 and transferred to new iMEF. Reprogrammed sarcomas were
characterised by observation on morphology, alkaline phosphatase and pluripotency markers expression, embryoid body formation and directed differentiation
into adipocytes and osteocytes. All four osteosarcoma cell lines, Saos-2, MG-63,
G-292 and U-2 OS, were reprogrammed to pluripotency. Embryonic stem cell
(ESC)-like clusters started to appear between 15 to 20 days post transduction for
all four cell lines. Morphology of the colonies resembles ESC colonies with
defined border and tightly packed cells. We then characterised our reprogrammed
sarcomas and found all the reprogrammed sarcomas expressed alkaline phosphatase and pluripotency markers, OCT4, SSEA4, TRA-1-60 and TRA-1-81,
similar to ESC. In our observation, all reprogrammed sarcomas could form
embryoid body-like spheres when cultured in suspension condition in a low
attachment dish for up to 10 days. We further test the differentiation capacity of
our reprogrammed sarcomas by performing directed differentiation into adipocytes and osteocytes. Our directed differentiation results showed that all four
reprogrammed sarcoma could differentiate into adipocytes as shown with Oil Red
O staining. While, only reprogrammed Saos-2-REP, MG-63-REP and G-292REP could differentiate into osteocytes as shown by Alizarin Red staining. These
results support the ability of cancer cells to be reprogrammed. However, further
works need to be done to fully characterise the reprogrammed sarcomas.
222
DUX4 EXPRESSION DURING OSTEOGENIC DIFFERENTIATION
IN MESENCHYMAL STROMAL CELLS (MSCS)
L de la Kethulle de Ryhove1, E Ansseau1, M Geens3, F Coppee1,
KD Sermon3, L Lagneaux2, A Belayew1
1
Laboratory of Molecular Biology, University of Mons, Mons,
Belgium, 2Laboratory of Clinical Cell Therapy, ULB, Brussels,
Belgium, 3Department of Embryology and Genetics, VUB, Brussels, Belgium
Our group has identified the Double Homeobox 4 (DUX4) gene within
repeated DNA elements in the 4q35 chromosome region linked to the FSHD
S65
muscular dystrophy. In healthy individuals Dr. S. Tapscott’s group has detected
a full length DUX4 mRNA (fl-DUX4) in induced pluripotent stem (iPS) cells
and human testis, and a longer mRNA where the gene contains 4 additional
exons and a more distal polyadenylation signal than in FSHD muscles. Our
preliminary data suggested DUX4 was expressed at a very low level in MSC
isolated from Bone Marrow (BM-MSC) and more abundant in Wharton jelly
(Wj-MSC). We wanted to evaluate whether DUX4 expression changed during
BM-MSC differentiation. We added an osteogenic differentiation medium to
BM-MSCs cultures, collected cells after 0, 7, 14 and 21 days and performed an
immunodetection on western blot. To confirm the differentiation process we
stained calcium deposits in the cell culture dishes with alizarin red. We
observed an increase of DUX4 expression after 14 and 21 days. We then
investigated whether DUX4 was involved in the differentiation process. We
transfected MSCs with antisense oligonucleotides (2’O Methyl phosphorothiate, DUX4-AO) targeting the DUX4 mRNA and previously shown to
interfere with the protein expression3. The cells transfected with a DUX4-AO
presented weaker alizarin red staining after switch to differentiation medium
(Fig.1). In conclusion we show that DUX4 expression is increased upon MSC
differentiation to osteoblasts. This observation is in contrast with the data
published about iPS cells differentiation to embryoid bodies in which DUX4-fl
expression disappeared. However Dr. M. Kyba’s group has recently shown
DUX4 implication in neurogenesis. They transfected murine Embryonic stem
cells with a DUX4 inducible vector and observed after DUX4 induction that
differentiated cells expressed neuronal expression markers. We hypothesize
that DUX4 could generally be implicated in the mechanism of early
differentiation.
223
MYELOID DERIVED SUPPRESSOR CELLS ARE EXPANDED IN
PATIENTS WITH MULTIPLE MYELOMA, INDUCE TREG CELLS
AND DELAY T-CELL RECOVERY POST TRANSPLANTATION
K Zarkos, J Favaloro, T Liyadipitiya, R Brown, S Yang, H Suen,
C Weatherburn, J Gibson, P Ho, D Joshua
rpa hospital, institute of haematology, Sydney, New South Wales, Australia
Background: Myeloid derived suppressor cells (MDSC) are a heterogeneous population of cells expressing immature myeloid markers and have
been implicated as inhibitors of lymphopoiesis. We determined the number
of MDSC in patients with multiple myeloma (MM), the impact of G-CSF
on MDSC prior to stem cell collection, the ability of MDSC to induce
Treg cells and the impact of MDSC on lymphocyte regeneration posttransplant.
Methods: MDSC were detected by flow cytometry as CD11b+CD33+HLADR lo/-. Treg cells were identified as CD4+CD25+CD127 low/neg. pSTAT3
was determined by phosphoflow.
Results:
Granulocytic
MDSC
(G-MDSC:
CD14-CD15+,
CD33+CD11b+HLA-DRlo/-) were significantly higher in the blood of patients with MM (n¼25; mean: 9.2%) compared to age matched controls (n¼11;
mean: 2.1%) (U¼78.5; p¼ 0.04) and greater still in patients with active disease
(n¼9; mean: 50.1%) (U¼0.0; p¼0.0002). Flow-sorted MM MDSC (n¼7) cocultured 1:1 with autologous mononuclear cells induced a greater proportion of
Treg cells (p¼ 0.05) than MDSC from age matched controls (n¼4). G-MDSC
were significantly increased in the blood of patients undergoing autologous
transplant following G-CSF administration with a pre G-CSF mean¼3.8% and
post G-CSF mean¼16.4%). The proportion of G-MDSC in the PBSC (mean
¼ 18%) was significantly higher than in matched blood samples (t¼3.24; p¼
0.018). Although the number of G-MDSC infused had no apparent influence
on lymphocyte recovery, there was a correlation (R2¼0.59; p<0.01) between
pSTAT3 expression in G-MDSC in the PBSC collection reinfused and the
lymphocyte count rising above 0.0x109/L post-transplant suggesting it was the
activity of G-MDSC that delayed lymphocyte recovery.
Conclusion: MDSC are increased in the blood of patients with MM and
increase after G-CSF stem cell mobilisation. Their activation status as
demonstrated by pSTAT3 expression suggests that they contribute to the inhibition of lymphocyte regeneration post-transplant.
224
ISOLATION OF WHARTON’S JELLY MESENCHYMAL STEM
CELLS AND THEIR DIFFERENTIATION TO INSULIN
PRODUCING CELLS
DH Kassem1, MM Kamal1, HO El-Mesallamy1, A El-Kholy2
S66
Poster Abstracts
1
Biochemistry Department, Faculty of Pharmacy, Ain Shams University,
Cairo, Egypt, 2Obstetrics and Gynecology Department , Faculty of Medicine,
Ain Shams University, Cairo, Egypt
Introduction: Now, cell therapy for Diabetes Mellitus (DM) is under extensive
study. Recently, there has been much attention towards generation of insulin producing cells (IPCs) from stem cells. Mesenchymal stem cells isolated from umbilical
cord wharton’s jelly (WJ-MSCs) offer several advantages over other stem cells.
Objectives: We sought to investigate the outcome of differentiation of WJMSCs to IPCs using several extrinsic factors, since there is no standard method
for induction till now.
Materials and methods: WJ-MSCs were isolated and expanded for several
passages. Expression of surface markers and differentiation towards MSCs
lineages were used to verify MSCs identity. Afterwards, WJ-MSCs were
induced to differentiate into IPCs using several protocols, involving various
extrinsic
factors
and
induction
periods;
namely
nicotinamide,
bemercaptoethanol and exendin-4. Differentiated IPCs were assessed by
determining the expression of key markers of b-cells such as Pdx-1, Isl-1,
Nkx2.2, together with both MafA and MafB using qRT-PCR, and functionally
by measuring insulin secretion after glucose challenge; a hall mark of functional
b-cells.
Results and conclusions: WJ-MSCs differentiated successfully to functioning IPCs. Interestingly, nicotinamide together with exendin-4 had a synergistic effect during the induction process, resulting in higher expression levels
of b-cells markers, as compared to the use of each of them alone. Furthermore,
the levels of both MafA and MafB elevated during differentiation stages to
IPCs, which highlights the possible role MafB could be playing during development and function of human b-cells. Unexpectedly, the levels of Oct-4 were
found to be elevated in obtained IPCs, despite the significant decrease of
Nestin. In conclusion, WJ-MSCs represent a potential source for cell therapy of
DM. Yet, further research is warranted to understand differentiation mechanisms in order to improve maturation and therapeutic outcome of these cells.
225
COMPARING UMBILICAL CORD BLOOD STEM CELLS AND
WHARTON’S
JELLY
MESENCHYMAL
STEM
CELLS
REGARDING THEIR DIFFERENTIATION POTENTIAL TO
INSULIN PRODUCING CELLS
MM Kamal1, HO El-Mesallamy1, LN Hammad2, RF El-Demerdash2
1
Biochemistry Department, Faculty of Pharmacy, Ain Shams University,
Cairo, Egypt, 2Pharmacology and Toxicology Department, Misr International
University, Cairo, Egypt
Introduction: The number of patients suffering from Diabetes Mellitus (DM)
is growing in an alarming rate which makes DM the most prevalent and serious
metabolic disease. Now, cell therapy treatment options for diabetic patients are
under extensive study. Interestingly, umbilical cord (UC) has been proved to be
a good source of mesenchymal stem cells (MSCs), namely from umbilical cord
blood (UCB-MSCs) and Wharton’s jelly (WJ-MSCs).
Objectives: We thought to investigate the difference between these 2
important banking sources of stem cells and to compare their differentiation
potentials towards insulin producing cells (IPCs) in vitro and their potential use
for treatment of streptozotocin (STZ) induced diabetic rats invivo.
Materials and methods: Both UCB-MSCs and WJ-MSCs were isolated
from UC and expanded for several passages. Expression of typical MSCs surface antigens and adipogenic differentiation potential as an example of
mesenchymal lineage was used to verify MSCs identity. Afterwards, both
UCB-MSCs and WJ-MSCs were induced to differentiate into IPCs, then the
differentiated cells were assessed both genetically by determining the expression of Nestin, as stem cell marker and key markers of mature b-cells such as
Pdx-1, Mafa and Nkx2.2 using qRT-PCR, and functionally by measuring insulin secretion after glucose challenge (Glucose stimulated insulin secretion;
GSIS); a hall mark of functional b-cells.
Results and conclusions: WJ appeared to be a much more homogenous
and potential source for MSCs as compared to UCB. Interestingly, both UCBstem cells and WJ-MSCs were successfully differentiated to IPCs. Yet, the
resulting IPCs from WJ-MSCs were to a limited extent functioning better than
those obtained from UCB-MSCs. Both cell types were able to decrease fasting
blood glucose level transiently in STZ induced diabetic mice. Taken together,
we can conclude that WJ could represent a potential source of cells in the field
of DM cell therapy rather than UCB.
226
QUEST FOR CIRCULATING MESENCHYMAL STEM CELLS IN MAN
MJ Hoogduijn1, AU Engela1, MM Verstegen2, SS Korevaar1,
M Roemeling-van Rhijn1, M Franquesa1, J de Jonge2, JN IJzermans2,
W Weimar1, M Betjes1, LJ van der Laan2, CC Baan1
1
Internal Medicine, Erasmus Medical Center, Rotterdam,
Netherlands, 2Surgery, Erasmus Medical Center, Rotterdam, Netherlands
Mesenchymal stem cells (MSC) are present in the bone marrow, from where they are
thought to migrate via the bloodstream to sites of injury. On the other hand, virtually
all tissues contain resident MSC that may contribute to local regenerative and
immunomodulatory processes, thereby hypothetically pre-empting the need for
recruitment of MSC via the bloodstream. Although there is some indication from
animal models, the question remains whether there is solid evidence for the mobilization and migration of MSC in human. In the present study we investigated
whether circulating MSC were present in the peripheral blood of healthy individuals
and patients with organ injury. We were unable to detect MSC in the blood of
healthy individuals by flow cytometry and cell culture techniques. We then analyzed
the presence of MSC in the blood of patients with end-stage renal disease (n¼10),
end-stage liver disease (n¼10) and in heart transplant patients with biopsy proven
rejection (n¼8), by culturing of mononuclear cells under MSC-supporting culture
conditions. In none of these patients MSC were identified in the blood. In the
stromal vascular fraction of adipose tissue and in liver transplant perfusion fluid we
were able to detect MSC, indicating that the methods used enabled the detection of
MSC. The conclusion of this study is that MSC are not detectable in the circulation
in patients with injured solid organs and during aggressive immune responses.
227
HUMAN MESENCHYMAL STEM CELLS SELECTIVELLY
RECOGNIZE AND ADHERE TO APOPTOTIC ENDOTHELIAL
CELLS
S Doronin1, IA Potapova2, IS Cohen3
1
Physiology and Biophysics, Stony Brook University, Stony Brook, New York,
United States, 2Physiology and Biophysics, Stony Brook University, Stony
Brook, New York, United States, 3Physiology and Biophysics, Stony Brook
University, Stony Brook, New York, United States
The majority of clinical applications of mesenchymal stem cells rely on their ability
to home to sites of injury. However, little is known about the mechanism of
mesenchymal stem cell homing to injured tissues. We investigated the adhesion of
human mesenchymal stem cells to apoptotic endothelial cells in vitro. Our results
show that the development of apoptosis in endothelial cells stimulates endothelial
cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells
to apoptotic endothelial cells depends on the activation of caspases and p38 MAPK
in endothelial cells. Inhibition of p38 MAPK in endothelial cells completely
abolishes the stimulation of the mesenchymal stem cell adhesion. The inhibition of
caspases in endothelial cells partially inhibits the stimulation of the mesenchymal
stem cell adhesion. We hypothesize that the activation of caspases potentiates p38
MAPK-dependent adhesion of human mesenchymal stem cells to apoptotic
endothelial cells. Overall, our study demonstrates that human mesenchymal stem
cells selectively recognize and adhere to distressed/apoptotic endothelial cells.
228
ENHANCED IDO ACTIVITY AND REDUCED IL8 PRODUCTION
OF
HBM-MSC
IN
INFLAMMATORY
CONDITION
BY
PRETREATMENT
WITH
CYTOKINES
INCLUDING
INTERFERON-GAMMA
J Jang1, S Suh2, J Kim2
1
School of Medicine, The Catholic University of Korea, Seocho-Gu, Seoul,
Korea, Republic of, 2Catholic Institute of Cell Therapy, Catholic Medical
Center, Seocho-Gu, Seoul, Korea, Republic of
Mesenchymal stem cells (MSCs) have recently been studied and used in many
fields of regenerative medicine including regeneration of body tissues such as
bone, cartilage and heart, enhancement of engrafting efficiency of hematopoietic stem cells, amelioration of allograft rejection, and alleviation of inflammatory responses in autoimmune diseases including rheumatoid arthritis.
Although MSCs have been reported to produce anti-inflammatory paracrine
factors in vitro, results from in vivo researches were not always successful.
Some research reported that MSCs, in some situations, may produce pro-inflammatory cytokines and sometimes deteriorate the inflammatory symptoms.
In this study, we defined an in vitro inflammatory condition consisting of
interleukin-beta and tissue necrosis factor-alpha and showed that naive MSCs
in the inflammatory condition, could be turned into pro-inflammatory cells.
We investigated pre-treatment conditions to improve MSCs’ anti-inflammatory properties and we found the use of cytokines including INF-gamma could
enhance indoleamine 2,3-dioxygenase(IDO) activity and relieve pro-inflammatory changes when encountered the in vitro inflammatory condition. We
believe that this result could contribute in the development of cell therapeutics
using mesenchymal stem cells with enhanced anti-inflammatory properties.
229
MODULATORY EFFECT ON B CELL FUNCTIONS OF
MESENCHYMAL STROMAL CELLS
E Amati1,2, G Bassi1, M Di Trapani1, F Liotta3, F Annunziato3, O Perbellini1,
M Ricciardi1, G Pizzolo1, M Scupoli2, M Krampera1
1
Laboratory, University of Verona, Verona, Veneto, Italy, 2Interdepartmental
Laboratory for Medical Research (LURM), University of Verona, Verona,
Veneto, Italy, 3Department of Internal Medicine and DENOTHE Center,
University of Florence, Florence, Toscana, Italy
Human bone marrow Mesenchymal Stromal Cells (MSC) are potent modulators
of T cell activation and proliferation, mainly through the production of partially
defined soluble factors, including the IFN-g-induced tryptophan-degrading
enzyme IDO, a key immunosuppressive effector pathway. Actually, MSC may
affect the functions of virtually all immune effector cells, including B cells. However, current literature concerning MSC immunomodulatory activity on B cells is
still controversial, due to both biological peculiarities of B cells, which do not
produce IFN-g, a key MSC-triggering cytokine, and to different and poorly
comparable experimental approaches. Human purified B cells, either resting or
activated for 4 days with a specific stimulation cocktail were co-cultured with MSC,
either at resting conditions or following inflammatory priming (MSC pre-incubation with IFN-g + TNF-a for 48 hours), or with MSC supernatants. CD27positive (memory) and CD27-negative (naïve) B cell survival, proliferation, and
intracellular activation status (through signaling network analysis by Phosphoflow)
were assessed. Our results showed that MSC are normally supportive cells, not
intrinsically capable of suppressing B cell proliferation, and require inflammatory
priming to acquire B cell inhibitory potential. Inflammatory-primed MSC impair
significantly activated B cell growth in a cell contact-independent manner. B cell
inhibition by MSC is not related to either induction of B cell apoptosis or early
signaling events necessary for B cell activation. In addition, IDO pathway triggered
in IFN-g-primed MSC seems to have a role also in B cell inhibition. Overall, B cell
behavior following the interaction with MSC depends on the functional state of
both B cells and MSC. The role of IDO in B cell regulation needs further investigation, as it may be relevant to develop new therapeutic approaches in pathological conditions related to B cell hyper-activation.
230
IMPORTANT ROLE OF THE IMMUNE ENVIRONMENT ON BMMSC
AND
ADSC
FUNCTION:
MODULATION
OF
IMMUNOSUPPRESSIVE
CAPACITIES
AND
SECRETORY
PROFILES OF MSC BY MACROPHAGES
N Espagnolle1,2, A Balguerie1,2, L Sensebe1,2, A Varin1,2
1
STROMALab, UMR CNRS 5273, U1031 Inserm, EFS-PM, Univ. P.
Sabatier, Toulouse, France, 2Etablissement Français du Sang Pyrenees
Mediterranee, Toulouse, France
During the last decade, adult mesenchymal stem cells (MSC) derived from
bone marrow (BM-MSC) or from adipose tissue (ASC) emerged as a new
source of cells for cell therapy and damaged tissue regeneration, based on their
multipotency and their capacity to differentiate on functional cell-type.
However, the demonstration of their immunomodulatory properties put forward that MSC could be used as treatment for new applications such as
autoimmune diseases and inflammatory pathologies. Studies demonstrated that
MSC modulated innate immune response but few studies were interested in the
role of immune environment on MSC properties. To determine the effect of
this environment and more precisely the role of innate immune cells on MSC
function, we co-cultivated BM-MSC and ASC with pro-inflammatory macrophages (M1-MF) or anti-inflammatory macrophages (M2-MF) for 24h.
After magnetic separation of the two cell types, we measured the capacity of
immunosuppression on T lymphocytes’ proliferation of the primed MSC. We
S67
demonstrated that M1-primed BM-MSC were more immunosuppressive than
non-primed or M2-primed BM-MSC. In contrast, M1-primed ASC stimulated
T lymphocyte proliferation compare to non primed or M2-primed ASC. In
both cases, the effect on the lymphocyte proliferation is dependent on the
MSC-MF contact and is not correlated with a different IDO expression.
Moreover, we demonstrated that contact with M1-MF modify the secretory
profile of MSC. For instance, M1-primed BM-MSC expressed more pro-inflammatory cytokines such as IL-6 and IL-8 as well as chemokine CCL2 and
Cox2 RNA. Interestingly, like M1-primed MSC, M1-primed ASC produced
more IL-6 but not IL-8. All together, our data establish that MSC function can
be modulated by macrophages present in the microenvironment and that the
response to this environment depends on the origin of MSC. Therefore, these
parameters have to be considered for future MSC-based therapies.
231
ADIPOSE-DERIVED MESENCHYMAL STEM CELLS EXPANDED
IN A COMPLETELY CLOSED HYPOXIC SYSTEM SHOW
REDUCED MARKS OF CELLULAR STRESS AND SENESCENCE
M Preti1,2, M Renoud1,2, S Dupuis-Coronas1,2, M Gadelorge1,2,
J Descamps1,2, L Sensebe1,2
1
STROMAlab, UMR CNRS 5273, U1031 Inserm, EFS-PM, Univ. P. Sabatier,
Toulouse, France, 2Etablissement Français du Sang Pyrenees-Mediterranee,
Toulouse, France
Due to their multipotency and immunosuppressive properties Mesenchymal
Stem/Stromal Cells (MSCs) are important tools for treatment of immune
disorders and tissue repair. Harvesting adipose tissue (AT) is easier than bone
marrow, moreover there are a higher MSCs’content in AT. On contrary of
classical culture conditions, in vivo whatever the tissues, MSCs are located in a
hypoxic environment. High oxygen level during cell culture may induce
oxidative stress, which may have an important role in cellular senescence.
Although previous works showed that culture in hypoxic conditions promotes
the maintenance of MSCs in an immature state, consequences on genetic
instability are controversial. For evaluating safety of clinically produced MSC
from AT (ASC) we compared genetic stability of three clinical grade productions of human ASCs, expanded in normoxia (20% O2) or hypoxia (1%
O2). All hypoxic culture steps were performed in a completely closed system.
Several parameters were tested, among which the expression of cell cycle
regulators (p53, p21, MDM2, p16, MYC, RB), p53 gene sequence, telomere
length and the presence of telomere elongation systems. Our results show that
hypoxia reduces expression of p53 and its target genes p21 and MDM2, and
expression of p16 in latest passages, indicating reduction of cellular stress and
senescence. Contrary to previous works, hypoxia doesn’t inhibit telomere
erosion, another marker of cellular senescence. Hypoxia doesn’t seem to promote ASC transformation: MYC expression decreases with passages, RB
expression is stable, no p53 gene mutations appear during cell culture, telomerase is inactive and no signs of alternative lengthening of telomeres are
detected. In conclusion, hypoxic culture conditions reduce stress and senescence of our ASCs, without signs of malignant transformation, even after longterm culture. The authors are supported by ANR RPIB 012 01: SAFE &
NOMASEC Region Midi-Pyrenees n 12050983.
232
HUMAN NATIVE BONE MARROW CD200 POSITIVE MESENCHYMAL STROMAL CELLS EXHIBIT CHARACTERISTICS OF
CELLS FORMING THE HEMATOPOIETIC STEM CELL NICHE
S Dupuis-Coronas, G Fabien, L Sensebe, F Deschaseaux
STROMALab, UMR CNRS 5273, Inserm U1031, EFS-PM, Univ. P.
Sabatier, Toulouse, France
Bone marrow (BM) mesenchymal stem/stromal cells are non-hematopoietic
(CD45-), non-endothelial (CD31-) multipotential cells capable to differentiate into osteoblastes, chondrocytes and adipocytes. In addition, different
subpopulations of MSCs and some of their derivatives (early osteoblastic
lineage cells) were shown to form HSC-niche. This makes a complex picture
of the relationship between MSCs and HSCs. Despite growing data in mice
model, few describe the human counterpart. The BM CD200+ and CD271+
fractions were previously shown to be enriched in native MSCs in human.
Herein, we found heterogeneity in expression of CD200 within CD45-/
CD31-/CD271+ human BM fraction. We thus selected CD200+ and
CD200- cells from CD45-/CD31-/CD271+ BM samples and we analyzed
S68
Poster Abstracts
their transcriptome and their capacities to generate multipotential CFU-f.
Compared to CD271+/CD200- fraction the CD271+/CD200+ population of
cells was highly enriched in CFU-f. By using in situ immuno-fluorescence
staining of BM samples we observed that CD200+ cells were localized at the
endosteal and endocortical positions suggesting an osteoblastic fate. Surprisingly, by analyzing the transcriptomic data we observed that HSC-niche
markers (VCAM1, CXCL12, PTHR1, ACVR1) were significantly increased
in CD200+ fraction compared to CD200- cells. However, we were unable to
discriminate the two types of populations regarding either the MSC (CD73,
CD90, CD44, CD105, CD146) or the mature osteoblastic (SP7, DLX5,
ALPL, iBSP, BGLAP) markers. Alternatively, early osteoblastic differentiation markers (CHD11, SOX9) and some of vascular smooth muscle lineage
markers (Metavinculin, CALD1, CCL2, IGF2, CTGF) were predominantly
expressed by CD200+ fraction. Altogether, these data suggest that CD271+/
CD200+ MSCs form a subpopulation gathering all markers of HSC niche.
Their endosteal position is not correlated with mature osteoblast phenotype
but rather with early differentiation markers of VSM and osteoblaste
lineages.
233
HSA-MIR152 MODULATE OSTEOBLASTIC AND ADIPOCYTIC
DIFFERENTIATION THROUGH INHIBITION OF THE
MITOCHONDRIOGENIC
FACTOR
PEROXISOME
PROLIFERATOR-ACTIVATED
RECEPTOR
GAMMA
COACTIVATOR 1A (PPRGC1A)
A Prel1, E Labat1, C Pontikoglou2, V Trichet3, A Langonne4, J Pagès5,
L Sensebe1, F Deschaseaux1
1
Sabatier, Toulouse, France, 2Department of Hematology, University of Crete
School of Medicine, Heraklion, Crete, Greece, 3Laboratoire de
Physiopathologie de la Resorption Osseuse et therapie des tumeurs osseuses
primitives, INSERM U957 EA3822 e Faculte de Medecine, Nantes,
France, 4Research, EFS-Centre Atlantique, Tours, France, 5INSERM U966 ,
Faculte de Medecine, Universite François Rabelais, Tours, France
MicroRNAs have a key role in post-transcriptional regulation of gene
expression. Few data reported the miRNA expressions and functions during the
conversion of bone marrow mesenchymal stem/stromal cells (MSCs) into fully
differentiated osteoblasts by using physiological osteoinducers. Therefore, we
studied the miRNome at different steps of the osteoblastic differentiation
process after induction by BMP4. MiRNAs were extracted from cells at day 7,
14 and 21 and miRNome was determined by using Agilent Whole Human
Genome Oligo Microarrays. 29 miRNAs were continuously increased
throughout the differentiation. One of them, mir152 was strongly induced and
in silico analyses showed that it could target the 3’UTR of PPRGC1A gene.
PPRGC1A is well known as mitochondriogenic factor and its expression
is enhanced during the differentiation processes. We thus confirmed by
QRT-PCR the increase of miR152 expression after differentiation of MSCs
into normal osteoblasts. We also confirmed that the 3’UTR of PPRGC1A gene
was specifically targeting by mir152. The osteoblastic cell line MG63 and
normal MSCs were transfected with plasmid allowing the overexpression of
mir152. After selection, we assessed the expression of PPARGC1A as well as
some osteoblastic and adipocytic gene markers. All of them were found to be
downregulated in miR152 transfected cells when compared to mock cell controls. Furthermore, the miR152-MSCs were not able to differentiate into adipocytes. Since miR152 decreases PPARGC1A expression, we transduced
MG63 by shRNA targeting specifically PPARGC1A gene. In this model
neither osteoblastic nor adipocytic markers were disturbed. In contrast, in
normal MSCs, the downregulation of PPARGC1A by shRNA prevented the
formation of adipocytes and blunted the osteoblastic differentiation. Therefore,
for the first time we demonstrate that expression of PPARGC1A can be
modulated by miR152. This highlights a new mechanism regulating the differentiation processes of MSCs.
234
BIOENERGETIC PROFILE ANALYSIS DURING MATURATION
OF EMBRYONIC STEM CELLS
M Vlaski1,2, P Brunet de la Grange1,2, Z Dolicanin1, J Chevaleyre1,2,
P Duchez1,2, H Boeuf2, Z Ivanovic1,2
1
Research and Development, EFS/aqli, Bordeaux, France, 2UMR 5164 CNRS,
University Bordeaux Segalen, Bordeaux, France
Upon withdrawal of leukemia inhibitory factor (LIF) self-renewing pluripotent mouse embryonic cells (mESC) undergo reversible commitment, followed by irreversible commitment and heterogeneous cell differentiation or
apoptosis, within three days. In this study we investigated bioenergetics
profile of these different maturation stages. For this purpose mESC were
cultivated in the presence or absence of LIF for 24, 48 and 72h and then
analysed. Mitotracker green staining showed greater mitochondrial mass in
the pluripotent than in the differentiated cells. To determine if this different
mitochondrial content provides bioenergetics differences between this cells
populations, we measured oxygen consumption rate (OCR) as indicator of
principal mitochondrial respiration parameters and extracellular acidification
rate as an indicator of glycolysis using XF24 analyzer. We found that basal
OCR and ECAR declined in the order: pluripotent cells (+LIF) > reversible
committed cells (-LIF 24h)> irreversible committed cells (-LIF 48h) >
differentiated cells (-LIF72h). This was associated with mitochondrial ATP
output, estimated as coupled rate (OCR linked to ATP synthesis), showing
the same downtrend. Likewise, + LIF cells possessed the highest maximal
respiratory capacity indicating their higher resistance to oxidative stress
comparing to maturating stages. In addition, inhibition of mitochondrial
respiration impaired proliferation of +LIF, but not eLIF 72h cells. Also,
suppression of mitochondrial ATP synthesis, provoked compensatory increase of glycolysis in the + LIF cells which is impaired in the course of the
maturation. Bioluminescent measurement of ATP content showed the
highest level in the eLIF 72h cells, implying lessen ATP turnover in the more
differentiated cells. Our results demonstrated that self-renewing pluripotent
mESC have bioenergetics advantage reflecting in the high OXPHOS and
glycolytic activity, which declines throughout their maturation.
235
ENGINEERING
NEW
CELL
EXPANSION
MEDIA
CONTROLLING BOTH IMMATURITY AND OSTEOGENIC
CELL FATE OF ADIPOSE TISSUE-DERIVED AND BONE
MARROW MSC
F Guilloton, V Rabani, L Sensebe, F Deschaseaux
Sabatier, Toulouse, France
Mesenchymal Stem/Stromal Cells from bone marrow (MSCs) or from adipose tissue (ADSCs) are now used in clinical trials including bone reconstruction. To date, all protocols employ MSCs after an in vitro expansion
using standard culture media before to be injected in patients. However,
these conditions induce aging and spontaneous differentiation which can
dampen their clinical use. We demonstrated previously that MSCs cultured
in a semi-synthetic medium (EGM2), displayed a pericyte progenitor
phenotype with a more immature phenotype compared to standard MSCs.
Herein, we addressed the question about the impact of the EGM2 medium
on ADSCs. As found for MSCs, ADSCs from EGM2 were phenotypically
more immature than ADSCs deriving from standard cultures (2% Platelet
Enriched Plasma, PLP). Indeed, expressions of the stemness markers OCT4,
NANOG and SOX2 were upregulated in EGM2-ADSC. We also compared
the gene expression profile of MSCs and ADSCs generated from standard
and EGM2 conditions. In silico analyses revealed a strong effect of media on
cell commitment. Whereas standard MSCs and ADSCs were phenotypically
close with a consistent expression of osteoblastic markers, EGM2-derived
cells expressed some adipogenic genes. Then, we used a strategy of omission
of growth factors constituting the EGM2 medium in order to determine key
factors causing this phenomenon. Removal of FGF2 was sufficient to
enhance osteoblastic markers notably the ALPL expression and mineralization. To optimize the system for a clinical use, we replaced the 2%FBS
(contained in EGM2 medium) by 0.8% PLP. Compared to EGM2-FBS, in
MSCs and ADSCs deriving from EGM2-PLP, the osteoblastic phenotype
was upregulated and the omission of FGF2 reinforced such effect. Thus,
FGF2 seems to have a great function in EGM2 properties. These data
emphasize the in vitro conditions controlling stem/progenitor commitment
that may help us to adapt the cell therapy protocols according the clinical
application.
236
CHARACTERIZATION OF THE IN VITRO IMMUNOMODULATORY PROPERTIES OF MICROVESICLES ISOLATED FROM
MESENCHYMAL STROMAL CELLS
A Conforti, M Scarsella, E Giorda, S Biagini, N Starc, GL Pira, A Proia,
R Carsetti, F Locatelli, M Bernardo
Onco-hematology, Bambino Gesù Children’s Hospital, Rome, Italy
Objective: Mesenchymal stromal cells (MSCs) are multipotent cells that
exert immunomodulatory effects; however, the mechanisms underlying
these effects have not been completely clarified. Aim of this study was to
compare in vitro the immunomodulatory properties of MSCs with those of
microvesicles (MVs) released in supernatants from the same MSCs.
Methods: MSCs were generated from bone marrow of 12 healthy donors
(HDs) and MVs were isolated from their supernatant by serial ultracentrifugation. Both MSCs and MVs were characterized by flow cytometry and cocultured with peripheral blood mononuclear cells (PBMCs) of 12 HDs and
stimulated in vitro with both PHA and CpG to induce T and B cell proliferation, respectively. Growth factors and cytokines were quantified in the supernatants by ELISA.
Results: MVs were identified as 1mm particles positive for CMFDA, CD107
and CD13 (suggesting a mechanism of membrane budding from MSCs). MSCs
inhibited PHA-induced T-cell proliferation up to 80% (SD 8.16; P¼0.0001
as compared with condition PBMCs/PHA) and 60% (SD 20.86; P¼0.0001)
with MSC:PBMC ratios 1:2 and 1:10 respectively, whereas MVs reduced it up
to 30% (SD 39.32; P¼0.01 as compared with condition PBMCs/PHA; MV
dilution of 1:2 in co-culture final volume). MSCs reduced CpG induced B-cell
proliferation up to 70% (SD 2.82; P¼0.002 as compared with condition
PBMCs+CpG) and plasma cell activation up to 50% (SD 10.59; P¼0.001;
MSC:PBMC ratio 1:10), whereas MV-induced inhibition was up to 60%
(SD 9.95; P¼0.02 as compared with condition PBMCs+CpG) and 30%
(SD 13.53; P¼0.02), respectively (same diluting conditions). In both T- and
B-cell cultures, MSC co-colture induced an increase of IL-6, IL-10, TGFb and
a decrease of IL-2 and IFN, whereas in MV co-culture no differences were
revealed in cytokines levels.
Conclusions: Our data indicate a lower in vitro immunomodulatory effect
on T and B cells of MVs, as compared to their cellular counterpart.
S69
that MSCsPTX, while not intrinsically damaged by priming with PTX,
maintain ability to display their in vitro immuneregulatory functions which
might be even increased by PTX.
238
HUMAN METABOLICALLY ACTIVE BROWN ADIPOSE TISSUE
DERIVED STEM CELLS
F Silva, D Holt, V Vargas, D Bull, AN Patel
United States
Background: Brown adipose tissue (BAT) plays a key role in the evolutionarily
conserved mechanisms underlying energy homeostasis in mammals. It is
characterized by fat vacuoles 5-10 microns in diameter and expression of
uncoupling protein 1 (UCP1), central to the regulation of thermogenesis. In
the human newborn, BAT depots are typically grouped around the vasculature
and solid organs. These depots maintain body temperature during cold exposure by warming the blood before its distribution to the periphery. They also
ensure an optimal temperature for biochemical reactions within solid organs.
BAT had been thought to involute throughout childhood and adolescence.
237
HUMAN MESENCHYMAL STROMAL CELLS PRIMED WITH
PACLITAXEL, APART FROM DISPLAYING ANTI-TUMOR
ACTIVITY, MAINTAIN THEIR IMMUNE REGULATORY
FUNCTIONS IN VITRO
N Starc, A Conforti, S Biagini, A Proia, F Locatelli, M Bernardo
MSCs loaded in vitro with Paclitaxel (MSCsPTX) have been shown to acquire
anti-tumor activity against both solid tumors and leukemia cell lines. We
assessed whether, apart from displaying anti-tumor activity, MCSsPTX
maintain immunomodulatory properties in vitro. MSCs were isolated from
bone marrow of 7 healthy donors and primed in vitro at passage 2 for 24
hours with Paclitaxel (2mg/ml). After 24h or 72h-incubation, conditioned
medium (MSCsPTX-CM) and MSCsPTX were collected for subsequent
analysis. PHA-induced allogeneic PBMC proliferation was measured either in
presence or absence of unprimed MSCs (as control) or MSCsPTX-CM or
MSCsPTX collected after 24h- and 72h-culture following PTX priming.
MSCsPTX displayed the typical mesenchymal morphology, proliferative capacity, immune phenotype and differentiation potential. The mean percentage of PBMC proliferation in the presence of MSCs was 12.7% (SD 6.8)
and 35.4% (SD 19.9) at MSCs:PBMCs ratios 1:2 and 1:10, respectively.
MSCsPTX collected after 24h-culture were able to prevent proliferation of
PBMCs with a mean percentage of proliferation of 6% (SD 4.4; P<0.001, as
compared with control MSCs; MSC:PBMCs ratio 1:2) and 15.3% (SD 12.6;
P¼0.05; MSCs:PBMCs ratios 1:10), whereas MSCsPTX collected after 72hculture reduced PBMC proliferation to 8.2% (SD 4.8; P¼0.002 as
compared with control MSCs) and 21.7% (SD 14.3; P¼0.13) with
MSCs:PBMCs ratios 1:2 and 1:10, respectively. While supernatant collected
from unprimed MSCs did not exert an inhibitory effect, MSCsPTX-CM
collected after 24- and 72-hour culture proved to have an inhibitory effect
with a mean percentage of proliferation of 46.1% (SD 4.1; P¼0.059 as
compared with condition PBMCs/PHA) and 36.9% (SD 3.8; P¼0.052 as
compared with condition PBMCs/PHA), respectively. Our results indicate
SEM Human Brown Adipose Stem Cells
Methods: Mediastinal adipose tissue depots were collected and studied in
situ to identify and characterize human brown adipose derived stem cells. We
define their phenotype and metabolic function.
Results: A resident stem cell population within depots of brown adipose tissue
from adult human mediastinum has been identified (age 25-95). Cells from this tissue
exhibit multi-lineage potential with capacities to undergo osteogenesis, chondrogenesis and both brown and white adipogenesis. Directionally differentiated brown
adipocytes exhibit a distinct morphology and gene expression profile, with functional
properties characteristic of brown adipose tissue in vivo. In a small animal model this
population of cells was capable of ameliorating the symptoms of metabolic syndrome
by improving glucose tolerance tests and decreased cholesterol.
Conclusions: Our study defines a new target stem cell population that can
be activated to restore energy homeostasis in vivo for the treatment of obesity
and related metabolic disorders.
239
NOVEL METABOLICALLY ACTIVE HUMAN RENAL MULTIPOTENT CELLS
D Bull, V Vargas, F Silva, AN Patel
Surgery, University of Utah, Salt Lake City, Utah, United States
Background: White and brown adipose tissue depots are viewed as part of the
endocrine system that secrete biologically active compounds with local and/or
systemic mechanism named adipokines. These adipokines help regulate metabolic
S70
Poster Abstracts
homeostasis in humans. Recent reports have demonstrated that renal glomerular
endothelial cells (HRGECs) are able to synthesize adiponectin, an adipokine that
has anti-inflamatory, anti-atherogenic, anti-diabetic and insulin-sensitizing effects.
In this report we sought to demonstrate that adult derived human renal cells were
capable of in vitro metabolic activity (basal oxygen consumption, glycolysis rates,
ATP production, and respiratory capacity) and whether they were capable
of ameliorating the symptoms of metabolic disorders in high-fat diet fed mice.
Methods: Fresh human renal tissue was collected and studied in situ to
identify and characterize a multi-potent renal cell population. We define their
phenotype and metabolic function. Cells were cultured in vitro on biological
scaffolds and transplanted in mice with induced metabolic syndrome.
Results: Our results demonstrate for the first time that human renal cells
have metabolic activity in vitro and decreased glucose levels in mice that had
been fed a high-fat diet compared to controls. These cells have the ability to
differentiate down the osteo, chrondo, and adipogenic pathways.
Conclusions: These findings that non-adipose tissues play a role in metabolic homeostasis warrants further attention and study towards understanding
metabolic disorders and potential treatments.
240
NEURONAL
DIFFERENTIATION
OF
PROGRANULINTRANSDUCED HUMAN BONE MARROW-DERIVED MESENCHYMAL STEM CELLS
KM Tsang1, KS Tsang1, G LU2, HK Ng1
1
Department of Anatomical and Cellular Pathology, The Chinese University
of Hong Kong, Shatin, Hong Kong, 2Department of Surgery, The Chinese
University of Hong Kong, Shatin, Hong Kong
PGRN is expressed ubiquitously, however cellular expressions at different stages of
maturation and the effect on neuronal induction of MSC is not well defined. We
detected a progressive increase in PGRN expression from H1, H9 and H14 ESC,
embryoid bodies to human bone marrow-derived MSC, but a modest decline by
human fibroblast D551. None of 14 tumor cell lines of neuroblastoma, glioblastoma
and leukemia expressed a higher level of PGRN gene than that of MSC. Readouts
suggest that PGRN was actively involved in cell differentiation and up-regulated at
development. We transduced PGRN into MSC by using adenovirus transfection.
PGRN-transduced MSC displayed no morphologic change compared to nontransduced MSC, however a slower growth kinetics was noted. Q-PCR demonstrated down-regulations of mesodermal genes, Brachyury and Gata-4, on PGRNtransduced MSC. Conversely there was no change of gene expressions of Sox-2,
Sox-1, Musashi-1, Pax-6 and Nestin in both PGRN-transduced and non-transduced MSC. PGRN-transduced MSC exhibited a mean of 1.5-folded increase of
the CNTF gene expression among the panel of neurotrophic factors and their
receptors. Upon completion of neuronal induction, the mean number of PGRNtransduced cells was comparable to that of control cultures, and similar numbers of
cells with neurite bifurcation were also noted. Immunofluorescence staining of btubulin III and tyrosine hydroxylase revealed no difference in positive cell numbers
among PGRN-transduced and non-transduced MSC in induction cultures, however protruding processes of derived cells from PGRN-transduced MSC were
noted to be significantly longer. An up-regulation of Pax-6, Musashi-1 and CNTF
gene expressions in neuronal differentiation cultures of PGRN-transduced MSC
was evident implying that forced expression of PGRN enhanced the neuro-ectodermal fate of MSC and paracrine signaling. Data suggest that PGRN-transduced
MSC might be beneficial to the neuro-regenerative therapy.
241
EXPOSURE TO IONIZING RADIATIONS AND STARVATION
CULTURE DOES NOT MODIFY PHENOTYPE, FUNCTIONS
AND GENETIC PROFILE OF MESENCHYMAL STROMAL
CELLS ISOLATED FROM BONE MARROW OF HEALTHY
DONORS
A Conforti1, S Biagini1, N Starc1, A Proia1, G Grisendi3, C Carella2,
M Dominici3, F Locatelli1, M Bernardo1
1
Onco-hematology, Bambino Gesù Children’s Hospital, Rome, Italy, 2Cell
Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy, 3Oncohematology, University Hospital of Modena and Reggio Emilia, Modena, Italy
Bone marrow (BM) exposure to ionizing radiations induces the rapid depletion of
hematopoietic precursors; however, radiation effects on mesenchymal stromal
cells (MSCs) have been poorly investigated. Moreover, little is known about
MSC behavior in starvation culture conditions. We examined morphology,
proliferative capacity, immunophenotype, differentiation potential, immunomodulatory properties (PHA-induced T-cell proliferation assay) and genetic
profile (conventional karyotype and array-CGH) of MSCs isolated from BM of
healthy donors (HDs) and exposed to ionizing radiations and starvation culture.
MSCs were isolated from 10 HDs (median age: 16 years; range: 5-32) and
expanded in culture medium supplemented with 5% platelet lysate (PL) up to
passage 2. Thereafter, MSCs were exposed both to escalating doses of ionizing
radiations (3000, 10000 and 20000 rad) and to starvation culture conditions (1%
PL instead of 5%). With escalating doses of radiations, MSCs lose their typical
spindle-shaped morphology, their boundaries become less regular and their
growth rate decreases (at 3000 rad) or even stops (at 10000 and 20000 rad).
Nonetheless, in the presence of 1% PL, although showing a slower growth rate as
compared with non irradiated MSCs, the effects on morphology are less evident,
thus suggesting that the lack of PL-derived growth factors may interfere with the
senescent process induced by ionizing radiations. Irradiated and starved MSCs
maintain the typical immunophenotype (positivity for CD105, CD90, CD13;
negativity for CD34, CD45, CD14), ability to differentiate into osteoblast and
adipocytes and to inhibit PHA-induced T-cell proliferation. The study of the
genetic profile did not show any chromosomal alteration in stressed MSCs. Our
data indicate that both irradiated and starved MSCs, although presenting altered
morphology and growth rate, maintain the typical characteristics of HD-MSCs
and do not display an increased propensity for malignant transformation.
242
LONG-TERM CULTURE OF HUMAN UMBILICAL CORD
MESENCHYMAL STEM CELLS IN DEFINED SERUM FREE
MEDIA
Z Han1, Y Wang1,2, Y Chi1, S Yan2, A Mao2, H Zhong-Chao1,2
1
Institute of Hematology and Hospital of Blood Diseases, Chinese Academy of
Medical Science & Peking Union Medical College, Tianjin, Tianjin,
China, 2National Engineering Research Center of Cell Products/AmCellGene
Co. Ltd, Tianjin, Tianjin, China
Mesenchymal stem cells (MSCs) are wildly used in clinical researches to treat various
diseases. Classic culture of MSCs, even for clinical using, contained fetal calf serum.
Because of the risk of contamination of infectious pathogens, serum-containing
medium (SCM) are a major obstacle for MSCs-related therapies. Some studies
demonstrated that MSCs could be cultured in serum free medium (SFM). However,
whether SFM would change the biological characteristics and safety issues of MSCs
has not been well answered. Therefore, we evaluated human umbilical cord
mesenchymal stem cells (hUC-MSCs) in a commercial serum-free, chemical defined
media. Both SFM- and SCM-cultured hUC-MSC maintained multipotency and
surface antigen expression which were used to defined human MSCs. SFM-derived
hUC-MSCs showed a clear different gene expression profile. Cell cycle, mitosis and
cell proliferation were down-regulated in SFM-derived hUC-MSCs. We did not
observe oncogenes of hUC-MSCs increased significantly in SFM. The expression of
hTERT could not be detected in SFM-derived hUC-MSCs. We utilized highthroughput aCGH to evaluate the effect of SFM or SCM on the genome stability of
UCMSCs in in vitro culture. We found that UCMSC gain copy number changes in
culture, no matter in SFM or in SCM. hUC-MCSs could be expanded in SFM safely
to obtain enough cells for clinical application. Nevertheless, hUC-MSCs cultured in
SFM were distinct from hUC-MSCs cultured in SCM.
243
ADIPOSE STROMAL CELLS EXPRESS FUNCTIONAL TOLL-LIKE
RECEPTORS
K Bieback, U Kraneburg, S Uhlig, S Elvers-Hornung, H Klueter
Institute of Transfusion Medicine and Immunology, Mannheim, Europe, Germany
Mesenchymal Stromal Cells (MSC) need to be licensed by immune cells or
inflamed tissue to exert their immunomodulatory capacity. Ligation of different
toll-like receptors (TLRs) has been demonstrated to polarize MSC towards an
anti- or a pro-inflammatory phenotype. This, however, has been demonstrated
so far only in MSC derived from human bone marrow, cultivated in fetal
bovine serum (FBS). First the expression of TLRs in adipose-tissue derived
MSC was assessed, comparing the effects of culture in FBS or human serum.
Second, functionality of TLRs was investigated by measuring IL6 and IL8
secretion after stimulation with TLR agonists. It was addressed whether adipokine secretion is induced by TLR ligation and finally whether immunoregulatory properties of ASC are chained. ASC expressed a number of TLRs,
however only TLR3 and 4 appeared to be functional, because only poly I:C and
LPS stimulation resulted in secretion of proinflammatory cytokines IL6 and
IL8. So far we were unable to recapitulate findings of polarization by different
TLR ligands, duration of stimulation or concentration of the agonist. Cultivation in human serum reduced the expression intensity of TLRs as assessed by
flow cytometry and led to a chemotactic cytokine profile. ASCs grown in FBS,
however, represented an inflammatory secretion profile. Secretion of proinflammatory adipokines was increased by TLR3 and TLR4 agonists, but adipose-derived hormones, such as adiponectin and leptin remained unchained.
Immunomodulatory capacities remained unaffected by TLR stimulation.
MSCs home to sites of injury where they will likely encounter TLR agonists.
The cultivation in humanized culture conditions in contrast to FBS may alter
the functional and thus therapeutic properties of ASCs/MSCs and needs to be
studied in detail to assess the impact of changing culture conditions.
244
CORD BLOOD DERIVED ENDOTHELIAL COLONY FORMING
CELLS EMERGE FROM A CD45DIM CD31D CIRCULATING
PRECURSOR
K Bieback1,2, S Elvers-Hornung1, S Uhlig1,2, H Klueter1
1
Institute of Transfusion Medicine and Immunology, Mannheim, Europe,
Germany, 2FlowCore Mannheim, Medical Faculty Mannheim, Heidelberg
University, Mannheim, Germany
Endothelial progenitor cells (EPC) serve as biomarkers or resource for cellbased therapies. However, a variety of different cell types are subsumed under
the term EPC. Culture adapted EPC have now been classified as colony
forming unit endothelial cells (CFU-EC), early outgrowth/ proangiogenic
(CAC) or late outgrowth/endothelial colony forming cells (ECFC). Especially
circulating EPC have been postulated as biomarkers, however their precise
phenotypic definition is heavily discussed. Authors propose different combinations of the markers CD34, CD133, VEGFR-2/KDR, CD31 and CD45
debating whether EPC are of hematopoietic origin or not. To gain insight into
the early phases of the differentiation cascade, the phenotypes of uncultivated
CD34+ mononuclear cells (CD34+ MNC), ECFC and HUVEC at primary
passage (p0) and p1 were analyzed. Already within p0 ECFC underwent a rapid
maturation from a CD45+ and CD31+ phenotype to a CD45-negative, and
endothelial marker-positive phenotype as defined by flow cytometry and
multiphoton imaging. In p0 ECFC colonies of homogenous cobblestone
morphology contained subpopulations expressing a mature endothelial
phenotype, but also subpopulations co-expressing CD45dim and CD31, but no
other hematopoietic marker such as CD14, CD41 or CD62p. Imaging revealed
that CD45 was dimly expressed at the cell surface - only in ECFC but not in
HUVEC. Interestingly, few ECFC showed perinuclear CD45 aggregation or
intracellular CD45 caps. Compared to HUVEC, ECFC showed a less concise
expression of CD31 at the cell-cell-contact sites. Finally our data confirm
ECFC as a unique cell population exerting high angiogenic and vasculogenic
capacity. Our study supports the notion that ECFC emerge from a CD45dim
CD31+ progenitor and very rapidly mature in culture. The data strengthen the
necessity to identify a set of markers capable of prospectively discriminating
endothelial from hematopoietic cells as well as progenitor from mature cells.
245
THE EFFECTS OF MICROSCALE AND NANOSCALE PATTERNS
ON THE PROLIFERATION OF UMBILICAL CORD BLOODDERIVED MESENCHYMAL STEM CELLS
M Seon1, M Lee1, D Kim2, K Lee3, Y Yang1, W Oh1, S Choi1, S Kwon1
1
Biomedical Research Institute, Medipost Co., Ltd, Seoul, Korea, Republic
of, 2Mechanical Engineering, POSTECH, Gyeongbuk, Korea, Republic
of, 3Biomedical Engineering, Korea University, Seoul, Korea, Republic of
Cell-material interactions play critical roles in regeneration, development, and disease. Recently, more and more studies were focused on the influence of micro and
nanoscale topographies on cell behaviors. Microscale and nanoscale topography of
the extracellular matrix (ECM) influences various cellular behaviors including cell
shape, adhesion, proliferation, and differentiation. Here, we screened several types of
patterns in micro and nano scale for the proliferation of human umbilical cord bloodderived mesenchymal stem cells (hUCB-MSCs). Several types of microscale and
nanoscale topographical patterns were fabricated using injection molding with AAO
nano template. The microscale patterns were used to investigate the effects of
different topological structures with line, rectangular and pillar/hole in micro scale.
The nanoscale patterns were used to screen the effects of different sizes of nanoscale
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structures. We found that specific microscale and nanoscale topography enhanced
hUCB-MSC proliferation when compared to non-patterned (control). In addition,
we investigate the effect of microscale and nanoscale on the hUCB-MSC stemness.
This study may provide a better understanding on the different roles of topographical cues on stem cell behavior.
246
PLASTICITY OF MUSCLE DERIVED STEM CELLS TO
UNDERGO
PLURIPOTENT
CONVERSION
WITHOUT
PLURIPOTENCY FACTORS OR SMALL MOLECULES
B Bose1, S Shenoy1, MM Reza1, CD McFarlane3, M Sharma2, R Kambadur1,3
1
School of Biological Sciences, Nanyang Technological University, Singapore,
Singapore, Singapore, 2Department of Biochemistry YLL School of Medicine,
National University of Singapore, Singapore, Singapore, 3Singapore Institute
of Clinical Sciences, A*STAR, Singapore, Singapore
Induction of pluripotency in somatic cells from multiple origins have been, in
vogue since almost a decade using pluripotency factors/genes, Oct3/4, Sox2,
Klf4, c-Myc, Lin28 and small molecules like inhibitors of BMP2, MAP kinase and
GSK3b. Here, we show the induced pluripotency of otherwise, multipotent,
muscle derived stem cells (MDSC) obtained from hind limb muscles of myostatin
null (Mstn-/-) mouse. The MDSC from Mstn-/-, in multipotent state, expressed
low levels of Nanog, Sox2 and high levels of Klf4. Furthermore, when these cells
were maintained in mouse embryonic stem cell (mESC) media containing
Leukemia Inhibitory factor (LIF) underwent pluripotent conversion over a span
of 60 days. Henceforth, we call these cells as Culture Induced Pluripotent stem
cells (CiPSC). On the other hand, multipotent MDSC from Wild type (WT)
mouse failed to exhibit a similar pluripotent conversion, when maintained in
mESC media. Upon microarray analysis, we found the Mstn-/- MDSC expressed
low levels of pluripotency associated genes like Sall4, LIF receptor, Teratocarcinoma derived growth factor1 (Tdgf1) and Sox2, in contrast to WT-MDSC.
WT- MDSC, in native form expressed Gosecoid (GSC), a mesodermal
patterning gene, in contrast to Mstn-/- MDSC. Finally, we confirmed the pluripotency of the CiPSC clones using q-RT-PCR, FACS analysis and teratoma
induction. Myostatin being a TGFb family member prompted us to study the
DNA methylation of TGFb family members of the parent Mstn-/- MDSC and
the derived corresponding CiPSC clones. Results revealed the presence of
methylated/ minimally active BMP2 loci in the parent Mstn-/- MDSC clones.
Also, BMP2 methylation was found to increase upon CiPSC conversion of
Mstn-/- MDSC, to a similar extent, to that of a standard mESC line. Gene and
protein expressions of BMP2 also corroborated the methylation data. Accordingly, this study indicates that the absence of myostatin makes MDSC amenable
to pluripotent conversion, via inhibition of BMP2.
Induced pluripotency without pluripotency factors or small molecules!
247
HYPOXIA AFFECTS PROLIFERATION AND PANCREATIC
DIFFERETIATION OF ADIPOSE DERIVED MESENCHYMAL
STEM CELLS
M Yoo, M Jung, H Shin, H Park, H Kim, J Ahn
Greencross labcell corp., Yongin, Korea, Republic of
Mesenchymal stem cells from adipose, bone marrow and umbilical cord blood
have been explored in use of cell therapy for various diseases. The adipose derived
mesenchymal stem cells(AMSCs) have also self renewal and multi lineage differentiation potential. Many strategies to differentiate adult stem cell into
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Poster Abstracts
pancreatic endocrine cell have been tried. Many studies showed that the AMSCs
can be differentiated into pancreatic endocrine cells in vitro. But the rate of transdifferentiation was low. Recent studies showed that hypoxia affects proliferation
and differentiation of mesenchymal stem cells, and controls the differentiation of
several cell types during development. And Hypoxia decreases the differentiation
of neural precursor cells, myogenic cells, adipocyte, pancreatic beta cells from
embryonic stem cells. In this study, we evaluated whether low oxygen level affects
the proliferation and trans-differentiation of AMSCs. When the AMSCs
cultured in 3% hypoxia condition, the AMSCs were increased cell number
compared with normoxia condition. In addition, hypoxia affected into pancreatic
differentiation. The expression of pancreatic transcription factors nkx6.1 were
increased in hypoxia culture compared with normoxia control. And the amount of
expressed endocrine cell marker was changed in hypoxia condition. But the
expression of another pancreatic transcription factors, pdx1 or neuroD have not
been changed. In our data suggested that hypoxia controls proliferation and affects trans-differentiation into pancreatic endocrine cells from AMSCs.
248
MYOGENIC PROPENSITIES OF CELLS OBTAINED FROM LIVER
BY PREPLATING
SP Shenoy1, B Bose1, M Sharma2, R Kambadur1,3
1
School of Biological Sciences, Nanyang Technological University, Singapore,
Singapore,, 2Department of Biochemistry YLL School of Medicine,, National
University of Singapore, Singapore, Singapore,, 3Singapore Institute of
Clinical Sciences, A*STAR, Singapore, Singapore
Liver is an organ which harbors cells from parenchymal and non parenchymal
origin. Different cell types from the liver are hepatocytes, kupffer cells,
endothelial cells and hepatic stellate cells. We cultured mouse liver homogenates from wild type and myostatin null genotypes. Thereafter, we sequentially
plated the cells, which appeared, initially in the nonadherent fraction. This
method is known as preplating and is commonly practiced by muscle biologists
for isolating muscle derived stem cells from muscle homogenates. In preplating, the initial nonadherent cell fraction, when plated separately, becomes
249
MICROPARTICLES ISOLATED FROM MESENCHYMAL STEM
CELLS TO INHIBIT HUMAN GLIOMA GROWTH
A Del Fattore, R Luciano, M Mirabella, E Giorda, M Paoletti, M Muraca
Children, Rome, Italy
Malignant glial tumors, including glioblastoma multiforme, account for
15-20% of all pediatric central nervous system malignancies. They are
most resistant to therapy and are associated with a poor prognosis. Given
the known ability of MSCs to suppress glioma growth, we investigated
the effects of microparticles (MPs) derived from mesenchymal stem cells
(MSCs) on the glioblastoma cells line U87MG. MPs were isolated from
culture media of MSCs from different sources, including bone marrow
(BM) and umbilical cord (UC), by ultrafiltration (100 kD) e ultracentrifugation (100.000 g) and added to U87MG culture. Results are
expressed as MeansSD, n¼5 when not specified. Confocal microscopy
analysis showed co-localization of MPs with tumor cells one hour after
addition of PKH26-labeled MPs to U87MG culture. FACS analysis
showed up to 32% reduced viability of U87MG cells following MP
administration (control: 93.492.16%; BM-MPs: 74.1211.83%*; UCMPs: 63.3215.74%*; *p<0.05 vs. control). MPs induced both early
apoptosis (control: 2.391.34%; BM-MPs: 6.202.86%*; UC-MPs:
7.700.90%*; *p<0.05 vs. control) and late apoptosis (control:
3.451.91%; BM-MPs: 9.963.13%*; UC-MPs: 14.603.73%*; *p<0.05
vs control). Cell cycle analysis by propidium iodide confirmed an increase
of subG1 population (control: 1.3020.48%; BM-MPs: 5.591.25%*;
UC-MPs: 6.550.32%*; n¼3; *p<0.05 vs control). Moreover, MP-treated
cells showed up to 45% reduced proliferation rate and a 2-fold increased
adhesion rate (n of cells attached to the plate/field; control: 40.337.64;
BM-MPs: 83.0010.54*; UC-MPs: 76.337.50*; n¼3; *p<0.05 vs control). These data show that MPs isolated from MSCs of different origin
exhibit antitumor activity on a glioblastoma cell line by inducing
apoptosis and reducing proliferation. Increased cell adhesion following
MPs treatment also suggests reduced tumor invasion ability.
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THE ROLE OF MONOCYTES IN THE IMMUNOREGULATORY
FUNCTION OF MULTIPOTENT STROMAL CELLS
S Melief, E Schrama, S Geutskens, M Tiemessen, M Brugman, W Fibbe,
H Roelofs
Leiden University Medical Center, Leiden, Netherlands
Fibrosis versus Myogenesis!
adherent cell population. The first nonadherent fraction is, thus, known as
preplate 1, the second one as, preplate 2. We cultured preplate 2 cells, which
were obtained in large quantities and had mesenchymal like morphology.
Furthermore, we FACS sorted the preplate 2 cells for CD146+/CD45-/
CD34-/CD56- to obtain a cell population, which was very similar to perivascular mesenchymal stem cells. Upon characterization, these cells were also
found to express bonafide markers of perivascular mesenchymal stem cells like
PDGFRb and NG2. Furthermore, these FACS sorted population expressed
myogenic marker Pax7, the marker for muscle stem cells (satellite cells).
Interesting differences in the FACS sorted cells were the fibrogenic and
myogenic properties of cells obtained from WT and Mstn-/- genotypes. In
vitro and in vivo myogenic differentiation of the FACS sorted cells from
Mstn-/- genotype further corroborated the pro myogenic nature of these cells.
Accordingly, we propose the muscle based cell therapy applicability of perivascular mesenchymal cells from liver.
Multipotent stromal cells (MSC) have already entered the clinical era despite
limited consensus on their mode of action. Many molecules have been appointed
key factors for the immunomodulatory function of MSC. Accumulating evidence indicates that the immunosuppressive properties of MSC can be stimulated and that effective immunosuppression by MSC might require the
involvement of accessory cells, especially monocytes/macrophages. In our
studies we have focused on the interaction between MSC and monocytes. Using
in vitro assays for differentiation of monocytes and for generation of regulatory
T cells (Treg), we have investigated the effect of MSC on monocytes. Using
inhibition and depletion strategies we searched for cellular and molecular
components that are essential in these interactions and for the functionality of
MSCs. Further, we performed expression analysis by array technology on
monocyte populations cultured in the absence or presence of MSC-derived
factors. We found that MSC inhibit the differentiation of monocytes towards
dendritic cells and rather skew monocytes towards an IL-10 producing cell
population with anti-inflammatory function. A key MSC-produced factor in this
process is IL-6. The generation of Treg is markedly enhanced by the presence of
MSCs and is critically dependent on the presence of monocytes. Also in this
system the presence of MSCs enhances IL-10 secretion and skews monocytes
towards a cell population with a macrophage type 2 phenotype. We show that,
upon interaction with MSCs, monocytes upregulate CCL18, which proved to be
essential for the observed Treg induction. These data enhance our knowledge
on the mechanism of immunomodulation by MSCs, supporting the improvement of MSC therapy for immunologic or inflammatory diseases. Such
improvement might entail patient selection based on more defined parameters
or improvement of the therapeutic product.
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POPULATION KINETICS OF MSC’S IN THE DEVELOPMENT OF
BIO-ARTIFICIAL BONE SCAFFOLDS
S Müller, L Nicholson, JR De Havilland, A Dickinson, X Nong-Wang
Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne,
United Kingdom
Our group is assessing the potential for Mesenchymal Stem Cells (MSC’s)
to populate synthetic biomaterials for manufacturing scaffolds to support
the growth and differentiation of MSC’s into osteocytes for bone repair in
osteoarthritis. We compared the use of non-haematopoietic medium with
MSC basal medium across three cultures of bone marrow derived MSCs
taken from the femoral heads of osteoarthritis patients. No difference was
found in doubling potential, and growth rate using either growth media.
Selecting the use of MSC growth media to promote growth of undifferentiated, plastic adherent MSC’s, we cultured MSCs through six passages,
testing their tri-lineage potential to differentiate into osteocytes, chondrocytes and adipocytes at early, mid and late time points. We also
assessed the surface phenotype by flow cytometry of undifferentiated
MSCs against the ISCT standard classification guidelines (>95% expression of CD73, CD90 & CD105 with <2% CD14, CD19, CD34, CD45
and HLA-DR). Results showed the MSC’s cultured could be differentiated
into the three specific lineages and demonstrated conformance with the
ISCT classification, except for CD34. Data showed no presence of CD34
in early cultures, but in two of three experiments, by day 50 in culture
CD34 positivity was 3% and 8% - exceeding the classification criteria by
this single parameter. By day 89 in culture, CD34 expression advanced to
15% and 46%. Those CD34+ cells remained >95% positive for CD73,
CD90 & CD105. The data show that in our culture conditions, there is
potential for MSC’s to upregulate CD34 expression, whilst retaining all
the other surface markers identifying them as MSC’s. The retention of
CD73 expression and the preferential culture medium used mitigates
against the suggestion that CD34+ cells are haematopoietic cells, and
instead suggests expansion of CD34+ MSC-like cells. The impact of this
occurrence in manufacturing cells for regenerative medicine therapies is
currently being assessed.
252
STEM CELL FUNCTIONAL MARKERS AS KEY QUALITY
ATTRIBUTES OF LARGE SCALE EXPANSION
J Murrell1, N Stankiewicz2, A Verma1, S Punreddy1, M Aysola1,
Y Trumpfheller2, J Loersch2, K Mann1, M Rook2
1
Stem Cell Franchise, EMD Millipore, Bedford, Massachusetts, United
States, 2Merck Millipore, Merck KGaA, Darmstadt, Germany
Human mesenchymal stromal/stem cells (hMSCs) are an attractive target for
clinical studies due to their easy accessibility, the potential for in vitro expansion and their immunomodulatory properties. The characterization of stem
cells and their specialized derivate cells is crucial for their use as therapeutic
agents. Within production processes of stem cells and at the end of expansion,
the potency status of stem cells can be monitored by the expression of distinct
markers. Here we report the development of several assays for the simultaneous, specific and sensitive detection of a variety of stem cell lineage markers
including intracellular proteins, cell surface structures and soluble factors.
Three panels focusing on lineage specific markers in hMSCs (adipocytes,
chondrocytes and osteoblasts) with a distinct temporal expression pattern will
allow a faster and easier assessment of the differentiation state of bone marrow
derived hMSCs. Application data of assays for the monitoring of differentiation
in hMSCs show a differential marker expression pattern typical of lineage
specific differentiation. However, this temporal expression pattern is absent in
samples from the bioreactor, confirming the usefulness of the bioreactor for
expanding large number of high quality cells that maintain an undifferentiated
cell state.
253
SINGLE USE EXPANSION AND HARVEST OF ADULT STEM
CELLS SUPPORTS LARGE SCALE MANUFACTURING
J Murrell, D Kehoe, M Aysola, D Jing, S Punreddy, A Verma, K Mann,
T Lawson, M Rook
Stem Cell Franchise, EMD Millipore, Bedford, Massachusetts, United States
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As more stem cell therapeutics progress through clinical testing, current in
vitro culture methods are cumbersome to scale. We previously demonstrated
an expansion paradigm that uses a scalable, single use, stirred tank bioreactor
with microcarrier scaffold for human mesenchymal stromal/stem cell (hMSC)
culture that enables direct monitoring for the specific characteristics of
hMSCs at any point during the culture, thus assuring product quality and
consistency. In addition, a bioreactor provides ease of use in handling and
lower medium volume requirements. Here we describe a full expansion,
harvest and characterization system for hMSCs. The cells were evaluated
using flow cytometry to assess a variety of markers that confirm identity and
purity. In addition to protein expression, we examined genetic variation using
karyotyping, comparative genomic hybridization and gene expression analysis. Differentiation potential was confirmed using traditional methods.
Finally, we evaluated multiple harvest technologies and found that they
provide good recovery of viable cells. In this work, we verified that cells
expanded in the single use stirred tank bioreactor and subsequently harvested
were identical in phenotypic and genotypic profile in comparison to flat
culture and maintained the desired cell characteristics of hMSCs, thereby
confirming the consistency, quality and reproducibility of large scale in vitro
systems for stem cell expansion.
254
MOBILIZING ENDOGENOUS NATIVE ADIPOSE STEM/
STROMAL CELL FROM ADIPOSE TISSUE
MG Ortega1, L Garidou2, C Barreau1, G Tavernier2, L Casteilla1,
C Sengenes1
1
STROMALab, CNRS 5273, Inserm U1031, EFS-Pyrenees-Mediterranee,
Universite de Toulouse, Toulouse, France, 2I2MC, Inserm U1048, Toulouse,
France
Adipose tissue (AT) is a source of multipotent progenitor cells, the adipose
stromal cells (ASCs) which display similar but not identical features with their
bone marrow counterparts, mesenchymal stromal cells (MSCs). Like MSCs,
ASCs are endowed with multilineage mesodermal differentiation potentials
and exhibit paracrine and immune-modulatory properties. Most current
research is directed at investigating their therapeutic properties after their
transplantation in damaged tissues. While some reports described their
homing into injured tissues after their injection into the circulation, their
endogenous mobilization from AT is not documented. In this study, we
aimed to examine the ability of AT to release ASCs in vivo. To do so, the ASC
content from various murine fat pads were investigated by flow cytometry
after the acute administration of CXCR4 antagonist AMD3100. To further
analyze the capability of ASCs to be mobilized from AT, we specifically
developed an ex vivo microperfusion method of an intact adipose depot. Our
data show that in vivo administration of AMD3100 induces the rapid decrease
in ASC content in a fat depot specific manner. Using the microperfusion
model, we definitively confirmed that such a reduction in ASC content was
attributable to the specific and time dependent egress of ASCs from AT. In
vivo, we also demonstrated that ASCs traffic through lymph fluid and are able
to colonize secondary lymphoid organs. Finally while preliminary our data
suggest that such ASC mobilization from AT can also be triggered after tissue
damage. Collectively, our findings propose that besides being a great reservoir of ASCs, AT could release this cell population in vivo according to the
needs of the organism. Moreover, since such a mobilization can be achieved
both physiologically and pharmacologically this might open a completely
unexplored and very promising approach regarding the regenerative potentials of ASCs.
255
DIRECT COMPARISON OF WHARTON’S JELLY AND BONE
MARROW MESENCHYMAL STEM/STROMAL CELLS
A Batsali1,2, CG Pontikoglou1, E Kouvidi1, A Damianaki1, M Kastrinaki1,
HA Papadaki1
1
Hematolgy, University of Crete School of Medicine, Heraklion, Greece,
2
Graduate Program “Molecular Basis of Human Disease”, University of Crete
School of Medicine, Heraklion, Greece
As compared to the Bone Marrow (BM) the Wharton’s Jelly (WJ) of the
umbilical cord is considered a more abundant and easily attainable source
of Mesenchymal Stem/Stromal Cells (MSCs). Yet, although WJ-MSCs
have been shown to display typical MSC characteristics, a head-to-head
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Poster Abstracts
comparison with their BM counterparts, which represent the most extensively studied source of MSCs, is still lacking. Since ex vivo MSC expansion is
a prerequisite for clinical MSC-applications, in the present study we seek to
comparatively investigate the characteristics of WJ- and BM-MSCs, cultured
under identical conditions. WJ-MSCs and BM-MSCs did not differ in their
shape or immunophenotypic characteristics. WJ-MSCs displayed higher
proliferative capacity throughout passages (P) and an increased proportion of
cycling cells at P4, without any significant difference in the percentage of
apoptotic cells at P4 as compared to BM-MSCs. The increased proliferation
of WJ MSCs could be, partly, attributed to the significantly higher Cyclin D1
gene expression (p<0.05). Significantly fewer SA-b-gal+ cells were observed
in WJ-MSC cultures at P10 (p<0.05). No chromosomal abnormalities were
observed in either WJ- or BM-MSCs during in vitro expansion. Compared
to BM-MSCs, WJ-MSCs displayed inferior capacity to differentiate into
adipocytes and osteoblasts, as shown by cytochemical stains and the expression of lineage-specific genes. The reduced differentiation potential of WJMSCs might be partly explained by the aberrant expression of Wnt-signaling
molecules. Notably, sFRP4 (inducer of adipogenesis) and Wisp1 (regulator
of osteogenesis) gene expression was significantly down-regulated in WJMSCs (p<0.05 and p<0.0001, respectively). In contrast, the gene expression
of DKK1 (inhibitor of osteogenesis) and that of Cyclin D1 (repressor of
adipogenesis) was significantly up-regulated in WJ-MSCs (p<0.05 and
p<0.05, respectively). The role of Wnt- pathway in WJ-MSC biology is
currently being explored.
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FGF2 PROTECTS MOUSE MESENCHYMAL STEM CELLS FROM
OXIDATIVE STRESS BY MODULATING A TWIST2-P53
SIGNALING AXIS
V Krishnappa, SV Boregowda, DG Phinney
Molecular Therapeutics, The Scripps Research Institute , Jupiter, Florida,
United States
Although bone marrow is a low oxygen environment few studies have
examined how oxidative stress alters MSC function. Previously, we reported that exposure of primary mouse MSCs to atmospheric oxygen
rapidly induced mitochondrial ROS generation by a p53-dependent
mechanism resulting in reduced cell growth and viability. In addition, we
showed that long-term exposure to atmospheric oxygen selected for
immortalized MSC clones with impaired p53 function but expansion of
cells in 5% oxygen using a closed system generated large numbers of
primary cells that retained a functional p53 protein. In this study we
demonstrate that atmospheric oxygen impairs adipogenic and osteogenic
differentiation of primary mouse MSCs and their ability to support hematopoiesis in vitro. Moreover, we demonstrate that FGF2 protects MSCs
from the detrimental effects of oxygen via a p53 dependent mechanism.
Specifically, culture of MSCs in FGF2-supplemented media blocked oxygen induced increases in mitochondrial ROS levels and p53 and BAX
protein expression. This effect of FGF2 was strongly correlated with induction of Twist2 mRNA and protein expression. Transfection of MSCs
with a Twist2-specific siRNA resulted in increased p53 protein expression
and a concomitant increase in intracellular ROS levels. Biochemical studies
demonstrated that Twist2 directly bound to p53 and enhanced its degradation. Moreover, knockdown of Twist2 but not Twist1 inhibited MSC
growth and CFU-F activity and augmented adipogenic and osteogenic
differentiation independent of p53. Additionally, sorting for oxidative stress
resistant sub populations enriched for multi-potent progenitors that express
high Twist2 and low p53 levels. These studies implicate Twist2 in selfmaintenance of the progenitor pool, demonstrate a functional link between
pathways that regulate MSC self-maintenance and oxidative stress, and
provide a means to enrich progenitors that does not rely on non-specific
surface markers.
257
MICRORNA IN MICROVESICLES RELEASED FROM MSCS
MEDIATES TROPHIC EFFECT
S Otsuru1,2, T Hofmann1, E Horwitz1,2,3
1
Division of Oncology/Blood and Marrow Transplantation, The Children’s
Hospital of Philadelphia, Philadelphia, Pennsylvania, United States, 2Center
for Childhood Cancer, The Research Institute at Nationwide Children’s
Hospital, Columbus, Ohio, United States, 3Division of Hematology/
Oncology/BMT, Nationwide Children’s Hospital, Columbus, Ohio, United
States
Mesenchymal stromal cells (MSCs) have been utilized as cell therapy for a
wide variety of diseases. Due to their in vitro multipotency, MSCs had been
expected to regenerate the damaged tissue by differentiating into resident
tissue cells. However, in most of the cases, the engraftment of MSCs in the
target tissue is insufficient to account for the mechanism of therapeutic effect.
MSCs are more likely to support the damaged tissue by secreting trophic
factors. An emerging body of literature suggests that microvesicle (MVs),
which play an important role in intercellular communications, are mediators of
these MSC-associated trophic effects. We have conducted series of clinical
trials of MSC cell therapy for patients with osteogenesis imperfecta (OI), a
genetic bone disorder. The patients demonstrated a remarkable acceleration of
growth after the MSC therapy, however the engraftment of donor MSCs as
osteoblasts was minimal (<1%), suggesting that the bone growth after MSC
therapy was induced by a trophic effect. Further investigation using an animal
model of OI revealed that a soluble factor(s) from MSCs induces a release of a
chondrocyte proliferation factor(s) in serum from an as yet unrecognized tissue, which stimulates the proliferation of chondrocytes in the growth plate
resulting in linear bone growth. Recently, we found that MVs released from
MSCs initiate this trophic effect. Interestingly, RNase treatment of MVs,
which degrades RNAs in MVs, and transient knockdown of Drosha in MSCs,
which blocks the biogenesis of microRNA, abrogated thistrophic effect,
indicating that the factor in MVs released from MSCs is most likely to be
microRNA. These findings will give us the way to develop a novel cell-free
therapy, which can potentially exclude safety issues of cell therapy. Additionally, the further elucidation of the microRNA may lead to a new therapy using
pharmaceutically synthesized microRNA without biological products from
MSCs.
258
NOT ALL STEM CELL ARE CREATED EQUAL: A COMPARATIVE
ANALYSIS OF OSTEOGENIC POTENTIAL IN COMPACT BONE,
ADIPOSE, AND BONE MARROW DERIVED MESENCHYMAL
STEM CELLS
J Fernandez-Moure1,2, J Van Eps1,2, B Corradetti1, P Chan1, P Ramehswar3,
B Weiner1,2, E Tasciotti1
1
Surgical Advanced Technologies Lab, Houston Methodist Research Institute,
Houston, Texas, United States, 2Surgery, Houston Methodist Hospital,
Houston, Texas, United States, 3Medicine, Rutgers, Newark, New Jersey,
United States
Mesenchymal stem cells (MSCs), readily harvested from bone marrow and
adipose tissue, hold great promise for regenerative therapies in the musculoskeletal system. While particular sources of MSCs are predisposed to immune
functions, other sources are excellent for tissue regeneration. To this end, we
identified a novel source of MSCs by isolating compact bone MSCs (cbMSCs)
from human spinal lamina and studied their in vitro osteogenic potential
compared with adipose-derived MSCs (adMSCs). These studies were conducted with the hypothesis that, in osteogenic conditions, cbMSCs would have
a greater biosynthetic and transcriptional activity as compared to adMSCs.
cbMSCs were isolated from ten patients undergoing laminectomy. Adipose and
bone marrow derived MSCs were obtained from three independent donors.
Expanded MSCs were subjected to phenotypic analyses consistent with MSCs
literature and then subjected to osteogeneic differentiation for 2 and 4 weeks.
We quantified biosynthetic activity using Von Kossa staining for calcium and
fluorescent staining of alkaline phosphatase (ALP). Transcriptional activity was
assessed in hypoxic and normoxic conditions by real-time PCR (RT-PCR) for
genes linked to osteodifferentiation : RUNX2, ALP, osteopontin, and osteocalcin,. We observed greater calcium deposition at wks 2 and 4 and greater
ALP activity at wk 2 by cbMSCs as compared to adMSCs. These functional
observations correlated with the RTPCR for osteodifferentiation markers. In
hypoxic conditions cbMSC demonstrated the greatest increase in osteogenic
gene expression In conclusion, our novel isolated cbMSCs showed a greater in
vitro osteogenic potential than adMSCs and bmMSCs in normoxic and hypoxic
conditions. This finding adds to our understanding of the therapeutic potential
for this novel source of MSCs and seems to poise cbMSCs as the preferred cell
type for orthopedic tissue engineering applications.
259
EQUIVALENT MSC PREPARATIONS USING TWO ISOLATION
METHODS
S Otsuru1,3, T Hofmann1, P Raman2, T Olson1, E Horwitz1,3,4
1
Division of Oncology/Blood and Marrow Transplantation, The Children’s
Hospital of Philadelphia and the University of Pennsylvania Perelman School
of Medicine, Philadelphia, Pennsylvania, United States, 2Center for Biomedical
Informatics, The Children’s Hospital of Philadelphia, Philadelphia,
Pennsylvania, United States, 3Center for Childhood Cancer, The Research
Institute, Nationwide Children, Columbus, Ohio, United States, 4Division of
Hematology/Oncology/BMT, Nationwide Children’s Hospital, Columbus,
Ohio, United States
Bone marrow derived mesenchymal stromal cells (MSCs) are currently being
studied as cell therapy for various indications. At the 2012 ISCT annual
meeting, we introduced a novel closed system device (KANEKA CORPORATION, Japan) that allowed us to isolate up to 3.4-fold more MSCs
compared to a conventional density-gradient method from same donor bone
marrow (n¼5). This device is attractive especially in cell therapy, such as our
MSC therapy for osteogenesis imperfecta (OI), a genetic bone disorder that
requires many MSCs. Even though the MSCs isolated with the device were
shown to have similar phenotype and differentiation ability to the conventionally isolated MSCs, it is still unclear if they have equivalent therapeutic
effect. In this report, we compared gene expression profiles of MSCs isolated
with the device or by conventional methods. Heat map and clustering
analysis of microarray data showed that there were slight differences in the
gene expression profiles over 30,000 genes between donors, however the
profiles between MSCs isolated from the same donor but using the two
different isolation methods were essentially identical (Correlation Coefficient : R¼0.9988). Furthermore, we examined the therapeutic effect of these
MSCs on OI. Previously, we demonstrated that MSC infusion induced
linear bone growth by stimulating chondrocyte proliferation in the growth
plate and we successfully established an in vitro chondrocyte proliferation
assay to evaluate the therapeutic activity of MSCs. Using this functional
assay system, we assessed MSC activity to induce chondrocyte proliferation.
Consistent with the microarray analysis, MSCs from different donors had
different levels of activity. However, MSCs isolated from the same donor by
the two methods had equivalent therapeutic effect on chondrocyte proliferation. Therefore, this novel device can isolate significantly more MSCs
with functional equivalency to conventionally isolated MSCs and will
expedite clinical investigations of MSC therapy for OI and possibly other
disorders.
260
261
DEVELOPMENT OF GMP-GRADE MESENCHYMAL STEM
CELLS DERIVED FROM BONE MARROW OF HEALTHY
DONORS
J Jang1,2, S Suh2, J Kim2
1
School of Medicine, Catholic University of Korea, Seocho-Gu, Seoul, Korea,
Republic of, 2Catholic Institute of Cell Therapy, Catholic Medical Center,
Seocho-Gu, Seoul, Korea, Republic of
Mesenchymal stem cell (MSC) is one of the most promising cell types in the
field of cell therapy, thanks to their potentials of extensive in vitro proliferation, multi-differentiation, immune modulation. For the translational
research and development of cell therapy technologies using MSCs, GMPgrade healthy human MSCs may be considered as one of the indispensible
prerequisites. However, any individual researcher can hardly meet all the
relevant regulational, ethical issues and cannot guarantee consistent supply
of quality controlled cells in sufficient number with GMP-compliant
documentation. We have obtained human bone marrow biopsies from
healthy donors after IRB approval and established GMP-grade MSCs
(Catholic MASTER cells) by isolating plastic-adherent cells and expanding
cell number through passaging, with proper quality control in the institutional GMP facility. We established extensive quality tests for the cultureexpanded mesenchymal stem cells, which include microbial sterility, endotoxin, mycoplasma, multi-differentiation potentials (osteoblasts, adipocytes,
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chondroblasts), and cell surface markers as proposed by ISCT. The Catholic
MASTER cells were shown to meet all the above-mentioned criteria. The
cells also showed additional characteristics of the MSCs in terms of potentials of extensive proliferation and secretion of paracrine factors. All the
protocols and records were established in compliance with Good
Manufacturing Practices (GMP) requirements. We believe that these
mesenchymal stem cells could be used as the noble source for the research
and development of new regenerative medicine to treat many non-curable
diseases.
262
LONG-TERM CULTURED MESENCHYMAL STEM CELLS
FREQUENTLY DEVELOP GENOMIC MUTATIONS BUT DO
NOT UNDERGO MALIGNANT TRANSFORMATION
Y Wang1,2, Z Han1, Z Zhang3, Y Chi1, Z Yang1, S Yang1, S Yan4, A Mao4,
J Zhang4, F Xu1, L Liang4, Q Zhang3, Y Yang3, S Wang3, L Meng1,2, J Cui1,
Y Ji1, X Fang3, H Zhong-Chao1,2,4
1
State Key Laboratory of Experimental Hematology, National Engineering
Research Center of Stem Cells, Institute of Hematology and Hospital of Blood
Diseases, Chinese Academy of Medical Science & Peking Union Medical
College, Tianjin, Tianjin, China, 2TEDA Life Science and Technology
Research Center, Chinese Academy of Medical Science, Tianjin, China,
3
Laboratory of Disease Genomics and Individualized Medicine, Beijing
Institute of Genomics, Chinese Academy of Medical Science, Beijing, Beijing,
China, 4National Engineering Research Center of Cell Products/AmCellGene
Co. Ltd, Tianjin, Tianjin, China
Cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) are
being tested in several clinical trials, and encouraging outcomes have been
observed. To determine whether in vitro expansion influences the genomic
stability of hUC-MSCs, we maintained nine hUC-MSC clones in long-term
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Poster Abstracts
culture and comparatively analyzed them at early and late passages. All of the
clones senesced in culture, exhibiting decreased telomerase activity and
shortened telomeres. Two clones showed no DNA copy number variations
(CNVs) at passage 30. Seven clones had 1 CNVs at passage 30 compared
with passage 3, and one of these clones appeared trisomic chromosome 10 at
the late passage. No tumor developed in immunodeficient mice injected with
hUC-MSCs, regardless of whether the cells had CNVs at the late passage.
mRNA-Seq analysis indicated that pathways of cell cycle control and DNA
damage response were downregulated during in vitro culture in hUC-MSC
clones that showed genomic instability, but the same pathways were upregulated in the clones with good genomic stability. These results demonstrated that hUC-MSCs can be cultured for many passages and attain a large
number of cells, but most of the cultured hUC-MSCs develop genomic alterations. Although hUC-MSCs with genomic alterations do not undergo
malignant transformation, periodic genomic monitoring and donor management focusing on genomic stability are recommended before these cells
are used for clinical applications.
263
A XENO-FREE CULTURE SYSTEM FOR HMSC FROM VARIOUS
SOURCES
SUITABLE FOR INITIAL ISOLATION AND
EXPANSION TOWARD CLINICAL APPLICATIONS
M Genser-Nir, S Daniliuc, M Teverovsky, D Fiorentini
Biological Industries, Beit Haemek, Israel
Human mesenchymal stem cells (hMSC) are multipotent adult stem cells
present in a variety of tissue niches in the human body. hMSC have advantages over other stem cell types due to the broad variety of their tissue
sources, since they are immuno-privileged, and for their ability to specifically
migrate to tumors and wounds in vivo. Due to these traits hMSC have
become desirable tools in tissue engineering and cell therapy. In most clinical
applications, hMSC are expanded in vitro before use. The quality of the
culture medium and its performance are particularly crucial with regard to
therapeutic applications, since hMSC properties can be significantly affected
by medium components and culture conditions. To date, there is no efficient
xeno-free (XF) medium for the initial isolation of hMSC from various tissues.
In addition, most of the common culture media for growth and expansion of
hMSC, as well as auxiliary solutions (for attachment, dissociation, and cryopreservation), are typically supplemented with serum or other xenogenic
compounds. A defined serum-free (SF), XF culture system optimized for
hMSC isolation and expansion would greatly facilitate the development of
robust, clinically acceptable culture process for reproducibly generating
quality-assured cells. The present study evaluated a novel XF culture system,
comprising MSC NutriStemÒ XF culture medium and all the required
auxiliary solutions for the attachment, dissociation, and cryopreservation of
SF, XF culture system suitable for culturing hMSC from various sources
the cells. The system was evaluated for initial isolation of hMSC from various
sources, and for long-term culturing under SF, XF culture conditions suited
for clinical applications. Results show that the XF culture system for hMSC
efficiently supports initial isolation and optimal expansion of hMSC from
various sources, while maintaining hMSC features: typical fibroblast-like cell
morphology, phenotypic surface marker profile, differentiation capacity, selfrenewal potential, and genetic stability.
264
CULTURE OF INTESTINAL STEM CELLS IN SERUM-FREE
CONDITIONS
C Yao1, MS Mohamed2
1
Department of Chemical Engineering and Materials Science, Yuan Ze
University, Chung-Li city , Taiwan, 2Graduate School of Biotechnology and
Bioengineering, Yuan Ze University, Chung-Li city, Taiwan
Intestinal stem cells (ISCs) are located at the bottom of the intestinal crypts.
Additionally, intestinal stem cells have the ability of differentiation into
transit amplifying cells which in turn will give rise to all the mature
epithelial cells. Recently, intestinal stem cells have taken a great attention as
a promising stem cell therapy for many intestinal diseases such as small
bowel syndrome. In addition, many challenges were facing the study of ISCs
because of the lack of definitive markers and definitive isolation and culture
methods. Thus, developing a definitive culture system and serum-free medium of intestinal stem cells will be promising in the clinical applications and
may help in the small intestine tissue engineering. In the present study, we
are showing ISCs isolation from mouse small intestine. Furthermore, we
have selected five significant growth factors which could enhance ISC
proliferation in vitro and replace the serum components. Moreover, our
optimum growth conditions could maintain the ISC growth in 3D culture
and enhanced the enteroid formation ability of the intestinal crypts in
matrigel. The results of gene expression analysis of some ISC markers
including Lgr5, Bmi1, Ascl2 and PTEN have confirmed that our optimum
medium could maintain the stem cell state in this culture system. In a
conclusion, these results may help in the enhancement of ISC expansion and
understanding the major signaling pathways which maintain the ISC selfrenewal and differentiation. In addition, this serum-free medium will be a
good tool in the clinical applications.
265
UMBILICAL CORD-DERIVED MESENCHYMAL STEM CELL
INFUSION IMPROVES LIVER FUNCTION IN LIVER CIRRHOSIS
AND IS ASSOCIATED WITH VIRAL LOAD REDUCTION
S Chin1, Z Cheng2, K Then3, S Cheong4
1
Beverly Wilshire Medical Centre, Kuala Lumpur, Malaysia, 2Cytopeutics,
Cyberjaya, Selangor, Malaysia, 3Cryocord, Cyberjaya, Selangor,
Malaysia, 4Tunku Abdul Rahman University, Kajang, Selangor, Malaysia
SF, XF culture system optimized for the Initial Isolation of hMSC
Background: Mesenchymal stromal cells (MSC) may attenuate inflammation
and T-cell mediated injury. MSC has also been proven to differentiate into
functioning hepatocytes. These properties may be useful for the palliative
treatment of patients with end-stage liver failure and cirrhosis.
Methods: Five consecutive patients (4 men; mean age 59 years) with the
condition were recruited from a medical clinic. Two patients presented with
decompensated liver encephalopathy. The aetiologies were viral hepatitis
(n¼3), alcohol-induced (n¼1), and autoimmune/idiopathic (n¼1). Liver
cirrhosis was confirmed by abdominal ultrasound. Three patients had portal
hypertension with splenomegaly. All received umbilical cord-derived mesenchymal stem cells (MSC) via intravenous infusion. Blood samples were taken at
baseline, 6 weeks and 3 months after cell treatment and sent for haematology,
liver function test and prothrombin time.
Results: All patients tolerated the procedure well. There was generally
improvement in all blood parameters at 6 weeks, sustained at 3 months. Specifically two patients with anaemia and thrombocytopenia, presumably due to
splenomegaly, demonstrated significant improvement. Hepatitis viral load by
PCR also improved significantly in two out of three patients.
Conclusion: MSC infusion improves liver function tests in patients with
hepatitis and may potentially play a role in management of end-stage liver
failure and cirrhosis. The association between MSC infusion and viral load
reduction warrants further investigation.
266
THE EFFECT OF ADIPOSE TISSUE DERIVED MESENCHYMAL
STEM
CELS
ON
B
CELL
PROLIFERATION
AND
DIFFERENTIATION
1
1
2
1
1
M Franquesa , F Mensah , R Huizinga , M Betjes , W Weimar , CC Baan1,
MJ Hoogduijn1
1
Nephrology and Transplantation, Erasmus MC, Rotterdam, Netherlands,
2
Immunology, Erasmus MC, Rotterdam, Netherlands
Background: Mesenchymal stem cells (MSC) have proven immunomodulatory capacity which makes them a promising therapeutic tool in transplantation. While the immunosuppressive effect of MSC on T cell-mediated
effector mechanisms has been well studied, less is known about the effects of
MSC on B cell-mediated immune responses.
Methods: MSC were isolated from subcutaneous fat tissue from kidney
transplant donors. Resting mature B cells from tonsils were obtained by CD43
negative selection with Magnetic Activated Cell Sorting (MACS). MSC were
co-cultured with CFSE-labeled B cells stimulated in a T cell-like fashion (antiIgM + anti-CD40 + IL2) or by PMA/ionomycin activated CD4 T cells. Proliferation and B cell phenotype were analyzed by Flow Cytometry, and IgG
production quantified by ELISA.
Results: Proliferation of B cells activated in a T cells-like manner (antiIgM + anti-CD40 + IL2) was not affected by the presence of MSC, while
MSC decrease the proliferation of B cells stimulated with activated T cells. An
induction of plasmablasts (CD19+ CD27high CD38high) occurred when B
cells were stimulated in a T cell dependent manner or in the presence of
activated CD4 T cells. MSC abolished the differentiation into plasmablasts
completely, which was correlated with decreased IgG production. Furthermore, MSCs induced an increase in the percentage of CD19+ CD27CD38high CD24high regulatory-like B cells when stimulated in a Tcell-like
fashion.
Conclusion: MSC inhibit B cell differentiation while increasing the
proportion of regulatory-like B cells. The reduction of B cell proliferation
by MSC is T cell-dependent. These results suggest a therapeutic role
of MSC for the treatment of patients suffering from B cell mediated
alloreactivity.
267
AUTOLOGOUS BONE MARROW-DERIVED MESENCHYMAL
STEM CELL TRANSPLANTATION IMPROVES CLINICAL
DISABILITY IN PATIENTS WITH ACUTE MIDDLE CEREBRAL
ARTERY INFARCT
NM Ibrahim1, H Tan1, N Ismail2, S Chin4,7, SS Abdul Wahid1,2, S Muda3,
S Dan4, K Then5, S Cheong6
1
Department of Medicine, Faculty of Medicine, Univesiti Kebangsaan
Malaysia Medical Centre, Kuala Lumpur, Malaysia, 2Cell Therapy Centre,
Faculty of Medicine, Univesiti Kebangsaan Malaysia Medical Centre, Kuala
Lumpur, Malaysia, 3Department of Radiology, Faculty of Medicine, Univesiti
S77
Kebangsaan Malaysia Medical Centre, Kuala Lumpur, Malaysia, 4Cytopeutics,
Cyberjaya, Selangor, Malaysia, 5Cryocord, Cyberjaya, Selangor, Malaysia,
6
Tunku Abdul Rahman University, Kajang, Selangor, Malaysia, 7Beverly
Wilshire Medical Centre, Kuala Lumpur, Malaysia
Background: Stroke involving the middle cerebral arteries (MCA) confers
significant mortality and morbidity due to irreversible neuronal damage.
Studies, animal and clinical have shown that bone marrow-derived mesenchymal stem cells (BMMSCs) improve disability in stroke. In this study, we
studied the efficacy of autologous BMMSCs in patients with acute MCA
infarct.
Methods: In this open-label series, 5 consecutive patients with acute
MCA infarct, aged 30-75 years, stroke onset between 2 weeks to 2 months,
and National Institutes of Health Stroke Scale (NIHSS) score 10-35 were
recruited. Autologous BMMSCs were isolated, expanded in vitro, and
infused intravenously. Patients were serially assessed using NIHSS, Barthel
Index (BI), Modified Rankin Scale (mRs) at baseline, 3 months, 6 months
and 12 months, and magnetic resonance imaging (MRI) at baseline and 12
months.
Results: Mean age of patients was 59 years and mean duration of stroke was
1.10.6 months. At baseline the mean NIHSS score was 14.42.7, the BI was
31.030.1, and the mRs was 4.40.6. Following infusion, there were significant improvements in the NIHSS score and BI at 3 months (NIHSS: 7.02.6;
p<0.01, BI: 80.018.4; p<0.01); NIHSS and BI at 6 months (NIHSS: 5.43.2;
p<0.01, BI: 85.0 11.7; p<0.01); and NIHSS, BI and mRS at 12 months
(NIHSS: 3.02.2; p<0.01, BI: 91.37.5; p<0.01, mRs,: 2.01.2; p<0.05)
compared to baseline scores. MRI at 12 months post-BMMSCs treatment
showed no significant changes in infarct size compared to baseline. No adverse
events were reported.
Conclusion: Our findings suggest that BMMSC infusion is efficacious in
reducing clinical disability as early as 3 months with no adverse effects.
Autologous BMMSCs infusion appears safe and feasible in improving clinical
disability in patients with acute MCA infarct. Future trials involving larger
samples are needed to confirm our findings.
268
EFFICACY OF AUTOLOGOUS BONE MARROW MONONUCLEAR
CELLS
PLUS
MESENCHYMAL
STEM
CELL
VERSUS
AUTOLOGOUS BONE MARROW MONONUCLEAR CELL
ALONE IN ISCHEMIC FOOT ULCER
H Harunarashid1, MM Idris1, FM Yusoff1, S Chin3,6, S Shahari1, N Ismail2,
S Dan3, K Then4, S Cheong5, SS Abdul Wahid1,2
1
Department of Surgery, Faculty of Medicine, Univesiti Kebangsaan
Malaysia Medical Centre, Kuala Lumpur, Malaysia, 2Cell Therapy Centre,
Faculty of Medicine, Univesiti Kebangsaan Malaysia Medical Centre, Kuala
Lumpur, Malaysia, 3Cytopeutics, Cyberjaya, Selangor, Malaysia, 4Cryocord,
Cyberjaya, Selangor, Malaysia, 5Tunku Abdul Rahman University, Kajang,
Selangor, Malaysia, 6Beverly Wilshire Medical Centre, Kuala Lumpur,
Malaysia
Background: Non-healing ischemic foot ulcer has remained an important
clinical challenge. Studies have shown that Bone Marrow Mononuclear
Cells (BM-MNC) implantation may promote capillary network proliferation while Bone Marrow Mesenchymal Stem Cells (BM-MSC) may promote sturdier arteriolar formation and vascular regeneration. This process
of angiogenesis may help resolve ischemic foot ulcers and potentially
avoid limb amputation. The purpose of the study was to compare the
efficacy of intramuscular implantation of autologous BM-MNC plus BMMSC versus BM-MNC alone, in ulcer healing of patients with ischemic
foot ulcers.
Methods: Seven consecutive patients with non-healing ischemic foot
ulcer were recruited. Patients were divided into two groups with 3 patients
in BM-MNC plus BM-MSC group (Group A: mean age 40 years, all males;
2 former smokers), and 4 patients in BM-MNC alone group (Group B:
mean age 61 years, all female and non-smokers). BM-MNC was injected
into the affected leg at baseline (Group A and B) while BM-MSC was
injected 4 weeks later (Group A). Ulcer size was measured and recovery of
ulcer was recorded at baseline, 1 month and 6 months after the BM-MNC
injection.
S78
Poster Abstracts
Results: The baseline mean ulcer size for Groups A and B were
35.618.1 cm2 and 28.115.0 cm2 respectively and mean ankle brachial
index (ABI) were 0.66 and 0.76 respectively. In Group A all three patients
(baseline ulcer size 28.6 cm2, 56.1 cm2 and 22.0 cm2 ) reported complete
healing of ulcer at 6 months. In Group B, two patients (baseline ulcer size
9.7 cm2 and 22.7 cm2 ) reported complete healing of ulcer at 6 months while
another two patients (baseline ulcer size 36.9 cm2 and 47.6 cm2 )’ reported
worsening of ulcer.
Conclusion: The combination of BM-MNC and BM-MSC injection is
better than BM-MNC alone in promoting ulcer healing. Larger studies
involving larger samples are needed to confirm the findings.
269
AUTOMATED IMAGE ANALYSIS IS USEFUL FOR SCREENING
CELL AGING IN MESENCHYMAL STROMAL CELL CULTURES
S Oja1,2, P Komulainen1,3, J Nystedt1, M Korhonen1,3
1
Research and Cell Therapy Services, Finnish Red Cross Blood Service,
Helsinki, Finland, 2Department of Biosciences, University of Helsinki,
Helsinki, Finland, 3Institute of Biomedicine, University of Helsinki, Helsinki,
Finland
Background: Senescent cells are undesirable in therapeutic cell products
due to reduced therapeutic activity and risk of aberrant cellular effects. It
has been known for long that early passage mesenchymal stromal cells
(MSC) are small and spindle shaped but become enlarged and rounded
upon cell aging. However, these changes have not hitherto been quantified.
We have developed a screening method based on automated image analysis
of cell surface area for identifying senescent MSCs from therapeutic cell
cultures.
Methods: MSCs were cultured from bone marrow aspirates to senescence.
The cell nuclei and cytoplasm were stained from passage 1, 3, 5, 7 and 8 cells.
Fluorescent signals were acquired on different channels using an automated
high-throughput microscope and resulting images were analyzed. Several
morphometric indices were analyzed for their ability to differentiate between
early and late passage cells. Telomere lengths from corresponding cell passages
were measured using terminal restriction fragment analysis (TRF) and the
expression of cell cycle regulating proteins p16INK4a and p21Cip1/Waf1 was
studied by western blot analysis.
Results: The average cell area in passage 1 cells at 143 population doublings (PD) was 419 26 mm2. A dramatic increase in the cell surface was
evident after passage 3 (253 PD), resulting in an average cell size of 2838 292 mm2 at 326 PD’s. A plateau phase in growth kinetics, telomere shortening and increased p16INK4a expression accompanied the enlargement of the
cell surface area. However, even upon aging the cell cultures maintained a small
but diminishing population of small cells.
Conclusions: Our findings suggest that MSCs used for cell therapies are of
uniform small size at early passages. Automated analysis of the cell surface area
may be used as a screening tool of cell aging for therapeutic MSC cultures.
270
LARGE NUMBERS OF HUMAN MESENCHYMAL STROMAL
CELLS ISOLATED DIRECTLY FROM HUMAN OSSEOUS
TISSUES
USING
GMP
COMPLIANT
MICROBEAD
TECHNOLOGY
R Cuthbert1, E Jones1, D Pawson2, A Lubenko2, P Giannoudis1,3,
D McGonagle1
1
Leeds Institute of Rheumatic and Musculoskeletal Disease, University of
Leeds, Leeds, West Yorkshire, United Kingdom, 2Blood and Tissue Service,
National Health Service, Leeds, West Yorkshire, United
Kingdom, 3Department of Trauma and Orthopaedics, Academic Unit,
University of Leeds, Leeds, West Yorkshire, United Kingdom
Introduction: Non-expanded, mesenchymal stromal cells (MSCs) represent
attractive candidates as therapeutic agents. Avoiding culture-expansion in
vitro minimizes manufacturing cost and safety concerns related to potential
chromosomal instability associated with culture expanded MSCs. However
the use of non-expanded MSCs has been limited due to their low frequency
in bone marrow (BM) aspirates the need to concentrate BM derived MSCs
and the lack of effective GMP compliant enrichment technologies. Here we
evaluated a MSC isolation technology utilising the MSC specific marker
CD271.
Methods: Using a clinical grade CD271-conjugated magnetic bead and the
CliniMacs cell separation system (Miltenyi Biotec, Germany) we enriched
MSCs from three intra-osseous sources. These were whole BM aspirates (n¼3,
20ml), discarded irrigation fluids from long bone reaming procedures (RF,
n¼3) and collagenase-released cell suspensions from discarded osteoarthritic
femoral heads (FHs, n¼3). The results were analysed by a combination of flow
cytometry and colony forming unit fibroblast assay.
Results: MSCs were enriched from all 3 sources; BM, FHs and RF
yielded mean 4.0x104 (3.1-4.9x104), 1.5x106 (0.9-2.4x106) and 2.5x105 (1.43.8x105) MSCs , respectively, based on measurement of the CD45-/low+
CD73 CD271bright cell population by flow cytometry, at a purity of 21.8%
(4.6-30.8), 55.8% (44.8-69.6) and 35.9% (34.5-36.6), respectively. Enriched
suspensions from FH contained on average 2.4x105 colony forming units
whereas Enriched RF suspensions yielded on average 1.2x104 colony
forming units.
Conclusion: Ready availability of femoral heads from hip replacement
surgery and the high yield of MSCs from this tissue makes this technology a
viable prospect for allogeneic therapy development. However MSCs isolated
from reaming waste fluid represent the best prospect for autologous therapy in
the orthopaedic setting.
271
COMPARATIVE
STUDY
OF
IMMUNE
REGULATORY
PROPERTIES OF STEM CELLS DERIVED FROM DIFFERENT
TISSUES
M Di Trapani1, G Bassi1, M Ricciardi1, E Fontana1,2, F Bifari1, L Pacelli1,
L Giacomello2, M Pozzobon3, F Feron4,5, P De Coppi3,6, P Anversa7,
G Fumagalli8, I Decimo8, C Menard9,10, K Tarte9,10, M Krampera1
1
Surgery, Pediatric Surgery Unit, Laboratory of Translation Surgery,
University of Verona, Verona, Veneto, Italy, 3Laboratory of Stem Cells and
Regenerative Therapy, Istituto Ricerca Pediatrica Fondazione Citta` della
Speranza di Padova, University of Padua, Padua, Veneto, Italy, 4CNRS,
NICN, UMR 7259, 13015, Aix Marseille Universite, Marseille, France,
5
Centre d’Investigations Cliniques en Biotherapie CIC-B 510, AP-HM,
Institut Paoli Calmettes, Inserm, Marseille, France, 6Department of Surgery,
Great Ormond Street, Hospital for Children, London, United Kingdom,
7
Division of Cardiovascular Medicine, Departments of Anesthesia and
Medicine, Brigham and Women’s Hospital, Harvard Medical School,
Boston, Massachusetts, United States, 8Department of Public Health and
Community Medicine, Section of Pharmacology, University of Verona,
Verona, Veneto, Italy, 9SITI Laboratory, Etablissement Francais du Sang
Bretagne, Rennes, France, 10INSERM U917, Universite Rennes 1, Rennes,
France
Allogeneic stem cell (SC)-based therapy is a promising tool for the treatment
of a range of human degenerative and inflammatory diseases. Many reports
highlighted the immune modulatory properties of some SC types, such as
mesenchymal stromal cells (MSCs), but a comparative study with SCs of
different origin, to assess whether immune regulation is a general SC property, is still lacking. To this aim, we applied highly standardized methods
employed for MSC characterization to compare the immunological properties of bone marrow-MSCs, olfactory ectomesenchymal SCs, leptomeningeal
SCs, and three different c-Kit-positive SC types, that is, amniotic fluid SCs,
cardiac SCs, and lung SCs. We found that all the analyzed human SCs share a
common pattern of immunological features, in terms of expression of activation markers ICAM-1, VCAM-1, HLA-ABC, and HLA-DR, modulatory
activity toward purified T, B, and NK cells, lower immunogenicity of inflammatory-primed SCs as compared to resting SCs, and indoleamine-2,3dioxygenase-activation as molecular inhibitory pathways, with some SC typerelated peculiarities. Moreover, the SC types analyzed exert an anti-apoptotic
effect toward not-activated immune effector cells (IECs). In addition, we
found that the inhibitory behavior is not a constitutive property of SCs, but is
acquired as a consequence of IEC activation, as previously described for
MSCs. Thus, immune regulation is a general property of SCs and the
characterization of this phenomenon may be useful for a proper therapeutic
use of SCs.
272
SCALABLE
MICROCARRIER-BASED
STIRRED
CULTURE
SYSTEM FOR HUMAN INDUCED PLURIPOTENT STEM CELL
EXPANSION IN CHEMICALLY-DEFINED CONDITIONS
SM Badenes1, TG Fernandes1, A Pusch2,3, S Haupt2,3, C Rodrigues1,
M Bear4, M Hervy4, M Diogo1, O Brüstle2,3, JS Cabral1
1
Department of Bioengineering and IBB - Institute for Biotechnology and
Bioengineering, Instituto Superior Tecnico, University of Lisbon, Lisboa,
Portugal, 2Institute of Reconstructive Neurobiology, University of Bonn,
Bonn, Germany, 3LIFE&BRAIN GmbH, Bonn, Germany, 4CORNING
Incorporated, CETC, Avon, France
Envisaging the scale-up of human induced pluripotent stem cell (hiPSC)
expansion to fulfil the large cell numbers requirement for cellular therapy, a
stirred culture system using xeno-free synthetic peptide-acrylate surface
(PAS) [1] hydrogel microcarriers in chemically-defined medium was developed. Controlled single-cell strategy for inoculation was optimized using a
chemical inhibitor of Rho-associated kinase pathway. In static PAS-microcarrier-based culture, 5-fold expansion of initially inoculated cells was
attained after 5 days. The expansion of hiPSCs under dynamic conditions on
PAS-microcarriers was established in 15 mL-laboratory-scale stirred spinner
flask as the first step towards the development of a robust process for hiPSC
culture in controlled large-scale bioreactor. An almost 14-fold increase in
relation to initially attached cells was achieved within 7 days. To improve
the scalability of the culture, multipassage expansion on PAS-microcarriers
was attained using confluent microcarriers as inoculum. After 4 passages, a
cumulative 214-fold expansion was attained over 15 days of culture. In static
and dynamic cultures, cells maintained their typical morphology and pluripotency-associated marker-expression. Moreover, maintenance of their
differentiation capability was verified by spontaneous differentiation through
embryoid bodies formation expressing markers of endoderm, ectoderm and
mesoderm. Direct neural commitment of expanded hiPSCs on PASmicrocarriers to neural precursor cells (NPCs) was successfully accomplished by the dual-SMAD inhibition protocol. Overall, we have developed
an efficient scalable microcarrier-based platform for hiPSC expansion with
the possibility of integrating controlled commitment for generation of
NPCs in therapeutically useful quantities. [1] Melkoumian, Z. et al “Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells” Nat Biotechnol
(2010), 28:606-10.
273
PHENOTYPICAL AND FUNCTIONAL CHARACTERIZATION OF
MESENCHYMAL STROMAL CELLS ISOLATED FROM BONE
MARROW OF A CYSTINOTIC PATIENT
N Starc, A Conforti, A Taranta, S Biagini, A Proia, F Emma, F Locatelli,
M Bernardo
Cystinosis is a rare recessive disease caused by mutations of the CTNS gene,
that encodes for a lysosomal cystine/H(+) symporter. Invalidation of the
Ctns gene in mice, causes intralysosomal cystine accumulation and progressive organ damage that can be, at least partially, reversed by infusion of
bone marrow (BM)-derived mesenchymal stromal cells (MSCs); however,
little is known on mesenchymal compartment in cystinotic patients. The aim
of the study was to investigate phenotypical/functional properties of MSCs
isolated from BM of a cystinotic patient (Cys-MSCs). BM-derived MSCs
were isolated and expanded ex vivo from one cystinotic patient and three
healthy donors (HD-MSCs). Morphology, proliferative capacity, immunophenotype, differentiation and immunomodulatory properties were analysed.
Despite lower proliferative capacity, Cys-MSCs displayed the characteristic
spindle-shaped morphology and the same immunephenotype as HD-MSCs.
Cys-MSCs and HD-MSCs prevented proliferation of PHA-stimulated
allogeneic peripheral blood mononuclear cells to the same extent: mean
percentage of proliferation 4.2% and 39.6% at MSC:PBMC ratios of 1:2
and 1:10, respectively. After in vitro induction into osteoblasts, Cys-MSCs
showed reduced alkaline phosphatase activity and calcium depositions
compared to HD-MSCs. Cys-MSCs were treated in vitro with increasing
doses of cysteamine (50-100-200 mM) during the differentiation assay.
This treatment was associated with a complete recovery of Cys-MSCs
S79
differentiation capacity into osteoblasts. No difference in adipogenic differentiation potential was observed between Cys-MSCs and HD-MSCs. Our
results demonstrate that Cys-MSCs, although presenting reduced proliferative capacity, maintain morphology, immunophenotype, adipogenic differentiative potential and immunomodulatory properties typical of HD-MSCs.
Cys-MSCs show a reduced capacity to differentiate into osteoblasts; however, this defect can be reverted after cysteamine treatment.
274
ROLE OF STROMAL CELL-MEDIATED NOTCH SIGNALING IN
HEMATOLOGICAL MALIGNANCIES
G Bassi1, PT Kamga1, AN Kamdje1,2, R Stradoni1, G Malpeli3, E Amati1,
I Nichele1, R Carusone1, Z Jasmina1, G Pizzolo1, M Krampera1
1
Laboratory, University of Verona, Verona, Veneto, Italy, 2Biomedical
Research Center, University of British Columbia, Vancouver , British
Columbia, Canada, 3Department of Pathology, Section of Pathological
Anatomy, University of Verona, Verona, Veneto, Italy
Stromal cells are essential components of the bone marrow (BM) microenvironment regulating and supporting the survival of different tumors, including
B-cell acute and chronic lymphocytic leukemia (B-ALL and CLL), and acute
myeloid leukemia (AML). In this study, we investigated the role of Notch
signalling in human BM-mesenchymal stromal cell (hBM-MSC)-promoted
ALL, CLL and AML survival and chemoresistance. The block of Notch signalling through g-secretase inhibitor (GSI) XII reverted the protective effect
mediated by co-culture with BM-MSC. The treatment with combinations of
anti-Notch neutralizing Abs resulted in the decrease of B-ALL cell survival,
either cultured alone or cocultured in presence of BM-MSC from normal
donors and B-ALL patients. The inhibition of Notch-3 and -4 or Jagged-1/-2
and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days,
similar to what is obtained by blocking all Notch signaling with the GSI XII.
The same Notch receptors are involved in CLL survival except for Notch-1
that, in CLL, mediates a synergistic effect with other Notch receptors in
inducing the anti-apoptotic phenotype. Some preliminary data showed that
Notch system is involved in survival and chemoresistance of acute myeloid
leukemia blasts. Overall, our findings show that stromal cell-mediated Notch
signaling has a role in promoting ALL, CLL and AML survival and resistance
to chemotherapy. Therefore, the target of Notch pathway activation may
represent a useful strategy to overcome drug resistance and improve the efficacy of conventional treatments.
275
MULTIPARAMETRIC FLOW CYTOMETRY ASSAYS FOR
THE EVALUATION OF MESENCHYMAL STROMAL CELL
IMMUNOMODULATORY ACTIVITY
M Corselli, R Hingorani, J Vidal, C Carson, N Emre
BD Biosciences, San Diego, California, United States
Although many studies have documented the immunomodulatory ability of
mesenchymal stromal cells (MSCs), factors like the tissue of origin, time in
culture and culture conditions are responsible for high batch-to-batch variability. Reproducible assays to determine the phenotype and potency of MSCs
are needed to obtain consistent results. We show how multicolor flow
cytometry can be used to i) assess the immunophenotype of resting and licensed
MSCs and ii) measure the inhibition of T-cell activation. Multiparametric flow
cytometry was used to investigate the co-expression of known surface and
intracellular immunomodulatory molecules by resting or licensed MSCs.
Resting adipose derived MSCs (ADSCs) displayed a more licensed phenotype
as compared to bone marrow derived MSCs (BM-MSCs), based on higher
expression of adhesion molecule CD54, IL-6 and PGE2-producing enzyme
COX2. Upon licensing with IFNg +/- TNFa, ADSCs and BM-MSCs similarly
up-regulated IL-6, IL-8, CXCL-10, CCL-2, CCL-5, COX2, CD54 and
CD274. We then optimized a multiparametric flow cytometry potency assay to
simultaneously investigate activation of both T-cells and resting MSCs in coculture. T-cells and MSC were discriminated based on size and surface marker
signature. Cell proliferation analysis revealed that the activation of T-cells was
significantly reduced in the presence of BM-MSCs and completely abrogated
by ADSCs, thus suggesting a possible correlation between licensed phenotype
and immunomodulatory activity. In the presence of activated T-cells, MSCs
S80
Poster Abstracts
increased the production of IL-6, COX2, CD54 and CD274. In conclusion,
multiparametric flow cytometry enables the simultaneous analysis of intracellular and surface markers to i) determine the licensed status of different batches
of MSCs, ii) measure the level of immunomodulatory activity, iii) identify the
source of cytokines in co-culture systems and iv) define biomarkers predicting
MSC potency.
276
CD117+ AMNIOTIC FLUID STEM CELLS VARY THEIR IMMUNE
REGULATORY PROPERTIES ACCORDING TO GESTATIONAL
AGE
M Di Trapani1, G Bassi1, E Fontana1,2, L Giacomello2, M Pozzobon3,
P V Guillot4, P De Coppi3,5, M Krampera1
1
Surgery, Pediatric Surgery Unit, Laboratory of Traslation Surgery, University
of Verona, Verona, Veneto, Italy, 3Istituto Ricerca Pediatrica Fondazione Città
della Speranza di Padova, Laboratory of Stem cells and Regenerative Therapy,
University of Padua, Padua, Veneto, Italy, 4Institute of Reproductive and
Developmental Biology, Imperial College, London, United Kingdom,
5
Department of Surgery, Great Ormond Street Hospital for Children,
London, United Kingdom,
Amniotic Fluid Stem Cells (AFSs) are pluripotent stem cells achievable
through the positive selection and ex-vivo expansion of CD117 (c-Kit)expressing cells derived from amniotic fluid. Given the broadly differentiation
potential toward adipogenic, osteogenic, myogenic, endothelial, neuronal and
hepatic lineages, AFS cells have raised great interest as new therapeutical tool.
However, their immunogenicity and immunomodulatory properties need to be
assessed before clinical use. To this aim, we analyzed the immunological effects
resulting from the interaction between AFS cells of different gestational age
and various immune effector cells, i.e. T, B and NK cells. Resting 1st
trimester-AFS cells showed lower expression of HLA class-I molecules and
NK-activating ligands than 2nd and 3rd trimester-AFS cells. This feature was
associated to lower sensitivity of 1st trimester-AFS cells to NK cell-mediated
lysis. Nevertheless, inflammatory priming of AFS cells by IFN-g and TNF-a
enhanced the resistance of all AFS cell types to NK cytotoxicity. AFS cells
modulated lymphocyte proliferation in a different manner according to
gestational age: 1st trimester-AFS cells significantly inhibited T and NK cell
proliferation, while 2nd and 3rd trimester-AFS cells were less efficient. In
addition, only inflammatory-primed 2nd trimester-AFS cells could suppress B
cell proliferation, which was on the contrary unaffected by the other AFS cells.
Indolamine 2,3 dioxygenase (IDO) pathway was not significantly involved in
1st trimester-AFS cells-mediated T cell suppression, while a different mechanism mostly related to the induction of apoptosis was evident. Overall, this
study revealed a number of significant qualitative and quantitative differences
among AFS cells of different gestational age in terms of phenotype, immunological functions and immunogenicity, which all have to be taken into
consideration in view of AFS cells clinical application.
277
CELL SURFACE ENGINEERING TO ENHANCE MESENCHYMAL
STEM CELL MIGRATION TOWARDS SDF-1 FOR ISCHEMIC
TISSUE
Y Won, F Silva, K Lim, S Kim, D Bull, AN Patel
United States
Background: Mesenchymal stem cell (MSC) therapy for myocardial infarction
(MI) has shown considerable promise but only 2% reach the ischemic
myocardium after systemic infusion. This is in part due to loss of the homing
signal on the MSC surface during culture. In the ischemic myocardium,
stromal-derived factor-1 (SDF-1) is expressed transiently after infarct. Current
approaches to induce CXCR4 expression, the natural ligand for SDF-1found
on MSC surfaces via genetic modification, hypoxic-preconditioning, and
cytokine treatment) requiring long-term MSC culture have not been optimal
for clinical translation. Our objective was to create a novel method to introduce
CXCR4 expression on the MSC surface within one hour to improve cell
transplantation.
Methods: The DMPE-PEG polymer provides a homogenous ultra-thin
coating on the cell surface without covalent bonds and electrostatic interaction,
both of which may cause severe cytotoxicity. In this study, we compared molecular weight of PEG and conjugation chemistry to attach rCXCR4 on the
PEG-lipid, and tested migration of MSCs toward the SDF-1 gradient along
with differentiation.
Results: MSC cell surface modification was accomplished using a 5 kDa
DMPE-linker. FACS analysis revealed that 100% cells are homogenously
modified through the hydrophobic interaction between the DMPE-PEG and
cell membrane. Migration of CXCR4+ MSCs toward the SDF-1 gradient was
determined visually and using cell counting kit (CCK). CCK assay also
demonstrated that MSCs modified with different amounts of DMPE-PEGCXCR4 migrated toward SDF-1 in a CXCR4 dose-dependent manner without
influencing MSC differentiation capabilities.
Conclusions: We have developed for the first time an MSC surface
modification method to incorporate recombinant CXCR4 protein (rCXCR4)
on a MSC membrane in 10 minutes and confirmed improved migration of
MSCs toward SDF-1. This approach may enable better MSC translation
requiring fewer cells with better homing and retention.
278
ROLE
OF
BONE
MARROW
DERIVED
ALLOGENEIC
MESENCHYMAL STROMAL CELLS (STEMPEUCEL Ò) IN
CRITICAL LIMB ISCHEMIA DUE TO BUERGER’S DISEASE e
EFFICACY AND SAFETY RESULTS OF NON-RANDOMIZED,
OPEN LABEL, MULTICENTRIC, DOSE RANGING, PHASE II
STUDY IN INDIA
PK Gupta1, A Chullikana1, M Rajkumar2, M Krishna3, S Dutta4, U Sarkar5,
S Desai6, R Radhakrishnan7, A Dhar8, S Balasubramanian1, U Kumar1,
U Baikunje1, K Prasanth1, N Anthony1, A Majumdar1
1
Stempeutics Research, Bangalore, Karnataka, India, 2Department of Vascular
Surgery, Madras Medical College, Chennai, Tamil Nadu, India, 3Department
of Vascular Surgery, Sri Jayadeva Institute of Cardiovascular Sciences &
Research, Bangalore, Karnataka, India, 4Department of Cardiovascular
Surgery, Nightingale Hospital, Kolkata, West Bengal, India, 5Department of
Cardiovascular Surgery, Health Point Hospital, Kolkata, West Bengal,
India, 6Department of Vascular Surgery, M.S Ramaiah Medical College &
Hospitals, Bangalore, Karnataka, India, 7Department of Vascular Surgery, Sri
Ramchandra Medical College, Chennai, Tamil Nadu, India, 8Department of
Vascular Surgery, All India Institute of Medical Sciences, New Delhi, Delhi,
India
Buerger’s disease is a nonatherosclerotic segmental inflammatory disease that
most commonly affects the small and medium-sized arteries. Its prevalence
among all patients with PAD varies from 0.5 to 5.6 percent in Western
Europe to 45 to 63 percent in India. CLI is a severe form of the disease
resulting in severe rest pain and non-healing ischemic skin lesions and finally
gangrene of the extremity. About 50% patients with CLI will lose their leg
within 6 e 12 months, and approximately 15% will require contralateral
amputation within 2 years. A phase II, non-randomized, multicentric, dose
finding study in 126 patients using three different dose levels (1, 2, & 4
million cells/kg e 36 patients in each dose) and control arm (n¼18) using
stempeucelÒ in Buerger’s disease is ongoing. Inclusion criteria: (i) Buerger’s
disease as diagnosed by Shionoya criteria (ii) Males or females in the age
group of 18-65 yrs (iii) Patients having infrapopliteal occlusive disease with
rest pain and ischemic ulcer/necrosis, who are not eligible for or have failed
traditional revascularization treatment (iv) Patients in Rutherford- III-6 if
gangrene extending maximally up to the head of metatarsal but limited to
toes. The primary efficacy end points of the study are - Relief of the rest pain
and healing of ulcerations in the target limb. Recruitment of two dose levels
S81
and the control arm patients is completed and the patients have completed six
months follow up. Results of six months follow e up: 30 patients in 1 million/
kg dose group showed reduction of rest pain by 5.55 cm; 88.23% ulcers
completely / partially healed and ABPI increased by 0.18. 23 patients in the 2
million/kg dose group showed similar trend with reduction of rest pain by
6.10 cm, 84.37% ulcer healed and ABPI increased by 0.18. The patients in
the control arm did not show similar trend of improvement. Conclusion e
stempeucel Ò administered intramuscularly has an efficacious role in patients
of CLI due to Buerger’s disease.
279
IMMUNOLOGICAL CHARACTERIZATION OF MULTIPOTENT
MESENCHYMAL STROMAL CELLS. THE INTERNATIONAL
SOCIETY FOR CELLULAR THERAPY (ISCT) WORKING
PROPOSAL
M Krampera1, J Galipeau2, Y Shi3, K Tarte4,5, L Sensebe6
1
Laboratory, University of Verona, Verona, Italy, 2Emory University Winship
Cancer Institute, Atlanta, Georgia, United States, 3Institute of Health Sciences,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences &
Shanghai Jiao Tong University School of Medicine, Shanghai, China,
4
Etablissement Français du Sang, Rennes, Saint-Denis, France, 5INSERM
U917, Universite Rennes 1, Rennes, France, 6UMR5273 STROMALab
CNRS/EFS/UPS e INSERM U1031, Toulouse, France
The large number of experimental approaches, culture conditions, qualitative
and quantitative methods, and in vitro and in vivo models employed so far to
assess immune regulatory properties of multipotent mesenchymal stromal cells
(MSC) has led to an excess of literature data that sometimes are poorly
comparable, redundant, and even contradictory. Thus, quite paradoxically, the
risk is that pre-clinical literature data may become eventually weak and scarcely
useful for supporting experimentally specific MSC-based clinical trials.
However, some data in this field appear more solid and reproducible and may
be generally accepted to suggest reproducible immunological assays to quantify
the differences in immune modulatory properties of MSCs produced according to Good Manufacturing Practice (GMP). The MSC Committee of the
International Society of Cell Therapy (ISCT) released a statement paper in
2006 that established the minimal criteria characterizing human MSC, without
focusing particularly on their immunological properties. To consolidate the
scientific research in this field, the MSC Committee of the ISCT is publishing
a working proposal paper aimed at stimulating the general discussion about the
need of shared guidelines for the immunological characterization of MSCs for
clinical use: 1. A standard immune plasticity assay should be implemented by
using IFN-g TNF-a 2. Functional analysis of an expanded cell product may
provide mechanistic insights on clinical response amongst patients 3. The use
of purified responders would be widely practicable 4. Interrogating the IDO
response as part of an in vitro licensing assay 5. Conclusions based on xenorecipient animal models should be drawn with caution 6. The prospective
hypothesis-driven analysis of lymphocyte populations in patients groups
treated with MSC should be encouraged 7. Clinical analysis should also
include the monitoring of whether injected MSCs are the target of an immune
response.
280
GENERATION OF INSULIN PRODUCING CELLS FROM HUMAN
BONE MARROW-DERIVED MESENCHYMAL STEM CELLS:
FURTHER IN VIVO MATURARTION.
AF Refaie1, MM Gabr2, MM Zakaria2, Sm Khater3, SA Ashamallah3,
AM Ismail4, MA Ghoneim5
1
Nephrology, Urology Nephrology Center, Mansoura, Egypt, 2Biotechnology,
Urology Nephrology Center, Mansoura, Egypt, 3Pathology, Urology
Nephrology Center, Mansoura, Egypt, 4Immunology, Urology Nephrology
Center, Mansoura, Egypt, 5Urology, Urology Nephrology Center, Mansoura,
Egypt
The proportion of differentiated insulin-producing cells (IPCs) from human
bone marrow-derived mesenchymal stem cells (HBM-MSCs) is modest and the
quantities of the released insulin & c-peptide are meager. Nevertheless, these
cells induced normoglycemia when transplanted in diabetic nude mice. The
aim of this study is to investigate the possibility of further in vivo maturation
of these cells. HBM-MSCs were obtained from 3 insulin-dependent type 2
diabetic volunteers, expanded then differentiated by trichostatin-A/glucagonlike peptide1-based protocol. Cells were evaluated by immunolabeling,
relative gene expression and human insulin & c-peptide release in response to
glucose challenge. A total of 1x106 cells were inserted under the renal capsule
of diabetic nude mice. Cured animals were euthanized at 1, 2, 4 and 12 weeks
after transplantation. IPCs-bearing kidneys, contralateral kidneys and pancreata were harvested. Immunolabed insulin-positive cells were counted.
Gene expression of relevant genes was determined. Values were compared to
the in vitro data. At the end of in vitro differentiation, all the pancreatic
endocrine genes were expressed. Insulin gene expression increased by more
than 1000 folds as compared to the undifferentiated cells. Nevertheless, the
proportion of insulin-positive cells was modest (w3%). Surviving transplanted animals became normoglycemic. Glucose tolerance testing and
concomitant human c-peptide levels were within normal. In vivo, the proportion of insulin-positive cells increased 20-30 folds reaching a maximum
after 2-4 weeks post transplantation (figure 1). Relative gene expression of
insulin, glucagon and somatostatin demonstrated a parallel amplification
(figure 2). For the first time, evidence was provided that further maturation
and/or differentiation continued in the in vivo milieu presumably offers a
favorable environment. The exact mechanism(s) involved have to be further
investigated.
281
DUAL EFFECTS OF HUMAN ADIPOSE TISSUE-DERIVED
MESENCHYMAL STEM CELLS ON NF-KB PATHWAY IN
TUMOR GROWTH OF A549 LUNG ADENOCARCINOMA CELLS
AND HT-29 COLON CANCER CELLS
J Rhyu1,2, E Kwon2, B Kang1,2
1
Graduate school of immunology, Seoul national university college of
medicine, Seoul, Korea, Republic of, 2Biomedical research institute, Seoul
national university hospital, Seoul, Korea, Republic of
Human adipose tissue-derived mesenchymal stem cells(hATMSCs) have a
great potential as therapies for various diseases and regenerative medicine.
However, emerging evidence suggests that human stem cells have both promoting and inhibitory effects on tumor growth. For the clinical use of
hATMSCs as a novel cell therapy, it is important to determine in which tumor
environment hATMSCs have tumor supporting effects or suppressing effects
and to understand the underlying mechanism. We investigated the effect of
hATMSCs on growth of 6 different types of tumor cell lines using in vivo
xenograft models of A-375, A-431, A549, NCI-N87, HT-29 and Capan-1.
The hATMSCs have an inhibitory effect on tumor growth of A549, but at the
same time, a promoting effect on growth of HT-29. We focused on A549 and
HT-29 tumor and performed further protein analysis. The activation level of
tumor-related proteins such as NF-kB p65, JAK3, STAT3 and b-Catenin was
S82
Poster Abstracts
29, and the correlation between the tumor growth and the expression level of
NF-kB. Despite the controversies concerning the effect on tumor progression,
the application of stem cells has broad prospective in many areas. Therefore,
we consider that it is required further research to understand the interactions
between stem cells and various types of tumors and those understandings will
provide a new clue for stem cell therapy.
282
THE SAFETY OF BONE-MARROW DERIVED MESENCHYMAL
STEM CELLS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS
P Adorable-Wagan, S Bernal, D Lavilles, M De Vera
The Medical City Hospital, Pasig City, Philippines
analyzed by western blotting. The results showed there is an obvious correlation between the tumor growth and the activation of NF-kB, which is the
expression level of phosphorylated NF-kB p65 was reduced in A549 tumor,
but the level was increased in HT-29 tumor. Moreover, those dual effects were
also supported by results from additional in vitro study using coculture and
flow cytometric analysis. Altogether, we demonstrated the dual effects of
hATMSCs, which is inhibiting effect on A549 and promoting effect on HT-
Background: The potential role of mesenchymal stem cells (MSCs) in the
management of Type 2 diabetes mellitus (T2DM) has been shown with varying
degrees of success in animal models and in clinical trials. Evidence shows that it
affects insulin resistance and secretory dysfunction of B-cells. It has also shown
potential effects on immune system dysregulation and inflammatory mediators,
both of which are involved in the basic pathogenesis of T2DM. The objective
of our study was to determine the effect of MSC infusion on patients with
advanced T2DM.
Methods: We performed a retrospective cohort analysis on patients with
T2DM who received MSC infusions. Eleven patients (aged 32-74) with long
standing T2DM, on oral hypoglycemic agents and/or daily insulin doses were
included. The patients received intravenous infusions of allogeneic bone
marrow derived MSCs that were harvested and cultured. Total dose range of
7.17-36 x 106 cells was given over 6 months. Primary outcome was safety,
observing for infusion-related adverse events. Secondary outcomes included
effects on fasting glucose levels (FBS), glycosylated hemoglobin (HbA1c), BMI
and changes in medication requirement before and after the 6-month period.
Results: There were no MSC infusion-related adverse events observed,
except for 1 patient who developed mild pruritus acutely during his first 2
infusions. This immediately resolved after 1 dose of antihistamine each time,
without any recurrence during subsequent infusions. There was an overall
decrease in the mean HbA1c and FBS values, but were not statistically significant. Three of the patients had decreased medication requirements to
different levels.
Conclusion: Bone marrow-derived allogeneic MSC infusion is relatively
safe for T2DM patients. There was no statistically significant improvement in
glucose control in this population of patients. Further prospective trials
involving more patients are needed to better characterize the effects of MSC on
DM control.
283
ANGIOPOIETIN-1 OF UMBILICAL CORD BLOOD-DERIVED
MESENCHYMAL STEM CELLS INHIBITS LIPOPOLYSACCHARIDEINDUCED INFLAMMATION
M Kim, H Jin, Y Bae, Y Yang, W Oh, S Choi
Biomedical Research Institute, MEDIPOST Co.,Ltd., Seoul, Korea, Republic
of
Umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) have been
considered for cell therapeutics in incurable diseases. Since paracrine action is
the main action of MSCs, we examined the anti-inflammatory activity of each
MSC under lipopolysaccharide (LPS)-induced inflammation. Co-culture of
UCB-MSCs with LPS-treated rat alveolar macrophage, reduced expression of
inflammatory cytokines including interleukin-1a (IL-1a), IL-6, and IL-8 via
angiopoietin-1 (Ang-1). Using recombinant Ang-1 as potential soluble paracrine factor or its small interference RNA (siRNA), we found that Ang-1
Aim: In our mesenchymal stem cell (MSC) bank, MSC are cryopreserved in
bags (100-200mL, Macopharma) at 1x10E6/mL and stored in liquid nitrogen
(vapor phase) for third-party use upon indication. For quality and validation
purposes, post-cryo viability is determined on accompanying vials. We studied
whether this is representative for bag viability and evaluated the viability loss
upon thawing as well as intra-vial variability and different methods of thawing.
Method: Using the trypan blue method, viability and living cell concentration were determined (counted in duplicate on 2 samples per product). Two
tailed p-values were considered statistically significant (paired t-test analysis
between bag and vial and between different thawing methods).
Results: Viability of thawed MSC vials (7110%) was not statistically
different from the post-cryo viability of corresponding thawed bags (7411%),
p¼0.26, n¼10. Also living cell concentrations were similar: 0.860.30 x10E6/mL
in MSC vials versus 0.830.30 x10E6/mL in MSC bags, p ¼ 0.55. This translates
in a positive correlation r ¼ 0.84 between vial and bag living cell concentration
and a viable cell loss of respectively 14% and 17% upon thawing. In addition,
thawed vials of 24 MSC products were analyzed in duplicate to evaluate the intravial variability. The mean difference between vials amounted 42% for viability
and 2623% for living cell concentration. Finally, the effect of dilution with
physiological buffer to diminish toxic DMSO exposure upon thawing was
compared between addition of +100% PBS versus +50% NaCL. Viability
(844% in case of +100%PBS versus 831% in case of +50% NaCl, p¼ 0.30) as
wel l as living cell concentration (1.180.17 versus 1.240.15 x10E6/mL
respectively, p¼0.15) were not statistically different between the thawing
methods.
Conclusion: Trypan blue cell counting revealed comparable thawed
viability and living cell concentration between MSC product frozen in bags and
corresponding vials.
285
OPTIMIZATION OF THE THERAPEUTIC EFFICACY OF HUMAN
UMBILICAL CORD BLOOD-MESENCHYMAL STROMAL CELLS
IN A NSG MOUSE XENOGRAFT MODEL OF GRAFT-VERSUSHOST DISEASE
Y Jang, M Kim, W Oh, Y Yang, S Choi
Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea,
Republic of
Background: Although in vitro studies have demonstrated the immunosuppressive capacity of mesenchymal stromal cells (MSCs), most in vivo studies on
graft-versus-host disease (GVHD) have focused on prevention, and the therapeutic effect of MSCs is controversial. Moreover, optimal time intervals for
infusing MSCs have not been established.
Methods: We attempted to evaluate whether human umbilical cord bloodMSCs (hUCB-MSCs) could either prevent or treat GVHD in an NSG mouse
xenograft model by injecting MSCs before or after in vivo clearance. Mice were
infused with either a single or multiple doses of 5 X 10 5 hUCB-MSCs (3- or
7-day intervals) before or after GVHD onset.
Results: Before onset, hUCB-MSCs significantly improved the survival rate
only when repeatedly injected at 3-day intervals. In contrast, single or repeated
injections after GVHD onset significantly increased the survival rate and
effectively attenuated tissue damage and inflammation. Further, the levels of
prostaglandin E2 (PGE2) and transforming growth factor-b1 (TGF-b1)
increased significantly, whereas the level of interferon-g (IFN-g) decreased
significantly in all MSCs treatment groups.
Discussion: These data establish the optimal time intervals for preventing
GVHD and show that hUCB-MSCs effectively attenuated symptoms and
improved survival rate when administered after the onset of GVDH.
Umbilical cord blood-derived marrow stromal cells (UCB-MSCs) with high
proliferation capacity and immunomodulatory properties are considered to be a
good candidate for cell-based therapies. But until now, little work has been
focused on the immunosuppression capacity of UCB-MSCs. Inflammatory
cytokines are critical in inducing the immune modulatory properties of MSCs.
However, the detailed regulatory mechanisms are yet to be fully understood.
Drug A is observed inhibition of inflammatory mediators due to its anti-inflammatory activity. The anti-inflammatory mediators such as IL-6, TGF-a
and HO-1 were significant increase in drug A group. IL-6 is often inhibits the
production of pro-inflammatory cytokines as IFN-g, IL-1 and TNF-a. Drug A
supplementation also resulted in a marginal increase in TGF-b levels.
Therefore, we investigated whether Drug A could synergize with MSCs in
suppressing immune responses. The functionality of the engrafted human
immune cells was verified using a mixed lymphocyte reaction (MLR) to measure T cell proliferation. Furthermore, we showed that the effect of drug A is
exerted through inhibiting inflammatory cytokines induced TNF-a and IFN-g
expression, and promoting anti-inflammatory cytokines induced PGE2
expression. Altogether, these data suggest that treatment with drug A in UCBMSCs enhanced the immunosuppressive capacity of UCB-MSCs. High capacity UCB-MSCs a very useful model for clinical application of allogeneic cell
therapies.
287
MSC AS CARRIER CELLS FOR A RHABDOVIRUS-BASED
ONCOLYTIC VIROTHERAPY OF OVARIAN CANCER
C Dold1, A Muik2,5, CU Rodriguez1, J Kimpel1, H Fiegl3, S Kuci4, C Marth3,
M Von Laer1
1
Division of Virology, Innsbruck Medical University, Innsbruck, Austria,
2
Applied Virology and Gene Therapy, Institute for Biomedical Research
Georg-Speyer-Haus, Frankfurt am Main, Germany, 3Department of
Gynecology and Obstetrics, Innsbruck Medical University, Innsbruck, Austria,
4
University Children’s Hospital III, Department of Hematology, Oncology
and Hemostaseology, Frankfurt am Main, Germany, 5Paul-Ehrlich-Insitut,
Langen, Germany
Ovarian Cancer is one of the leading causes of death from gynecological
malignancies in the western world. A promising new approach is the vesicular
stomatitis virus (VSV)-based oncolytic virotherapy as VSV is one of the most
potent oncolytic viruses and there is no pre-existing antiviral immunity in the
human population. However, VSV’s glycoprotein-mediated inherent
neurotoxicity has hindered clinical development so far. Furthermore, delivery
of virus to disseminated tumor cells and infiltration of solid tumor tissue has
to be optimized. To abrogate VSV’s neurotoxicity, we pseudotyped VSV
with the non-neurotropic glycoprotein of the lymphocytic choriomeningitis
virus (LCMV-GP). Oncolytic potency was tested in cancer cell cultures and
xenograft mouse models. Furthermore, toxicity was evaluated in tumorbearing immunocompromized NOD/SCID mice. VSV-GP exhibited a more
than 10 6-fold higher LD50 compared to VSV wild-type upon intracranial
injection in mouse brain, and the maximum tolerated dose was 10 6 fold
higher for VSV-GP upon intratumoral injection in immunocompromized
mice. Furthermore, effective oncolytic activity of VSV-GP could be
demonstrated in ovarian cancer cell cultures. Accordingly, intratumoral injection of VSV-GP into ovarian cancer s.c. xenografts led to tumor remission
in all animals. Relapsing tumors were still susceptible to VSV-GP mediated
oncolysis. To improve delivery of VSV-GP to distant tumor sides and to
circumvent recognition of the virus by the host immune system, we use
mesenchymal stem cells (MSC) as carrier cells. MSC were shown to be
effective carrier cells for VSV-GP as they can be infected easily and virus
replicates to high titers. The results of our in vitro and in vivo studies
demonstrate that LCMV GP-pseudotyped VSV exhibits a highly beneficial
toxicity and efficacy profile in ovarian cancer, which can be further optimized
using MSC as carrier cells.
ˇ
284
POST-CRYOPRESERVATION VIABILITY OF MESENCHYMAL
STEM CELLS
A Van Campenhout, L Swinnen, J Klykens, T Devos, G Verhoef
UZ Leuven, Leuven, Belgium
286
DRUG A ENHANCES THE IMMUNOSUPPRESSIVE PROPERTIES
OF
HUMAN
UMBILICAL
CORD
BLOOD-DERIVED
MECENCHYMAL STEM CELLS
Y Hong, S Choi, Y Yang, W Oh, E Jeon
Biomedical Research Institute, MEDIPOST Co., Ltd., Seoul, Korea,
Republic of
ˇ
secretion was responsible for this beneficial effect in part by preventing
inflammation. Intriguingly, secretion of Ang-1 was closely associated with upregulation of anti-inflammatory factors, which ultimately affect the outcome of
the paracrine effect by UCB-MSCs, providing new insights into the potential
clinical applications of MSC-based therapy.
S83
ˇ
S84
Poster Abstracts
288
ENHANCED CELL RECOVERY RESULTING IN HIGHER YIELDS
OF MSC WHEN USING THE COBE 2991 FOR CELL DENSITY
SEPARATION OF BONE MARROW MATERIAL: A VALIDATION
STUDY BETWEEN TWO MSC PRODUCTION CENTERS
Tden Bleker-Grijsen1, L Carlee1, E Steeneveld2, I Lommerse1, H Roelofs2,
J Zwaginga2, C Voermans1, D Thijssen-Timmer1
1
Laboratory for Cell Therapy, Department of Experimental
Immunohematology, Sanquin Research and Landsteiner Laboratory,
University of Amsterdam, Amsterdam, Netherlands, 2Department of
Immunohematology and Bloodtransfusion, Leiden University Medical Center,
Leiden, Netherlands
Bone-marrow derived mesenchymal stromal cells are multi-potent cells which
have immunomodulatory properties and the ability to migrate to sites of
inflammation. MSC infusions have been shown to be effective in patients
suffering from steroid refractory acute Graft versus Host Disease (GvHD),
irrespective of donor origin. Currently a multi-center, randomized phase III
trial (HOVON 113) is performed with MSC in steroid refractory GvHD patients. To this end we performed a validation study to compare cultureexpanded MSC generated at two production sites (Leiden and Sanquin). The
protocol for clinical grade MSC production was developed in Leiden and
implemented at Sanquin. Bone marrow of three healthy donors was split between the two production centers and cultured in parallel in the presence of
FBS until passage 2. Sanquin used a fully closed cell density separation procedure (Cobe 2991 cell processor) as an alternative to a ficoll procedure in
tubes, CellSTACKS with tubing systems instead of open flasks and recombinant TrypLE Select instead of Trypsine-Versene. All MSC cultures passed the
release criteria as described in Le Blanc et al. 2008. Cell recovery after the
density separation in the Cobe 2991 was two-fold higher compared to the
manual separation. In addition 20% more MSC were obtained after seeding the
same amount of mononuclear cells (MNC) obtained with the Cobe 2991.
Equal amounts of MSC were harvested in the two centers when corrected for
MSC numbers at Passage 0. No differences in viability were observed when
TrypLE Select was used. In conclusion Sanquin has validated the production of
clinical grade MSC in a more closed culture system and can now participate as a
production center in the HOVON 113 study. The use of the Cobe 2991 increases the MNC recovery from bone marrow resulting in higher yields of
MSC.
289
THE USE OF HUMAN BONE MARROW STEM CELLS REDUCES
ENDOTOXIN-INDUCED LUNG INJURY IN SHEEP
A Ting1, N Lehman1, N Cardenes2, E Kocyildirim3, M Romagnoli4, L Mroz2,
E Carceres2, J Tedrow2, C Bermudez4, M Rojas2
1
Regnerative Medicine, Athersys, Inc, Cleveland, Ohio, United States,
2
Simmons Center for Interstitial Lung Disease, Division of Pulmonary,
Allergy & Critical Care Medicine, University of Pittsburgh, Pittsburgh,
Pennsylvania, United States, 3McGowan Institute for Regenerative Medicine,
University of Pittsburgh, Pittsburgh, Pennsylvania, United States,
4
Department of Cardiothoracic Surgery, University of Pittsburgh, Pittsburgh,
Pennsylvania, United States
The objective of this study was to assess the use of an adult bone marrow
derived stem cell, MultiStemÒ, in an ovine model of Acute Respiratory
Distress Syndrome or ARDS. ARDS is the cause of 10-15% ICU admissions,
and is followed by poor survival and diminished quality of life. Although
existing therapeutic approaches have decreased mortality, they generally
aggravate lung injury. This presents the need for alternative therapeutic options. The infusion of mesenchymal stem cells (MSC) has been shown to be
extremely beneficial in the treatment of different murine models of induced
ARDS. Treatment with MSC improves lung function as measured by histology, decrease in lung water content and inflammatory cytokines in plasma and
bronchoalveolar lavage that result in restoration of alveolar fluid clearance and
increase in survival. However, the murine model is limited in that it is difficult
to measure gas exchange performance, systemic/hemodynamics, and ventilation/perfusion mismatch. Using sheep for the study of ARDS allows for these
measurements and sheep are similar to humans in global physiologic response
and sensitivity to endotoxin, a common cause of ARDS. Therefore, the ovine
model of ARDS has a larger clinical translational potential. Sheep were kept
under anesthesia and the chest was open to facilitate visual evaluation of lung
injury and sample biopsies at different time points. Sheep received a dose of
3mg/Kg E. coli endotoxin over 30 minutes. Pulmonary artery pressure (PAP)
was measured continuously and arterial blood gases were measured every 30
minutes to determine the onset of the acute phase of pulmonary hypertension.
The experimental group received 40 million human MultiStem by intratracheal
delivery 30 minutes after the end of the infusion of endotoxin. Our results
showed a significant recovery of PO2 and PCO2 levels in the group treated
with MultiStem within 1.5 hours of maximum lung injury.
290
MICRORNA PROFILING OF MESENCHYMAL STEM CELLS
(MSCS) PROVIDES A PUTATIVE, GENERAL MSC SIGNATURE
AND DISCRIMINATES CELLS DERIVED FROM DIFFERENT
TISSUES
D Mallison, D Olijnyk, S Paterson, S Ridha, D Dunbar, V O’Brien
Sistemic Ltd, Glasgow, United Kingdom
Background: Heterogeneity of MSCs, arising from the different tissue sources
and culturing techniques, presents a significant challenge if manufacturers are
to understand and preserve the favourable characteristics of these cells during
their production. To address the challenge there is a need for reference standards and reliable assays to characterise MSCs and provide insight into their
batch consistency and which are feasible ways to assess the potency of the final
product at release. MicroRNA (miRNA) profiling is a highly-informative and
broadly-applicable technique for cell characterisation: miRNA expression analyses provide sensitive assays of a stable analyte which can be used to assess the
comparability of cell populations. Furthermore, alterations in miRNA
expression can reveal deviations in cell phenotype which may crucially impact
on the potency of manufactured cell populations.
Methods: Total RNA was prepared using a column-based kit (Exiqon) and
miRNA expression profiles were analysed using microarray slides (Agilent
human miRNA 8*60K V16.0 of miRBase).
Results: MicroRNA profiles where derived for human monocytes, leucocytes and MSC populations. The MSC cells were from three different tissue
sources e adipose tissue, bone marrow and cord blood. In total, 55 datasets
were generated from the microarray analysis. High-level visualisation
demonstrated clustering of the cells based on lineage. Further analysis of the
MSC cells revealed a high degree of homogeneity in miRNA profiles (>40%
identity) between all MSCs irrespective of tissue origin. However, miRNA
differences were also identified which define the tissue of origin.
Conclusion: These data support the use of microRNA profiles to define:
(1). a microRNA signature which describes an MSC population irrespective of
tissue of origin and with potential as a standardisation assay; (2) miRNA biomarkers that permit the identification of the tissue of origin of the MSCs.
291
COMPARISON OF HUMAN PLATELET LYSATE AND FETAL
BOVINE SERUM FOR OPTIMAL CULTURE CONDITIONS OF
NEONATAL AND ADULT MESENCHYMAL STEM CELLS
CG Taylor, RN Dayment, MZ Albanna, EJ Woods
Cook General BioTechnology, LLC, Indianapolis, Indiana, United States
Fetal bovine serum (FBS) has significantly contributed to the large-scale
expansion of animal and human mesenchymal stem/stromal cells (MSCs) and
the rapid development of cell-based therapeutics; however, it poses several
regulatory and species cross-contamination challenges hindering the clinical
transition of most products. Serum-free media (SFM) supplemented with
several growth factors has been proposed as an alternative approach to FBS;
however, custom media development is often needed based on the cell type,
source, and species. Also, the high cost of SFM makes it an impractical option
for large-scale cell expansion. Hence, there is a pressing need to find a humanbased media additive. hPL is derived from human platelets and contains similar
growth factors and cytokines found in FBS at comparable levels. It has been
previously demonstrated that hPL supports the growth of various cells. The
focus of this study was to evaluate the ability of a serum converted hPL that
does not require the use of heparin (PL-NH) and standard hPL requiring
heparin (PL-H) to support attachment, proliferation, and maintenance of
multipotent properties of neonatal and adult MSCs from different tissues at
different concentrations of media. Variants of hPL used in this study (COOK
HPLÔ NH and H) are produced at an industrial scale (minimum lot size of 20
L) with high lot-to-lot consistency and purity. Preliminary results of both
versions of hPL at different concentrations (2.5, 5, and 10%) supported cell
attachment and proliferation of amniotic-, bone marrow-, adipose-, and cord
blood-derived MSCs to comparable levels to FBS at all concentrations. The
multipotency of MSCs expanded in hPL was maintained throughout several
passages. This study demonstrates the ability of using hPL as an alternative
media additive to FBS for large-scale expansion of adult and neonatal stem cells
for cell-based therapeutics.
292
COMPARISON OF CLINICALLY APPROVED HUMAN PLATELET
LYSATES FOR CULTIVATION OF MESENCHYMAL STROMAL
CELLS FROM BONE MARROW AND ADIPOSE TISSUE
J Tratwal, B Follin, RH Søndergaard, M Juhl, A Ekblond, J Kastrup,
M Haack-Sørensen
Cardiology Stem Cell Centre, Rigshospitalet, Copenhagen, Denmark
Background: New developments and progress in stem cell technology in
recent years has given rise to new therapeutic strategies for different degenerative diseases. Mesenchymal stromal cells (MSCs) have gained much attention for regenerative medicine because they are capable of self-renewal, can
differentiate to a variety of cell lineages, have trophic and immunosuppressive
effects, and have been shown clinically to alleviate symptoms of several diseases.
Clinical translation of MSC-based approaches often requires in vitro cultureexpansion to achieve sufficient number of cells.
Methods: MSCs from bone marrow (BMSCs) and adipose tissue-derived
stromal cells (ASCs) obtained from three donors were culture expanded in
three different human platelet lysates (hPL), manufactured differently, but each
fulfilling tracking criteria imposed by good manufacturing practice. BMSCs
and ASCs were cultured in 5% PLT-Max (Mill Creek), PL-H and PL-NH
(Cook HPLTM) with Minimum Essential Medium Eagle-alpha, and were
compared to standard culture conditions with 10% fetal bovine serum (FBS).
Cell morphology, proliferation, phenotype, chromosomal stability and lineage
differentiation of BMSCs and ASCs were analysed.
Results: BMSCs and ASCs cultured in all three hPL-media showed a significant increase in proliferation capacity compared to FBS culture. In general,
BMSCs and ASCs fulfilling the ISCT criteria regarding BMSC and ASC
phenotype. Comparative genomic hybridization was performed to assess the
level of genomic stability of the BMSCs and ASCs cultured in hPL media or
FBS medium. The BMSCs and ASCs were induced to differentiate into
osteogenic, adipogenic or chondrogenic lineages, and both BMSCs and ASCs
from all four original culture conditions were able to differentiate toward the
three lineages.
Conclusion: All three clinically approved human platelet lysates accelerate
proliferation of BMSCs and ASCs and meet the ISCT requirements without
exhibiting chromosomal aberrations.
293
294
STANDARDIZED HUMAN PLATELET LYSATE SUPPLEMENT
DEMONSTRATES TO BE AN EFFECTIVE, SERUM-FREE,
XENO-FREE, FBS REPLACEMENT FOR CULTURING AT-/BM-/
AND UC-MESENCHYMAL STEM CELLS
Y Tseng1, K Liu1, C Ku1, T Burnouf2,3
1
GwoWei Technology Inc., Vancouver, British Columbia, Canada, 2Human
Protein Process Sciences, Lille, France, 3Institute of Biomaterials and Tissue
Engineering, Taipei Medical Universit, Taipei, Taiwan
Fetal bovine serum (FBS) is not recommended for ex vivo culture of human
cells for transplantation into patients as there is risk of immunological reactions
and of transmitting bovine pathogens. FBS in clinical stem cell culture is
prohibited in Germany and may be prohibited in Europe, USA and other
countries in the future. Human platelet lysates contain a natural mixture of
growth factors including (PDGF-AA, -AB, and -BB), vascular endothelial
growth factor (VEGF), transforming growth factor-b (TGF-b1 and TGF-b2),
epidermal growth factor (EGF), basic fibroblast growth factor (FGF), and can
successfully replace FBS for enhanced cell culture of mesenchymal stem cells
(MSC) and other human cells. Gwowei R&D has applied their proprietary
platelet fractionation process to produce a standardized human platelet-derived
growth factors supplement to replace FBS supplement for human cell culture.
Gwowei’s process fractions allogeneic platelets (supplied by ARC & CBS in
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NA) to produce a functionally consistent human Platelet Lysate (hPL) supplement, which contains a mixture of primary growth factors, including:
PDGF-BB, PDGF-AB, FGF, TGF-b1. From collaborators in North America,
Europe and China, Gwowei R&D has extensive testing data demonstrating that
human AT-MSC, BM-MSC, and UC-MSC isolated and grown with this
standardized hPL supplement achieves; a sub-confluent layer (1), exhibit typical
spindle morphology (2) and maintain multi-potent capability (3). Gwowei’s 5%
hPL (v/v) supplement (i.e. 4 lots) consistently demonstrated superior performance compared to 10-20% defined FBS in both primary and expansion culture. This new 5% hPL (UltraGROÔ) is a promising, scaleable and
standardized supplement to consider for replacing 10-20% FBS in medium for
growing and producing mesenchymal stem cells derived from adipose tissue,
bone marrow and umbilical cords.
295
MULTIPLE
INTRA-ARTICULAR
TRANSPLANTATIONS
ENHANCES THE BENEFIT OF DENTAL TISSUE DERIVED
STEM CELLS THERAPY FOR THE TREATMENT OF CHRONIC
OSTEOARTHRITIS
R Bootcha1, J Temvilitr2, P Sriwattanakul3, S Petchdee4
1
Small Animal Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart
University, Nakorn Pathom, Thailand, 2Companion Animal Clinical Sciences,
Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand,
3
BioEden Asia Tooth cell bank, Bangkok, Thailand, 4Large Animal and
Wildlife Clinical Sciences Faculty of Veterinary Medicine, Kasetsart
University, Nakorn Pathom, Thailand
There are several types of osteoarthritis treatments in dogs. However, the
results of many surgical and medical treatments are still unsatisfactory. Cell
transplantation of dental tissue derived stem cell is a new treatment that could
restore the degenerated articular cartilage in dog with osteoarthritis (OA). In
this study, the production of canine dental derived stem cells and their possible
application in cellular therapy for dogs were evaluated. We hypothesize that
multiple doses of puppy deciduous teeth stem cells (pDSCs) might improve
clinical outcome of OA and can be replaced the drug therapy. The clinical
effects of multiple intra-articular injections of 5 millions pDSCs were evaluated
on 10 dogs with lameness associated with OA of the coxofemoral joints lasting
on average 3 months. The second doses of pDSCs were delivered within 28
days after the first transplantation. Clinical outcomes were evaluated by
radiographic evidence, gait changes including persistent lameness at walk or
trot, limited range of motion with pain and the responses to questionnaire from
the owners. Multiple injections of pDSCs showed that OA of coxofemoral
joints markedly improved with time. These findings suggest that multiple doses
of allogeneic dental tissue derived stem cells transplantations provide a significant potential for clinical uses in the treatment of lameness, and pDSCs
might be a novel strategy in the cell therapy for OA.
296
THE ACTIVATION OF DIRECTIONAL STEM CELL MOTILITY
BY GREEN LIGHT-EMITTING DIODE IRRADIATION
J Ho1,2
1
Taipei Medical University, Taipei, Taiwan, 2center for stem cell research,
Wan Fang Medical Center, Taipei Medical University, Taipei, Taiwan
Light-emitting diode (LED) irradiation is potentially a photostimulator to
manipulate cell behavior by opsin-triggered phototransduction and thermal
energy supply in living cells. Directional stem cell motility is critical for the
efficiency and specificity of stem cells in tissue repair. We explored that green
LED (530 nm) irradiation directed the human orbital fat stem cells (OFSCs) to
migrate away from the LED light source through activation of extracellular
signal-regulated kinases (ERK)/MAP kinase/p38 signaling pathway. ERK inhibitor selectively abrogated light-driven OFSC migration. Phosphorylation of
these kinases as well as green LED irradiation-induced cell migration was
facilitated by increasing adenosine triphosphate (ATP) production in OFSCs
after green LED exposure, and which was thermal stressindependent mechanism. OFSCs, which are multi-potent mesenchymal stem cells isolated from
human orbital fat tissue, constitutionally express three opsins, i.e. retinal
pigment epithelium-derived rhodopsin homolog (RRH), encephalopsin
(OPN3) and short-wave-sensitive opsin 1 (OPN1SW). However, only two
non-visual opsins, i.e. RRH and OPN3, served as photoreceptors response to
green LED irradiation-induced OFSC migration. In conclusion, stem cells are
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Poster Abstracts
sensitive to green LED irradiationinduced directional cell migration through
activation of ERK signaling pathway via a wavelengthdependent
phototransduction.
297
HAIR FOLLICLE DERMAL SHEATH DERIVED MESENCHYMAL
STEM CELLS: IN-VITRO CHARACTERIZATION AND EFFECTS
OF ITS CONDITIONED MEDIUM ON CUTANEOUS WOUND
HEALING
A Chua1, J Kua2, D Ma2, S Lee2
1
Skin Bank Unit / Department of Plastic, Reconstructive & Aesthetic Surgery ,
Singapore General Hospital, Singapore, Singapore, 2Department of Plastic,
Reconstructive & Aesthetic Surgery , Singapore General Hospital, Singapore,
Singapore
Aim: Characterize and evaluate the wound healing efficacy of dermal sheath
mesenchymal stem cell (DS-MSC)-derived conditioned medium in a healing
impaired murine excisional wound healing model.
Methods: DS-MSCs were isolated from hair follicles and cultured. In-vitro
studies were conducted to assess their proliferation capacity, colony forming
efficiency, expression of CD surface markers and differentiation potential. The
cytokine expression of DS-MSCs was determined through an antibody-based
protein array analysis. The effects of its conditioned medium on keratinocyte
proliferation, migration and angiogenesis were demonstrated. To investigate its
effect on cutaneous wound healing, the conditioned medium was applied to a
diabetic murine wound model and compared against a control and conditioned
medium derived from normal fibroblasts.
Results: In-vitro results revealed that DS-MSCs display a high proliferation
capacity and colony forming efficiency. They have a phenotype similar to that
of bone marrow derived MSCs (BM-MSCs) as they strongly stain for CD15,
CD29, CD49b and CD49d. They can also be differentiated into osteoblasts,
chondrocytes and adipocytes. Conditioned medium derived from these cells
had significant higher proportions of paracrine factors such as interleukin-6
(IL-6), IL-8 and growth-related oncogene. Keratinocyte proliferation as well as
endothelial cell migration and tube formation were enhanced by DS-MSC
conditioned medium and the overall wound healing time was shorter as
compared to fibroblast-derived condition medium and control.
Conclusion: The human hair follicle is a feasible source of MSCs which can
be easily harvested for wound therapy and tissue engineering. Conditioned
medium derived from these cells enhanced wound healing in both in vitro and
in vivo wound healing models. This could be attributed to elevated levels of a
milieu of paracrine factors involved in wound healing.
298
THE EFFICIENT FABRICATION OF EPIDERMAL CELL SHEETS
USING GAMMA-SECRETASE INHIBITOR
R Nakajima, S Takeda
Central Research Laboratory, Hitachi Ltd., Saitama, Japan
Epidermal cell sheets have been utilized for regeneration of skin defects and
prevention of esophageal stricture after endoscopic submucosal dissection.
Here, we developed a novel culture method to accelerate the fabrication of
epidermal cell sheets, that is; epidermal keratinocytes were cultured using gsecretase inhibitor during expansion of the cells to confluence and cultured
without g-secretase inhibitor during stratification. The proliferation of normal
human epidermal keratinocytes (NHEKs) on cell-culture inserts was promoted
using g-secretase inhibitor. However, NHEKs were not stratified completely
in the presence of g-secretase inhibitor. In contrast, NHEKs cultured using gsecretase inhibitor were stratified and differentiated by eliminating the inhibitor after reaching confluence. Real-time PCR analyses showed that the
gene expressions of putative epithelial stem/progenitor cell markers and
epidermis differentiation markers in the cell sheets fabricated by the novel
method were significantly higher than those in the cell sheets fabricated
without g-secretase inhibitor (control group). Immunofluorescence analyses
revealed that it was possible to fabricate well-differentiated epidermal cell
sheets efficiently by the novel method. The culture period was shortened to
67% comparing to that of control group. In feeder-free condition, stratified
epidermal cell sheets were also fabricated by using g-secretase inhibitor. Thus,
the novel culture method using g-secretase inhibitor can be effective for
fabricating epidermal cell sheets.
299
ASSESSMENT OF DIFFERENT VITAL STAINS FOR TRACKING
HUMAN MESENCHYMAL STEM CELLS: PHENOTYPICAL AND
FUNCTIONAL IN VITRO STUDIES
B Lukomska1, A Andrzejewska1, A Jablonska1, A Nowakowski1, M Janowski1,2
1
NeuroRepair Department, Mossakowski Medical Research Centre PAS,
Warsaw, Poland, 2Radiology and Radiological Science, Johns Hopkins
University, Baltimore, Maryland, United States
The success of cell therapy depends on the ability to monitor transplanted cells.
Our studies assess the effect of CMFDA, GFP and SPIO labeling of human
bone marrow derived mesenchymal stem cells (hBM-MSC) on their
morphology and functions.
Materials and Methods: hBM-MSC (Lonza) were cultured in MSCBM
medium +10% MCGS, L-glutamine, and gentamicin. Cell culture was maintained in 75 cm2 flasks at 37oC and 5% CO2, then hBM-MSC were transferred to 24-well plates and seeded at 5000 cells/well and labelled with
CMFDA, transfected with mRNA GFP or tagged with SPIO nanoparticles.
Vital observation of hBM-MSC morphology and intracellular persistence of
different agents used for labelling was performed over 14 days at various time
points. Proliferation potential of hBM-MSC was assessed by CCK-8 assay.
Concomitantly cell expression of different markers and adhesion molecules was
defined by immunocytochemistry.
Results: CMFDA, GFP or SPIO labelling did not affect the morphology
and baseline expression of CD-90, SSEA-4, CXCR-4, VLA-4 proteins of
hBM-MSC. No influence of any vital stains on hBM-MSC adipogenic differentiation potential was detected. Whereas with unlabelled cells CMFDAand GFP- but not SPIO tracking decreased proliferation rate of hBM-MSC.
Moreover, CMFDA and GFP are unsuitable for long detection of hBM-MSC.
The intracellular persistence of different agents was demonstrated for 7 days
after CMFDA labelling, up to 14 days after GFP transfection but more than 21
days after SPIO tracking.
Conclusions: SPIO particles provided efficient long lasting labeling of
human mesenchymal stem cells in vitro and did not modify viability, phenotype, proliferation potential or differentiation capability hBM-MSC. Our results indicate that SPIO particles are biocompatible with hBM-MSC and they
would be suitable labels for tracking cells to evaluate cell fate and distribution
after transplantation using MRI. Supported by NCR&D grant in ERA-NET
NEURON project: “MEMS-IRBI”.
300
RECONSTRUCTION OF DAMAGED CORNEAL EPITHELIUM
USING DENTAL TISSUE DERIVED STEM CELLS
N Pattanapol1, A Tyananupat2, P Sriwattanakul3, S Petchdee4
1
Small Animal Teaching Hospital, Faculty of Veterinary Medicine, Kasetsart
University, Nakorn Pathom, Thailand, 2Companion Animal Clinical Sciences,
Faculty of Veterinary Medicine, Kasetsart University, Bangkok, Thailand,
3
BioEden Asia Tooth cell bank, Bangkok, Thailand, 4Large Animal and
Wildlife Clinical Sciences, Faculty of Veterinary Medicine, Kasetsart
University, Nakorn Pathom, Thailand
Corneal ulceration and keratitis are leading causes of blindness. Even though
the therapeutic and management strategies for corneal injury have been studied, the effective treatment is still limited success. Potential application of cellbased therapy has been considered to provide effective treatment options for
corneal repair. The current study investigates whether subconjunctival transplantation of dental tissue derived stem cells (DTSCs) promotes corneal
regeneration and evaluates the efficacy of puppy deciduous teeth stem cells
(pDSCs) in the treatment of corneal ulcers in dogs. In the rabbit model of
corneal ulceration, 0.5 millions cells DTSCs were transplanted into subconjunctival space. The clinical data were confirmed by histological analysis, and
RT-PCR analysis of connexin 43 (Cx43). Transplantation of DTSCs showed a
significant difference in the epithelial cell layers and Cx43 change between
control and treatment groups, suggesting that DTSCs promotes corneal
reconstruction. In clinical trial, the same amount of pDSCs were injected into
subconjunctival space of 3 dogs, it was showed that corneal transparency of the
eyes that underwent pDSCs transplantation was improved throughout the
follow-up. These results suggest that pDSCs provide cell renewal in corneal
wound healing and demonstrated the potentials of pDSCs administration for
clinical applications.
301
EXPERIMENTAL AND CLINICAL BENEFIT OF DENTAL TISSUE
DERIVED STEM CELLS IN REGENERATIVE MEDICINE
S Petchdee1, R Bootcha2, N Pattanapol2, P Sriwattanakul3
1
Large Animal and Wildlife Clinical Sciences, Kasetsart University, Nakorn
Pathom, Thailand, 2Small Animal Teaching Hospital, Faculty of Veterinary
Medicine, Nakorn Pathom, Thailand, 3BioEden Asia Tooth cell bank,
Bangkok, Thailand
Cellular therapies and stem cells are one of the most promising areas of
regenerative medicine. The use of stem cells therapies in many untreatable
injuries and diseases has been a considerable research focus over the last
decade. Several stem cell types have been studied as the potential candidates to restore the structure and function of damaged tissues and organs.
The dental tissue derived stem cells (DTSCs) have revealed potential for
their use as a novel alternative resource in regenerative medicine and
dentistry. DTSCs have mesenchymal stem cell like (MSC) qualities,
including the capacity for self-renewal and multilineage differentiation
potential. In this study, we demonstrate the potential applications of
DTSCs as a tool to repair damaged tissues and organs. Diseases related to
chronic inflammation such as ischemic heart diseases, osteoarthritis and
ocular injury have been investigated through experimental and clinical trial
design to clarify the use of DTSCs therapies. Transplantation of DPSCs
provided good choice in terms of tissue regeneration and healing. Our
results suggesting that DTSCs may provide new approaches for the
treatment of diseases and tissue repair. However, the important points in
DTSCs biology, such as homing and immune-regulation are required a
further study of underlying mechanisms to support the application of
DTSCs in the future.
302
ANALYSIS OF CRITICAL FACTORS INFLUENCING PRIMARY
CELL QUALITY AND EXPANSION POTENTIAL OF ADSC
S Chen3, P Shen1, C Huang2, P Tseng2,1, W Lo2
1
MSC laboratory, HealthBanks Biotech Co., Ltd., Taipei, Taiwan, 2Research
and Development, HealthBanks Biotech Co., Ltd., Taipei, Taiwan, 3Division
of Plastic and Reconstructive Surgery, Department of Surgery, Tri- Service
General Hospital, National Defense Medical Center, Taipei, Taiwan
The feasible and abundant advantages of adult adipose-derived stem cells
(ADSCs) obtained by liposuction have made ADSCs an attractive MSC
source of autologous therapy for tissue engineering and regenerative medicine. To optimize the condition for preparation of adipose tissue and
manufacture of ADSC, we tried to identify the critical factors influencing
primary cell quality and expansion potential of ADSC. First of all, we
demonstrated that classical liposuction was the ideal collection technique to
get superior stromal vascular fraction (SVF) isolation efficiency when
comparing to water-jet liposuction (1.720.3*105 versus 0.30.12*105 cells/
ml adipose tissue). After collection, the processed lipoaspirate (PLA) tissue
should be storaged/transported at low temperature and set in subsequent
process within 48 hours. Furthermore, tissue/cell viability evaluated by XTT
assay was highly positively correlated to primary SVF isolation efficiency,
primary ADSC isolation efficiency and expansion efficiency. We also manifested the donor age was inversely proportional to both the tissue/cell
viability and the SVF/ADSC isolation efficiency. In conclusion, our results
validated the effective method for evaluation and revealed the critical factors
that influenced ADSC potential, which should be considered as the major
issue in manufacture and quality control of ADSC product for clinical
purpose.
303
LN-521 ENABLES DERIVATION, CLONAL CULTURE AND EASY
SINGLE-CELL PASSAGE OF HUMAN PLURIPOTENT STEM
CELLS WITHOUT ARTIFICIAL INHIBITORS
T Kallur, J Ericsson, K Tryggvason
BioLamina, Stockholm, Sweden
Laminins are a group of 16 protein isoforms found in the basement membrane
in the extracellular matrix. The natural environment for all stationary cells in
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the body consists of other similar cells and the basement membrane. Laminins are the only tissue-specific proteins in the basement membrane and
therefore one critical factor that differentiates one niche from another. LN521 is one of the first extracellular matrix proteins already expressed by the
cells of the inner cell mass in the blastocyst. Our data show that when using
LN-521 to create a niche on cell culture plates, human embryonic stem and
induced pluripotent stem (hPS) cells can be cultivated at clonal density and
grown indefinitely in a pluripotent state. In addition, LN-521 support, for the
first time, derivation of new human ES cell lines in a chemically defined and
xeno-free manner. This is the first xeno-free, defined and biologically relevant matrix that truly supports hPS cells in a clinically relevant, ethical and
robust way in cell culture. LN-521 also has growth factor like properties and
hPS cells cultured on LN-521 grow twice as fast compared to all other tested
matrices. Also due to the biological properties of LN-521, stem cells cultured
on LN-521 can be split 1:20 or up to 1:30 as single cells without the addition
of artificial ROCK inhibitor, which can push genetical variations. In
conclusion, we show that LN-521 is an optimal matrix for hPS cell culture
due to its biological relevance that allows derivation, clonal cultivation and
long-term pluripotent cell growth with single-cell passaging without any
artificial inhibitors that may modify the cell population.
304
EFFICIENT EXPANSION AND OSTEOGENIC DIFFERENTIATION
OF MESENCHYMAL STEM CELLS IN MICROCARRIER CULTURE
K Tan, E Sim, A Shekaran, A Chen, S Reuveny, S Oh
Stem cells, Bioprocessing Technology Institute, Agency for Science,
Technology and Research, Singapore, Singapore, Singapore
Mesenchymal stem cells (MSCs) can be expanded in vitro, and induced to
undergo osteogenic differentiation for potential clinical therapies, e.g. in repair
of bone fractures. Traditional cell expansion and osteogenic differentiation
occur in two-dimensional cell culture dishes. However, three-dimensional
culture on microcarriers in agitated culture offers the advantages of increased
space, material and labour efficiency. We carried out a comparison of
mesenchymal stem cell expansion in conventional monolayer culture vs culture
on three dimensional spherical microcarriers in spinner flasks, with subsequent
cell harvest and differentiation in monolayer culture. MSCs expanded in
monolayer or microcarrier culture continued to express MSC markers. However, integrin gene expression profiles were altered post-expansion, with
microcarrier-cultured MSCs expressing higher levels of integrins alpha 2 (5fold) and alpha 6 (10-fold), compared to monolayer cultured MSCs. After
MSCs were harvested from monolayer or microcarrier cultures, plated in
monolayers and cultured in differentiation media, osteogenesis proceeded with
different kinetics between monolayer- and microcarrier-expanded MSCs,
including alkaline phosphatase activity (50-70 fold higher in monolayerexpanded cells) and levels of other osteogenic genes measured by RT-PCR
(RUNX2, BGLAP, OPN). Despite differences in the kinetics of integrin and
osteogenic gene expression, monolayer- and microcarrier-expanded cultures
both supported high levels of mineralization at Day 28 of osteogenic differentiation (total incorporated calcium and alizarin red staining). These results
indicate that microcarrier culture allows for scalable expansion of mesenchymal
stem cells while maintaining equivalent cell quality in terms of proliferation and
differentiation capacity as compared to conventional culture methods, and may
thus represent a more commercially viable method of cell expansion and differentiation for future clinical therapy.
305
USE OF RECOMBINANT COLLAGENASES CLASS I AND II IN
OPTIMIZATION OF CELL PURIFICATION FOR TISSUE
ENGINEERING APPLICATIONS
M Salamone2,3, M Pampalone1, S Saladino1, G Ghersi1,3
1
Department of Sciences and Biotechnology, Chemistry and Pharmaceutics
(STEBICEF), University of Palermo, Palermo, Italy, 2Institute of Marine
Coastal Environment, Council of National Research, Capo Granitola (TP),
Italy, 3Abiel s.r.l., Palermo, Italy
Tissue dissociation/primary cell isolation and harvesting are main applications for enzymes in regenerative medicine and cell biology studies. Despite
the widespread use of enzymes for these applications, their mechanism of
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Poster Abstracts
action is not well understood. As a result, the choice of one technique over
another is based more on past experience than on an understanding of why
the method works and what modifications could lead to even better results.
Clostridium histolyticum is a Gram positive anaerobic pathogenic to humans.
It produces a variety of collagenase, which efficiently degrade the collagen
present in the connective tissue. The collagenases from Clostridium histolyticum are metalloproteases belonging to the family M9 having collagenolytic activity. The chromosome of C. histolyticum contains two distinct
genes, namely colG and colH, encoding collagenases isoforms class I and
class II respectively. Recombinant forms of class I and II collagenases were
synthesized. These isoforms were characterized by analyzing their relative
activities against the native insoluble collagen and synthetic substrates such as
soluble synthetic peptide FALGPA (2-furanacriloil-L-leuciglicil-L-prolyl-Lalanine) and the synthetic peptide PZ (4-phenylazobenzyloxycarbonyl-Lprolyl-L-leucyl-glycyl-L-prolyl-D-arginine). Class I enzymes have high
collagenase activity and a modest activity against peptides FALGPA and PZ,
while the enzymes of class II have a modest activity against native collagen
and a high activity of the peptides FALGPA and PZ. Additionally, collagenase class I are able to recognize different isoforms of collagen in native
form, since they possess two binding domains predisposed to interact with
collagen molecules; while those of class II recognize the linear sequence. In
conclusion, the cell extraction process is the result of the complementary
activity of both classes of enzymes.
306
COMPLETE RAT SPINAL CORD TRANSECTION AS A FAITHFUL
MODEL OF SPINAL CORD INJURY FOR HUMAN CELL
TRANSPLANTATION
S Erceg, D Lukovic, S Bhattacharya
CEll Therapy and Regenerative Medicine, CABIMER, Seville, Spain
Spinal cord injury (SCI) results in neural loss and consequently motor and
sensory impairment below the injury. There are currently no effective therapies
for the treatment of traumatic SCI in humans. Various animal models have
been developed to mimic human SCI. Widely used animal models of SCI are
complete or partial transection or experimental contusion and compression,
with both bearing controversy as to which one is more appropriate reproduce
the human SCI conditions. Here we present the widely used procedure of
complete spinal cord transection as a faithful animal model to investigate neural
and functional repair of the damaged tissue by the exogenous human transplanted cells. This injury model offers the advantage of complete damage to a
spinal cord at a defined place and time, is relatively simple to standardize and is
highly reproducible.
binding protein, etanercept (ET), resulted in significant reduction in CCTF
when compared results in pFUS only treated mice. Treatment with IB or ET
prior to pFUS and IV MSCs resulted in significant (ANOVA p<0.05)
reduction in homing to pFUS treated M or K similar to numbers of cells
located in the contralateral tissue. pFUS exposure in COX2-/- knockout mice
resulted in no differences in number of MSCs homing to treated tissue
compared to contralateral control. pFUS induces a transient molecular zip code
in treated tissue that can be used to evaluate drug-host interactions through
interference of pathways and have an important impact on cell homing and
therapy. 1. Ziadloo A, Stem Cells 2012;30:1216, Burks S, Stem Cells
2013;31:2551
308
MESENCHYMAL STROMAL CELLS (MSC) THERAPY RESTORES
RADIATION-INDUCED DYSFUNCTION: NEW INSIGHT FOR
PELVIC RADIATION DISEASE TREATMENT
N Mathieu, C Durand, L Moussa, C Demarquay, R Bessout,
MM Benderitter, A Semont
IRSN, Fontenay aux roses, France
Patients who undergo pelvic radiotherapy may develop high incidence of
undesirable chronic gastrointestinal complications considered as a new disease
the so called “pelvic-radiation disease”. The lack of curative treatment and the
potential severity of the disorder highlight the importance of novel and
effective therapeutic strategies. We used stem cell-based approaches using
mesenchymal stromal cells (MSC) from bone marrow and previously
demonstrated that MSC therapy reduces irreversible radiation-induced
colonic ulcers and prolongs animal survival (Semont et al, 2013). MSC are first
trapped by lung but also scarcely engrafted in colonic mucosa. MSC also
mobilize endogenous MSC that could endure the benefit over time. MSC act
through abscopal and/or local effects. Secretion of several factors by MSC is
associated to an enhancement of the proliferation of colonic epithelial progenitor/stem cells (SOX9+) and to a decrease of radiation-induced inflammation (Bessout et al, 2013). Chronic visceral pain is a cardinal feature of
radio-induced side effects and is very devastating for patients. New data obtained recently demonstrated that MSC reverse radiation-induced chronic
mechanical allodynia probably by a mast cell-dependent mechanism. This
work constitutes new insights on MSC ability to limit functional consequences
of radiation-induced toxicity in the pelvis. We highlight new mechanisms of
MSC action.
307
CYCLO-OXYGENASE OR TNF ALPHA INHIBITORS INTERFERES
WITH THE ENHANCED MESENCHYMAL STEM CELL HOMING
INDUCED BY PULSED FOCUSED ULTRASOUND: IMPLICATION
FOR REGENERATIVE MEDICINE
P Tebebi, S Burks, B Nguyen, S Kim, JA Frank
Frank Laboratory, Nat’’l Inst of Health, Bethesda, Maryland, United States
309
ISOLATION AND CHARACTERIZATION OF MESENCHYMAL
STROMAL CELL-DERIVED MEMBRANE VESICLES
G Bertolino1,2, A Andrade1, J Riewaldt1, J Karbanova3, D Corbeil3,
M Odendahl1, T Tonn1,2
1
Experimental Transfusion Medicine, German Red Cross Blood Donation
Service North-East, Technische Universität Dresden, Dresden,
Germany, 2Center for Regenerative Therapies Dresden, Technische
Universität Dresden, Dresden, Germany, 3Tissue Engineering Laboratories
(BIOTEC), Technische Universität Dresden, Dresden, Germany
Recent studies have demonstrated the utility of noninvasive pulse focused ultrasound (pFUS) in enhanced homing permeability and retention (EHPR) of
stem cells to targeted tissues (1). We show that pFUS targeted exposure to
skeletal muscle (M) or kidney (K) results in nondestructive micro-environmental changes associated with increased expression cyclo-oxygenase (COX2)
in tissues along with a cascade of cytokines, chemokines and trophic factors
(CCTF) and cell adhesion molecules (CAM) initiating within 10 minutes and
lasting up to 48 hours. pFUS induced a transient 4-6x increase in Tumor
Necrosis Factor alpha (TNFa) within 10 minutes after exposure that returned
to control levels of protein expression by 30 minutes. Following TNFa increases there was increases in both pro-inflammatory and anti-inflammatory
factors (e.g., Interleukin (IL) 1, IL2, IL6, IL10, IL13, IL15, IL12p40, MCP-1,
RANTES, VEGF, M-CSF, MIP-2, PDGF and FGF, EGF, TGFb) and CAM
expressed in pFUS treated M or K. Coupling pFUS with IV of mesenchymal
stem cells (MSC) after resulted in 5-8 times (ANOVA p<0.01) more of infused
cells homing to M or K as compared to contralateral tissue. Pretreatment prior
to pFUS with nonspecific COX inhibitor, ibuprofen (IB) or TNFa receptor
Introduction: Recently, several groups have reported the secretion of membrane vesicles (MVs) from various cell types, including mesenchymal stromal
cells (MSCs). Interest in MVs has intensified due to the recognition of their
role as an important component of the intracellular microenvironment.
Therefore, our work aimed to characterize MSC-derived MVs and test if they
share immunomodulatory properties with MSCs.
Methods: MVs were isolated from bone marrow (BM-MSCs, n¼3) and
adipose tissue-derived MSCs (AT-MSCs, n¼3) by conventional ultracentrifugation (UC-MVs) or ultracentrifugation on 30 % sucrose cushion (SC-MVs).
For their characterization, flow cytometric analysis (CD9, CD63 and CD81)
and Nanoparticle Tracking Analysis were performed. Immunomodulatory
capacity was assessed by Lymphocyte Proliferation Assay (MV concentrations:
10-100mg/mL).
Results: Analysis of total protein quantification demonstrated that UCMVs exhibited higher protein concentrations than SC-MVs, although UCMVs protein values did not correlate with the number of MV-secreting
MSCs. In SC-MVs, however, this correlation could be made. This finding
suggests that UC-MVs contain high amounts of protein not originating
from MVs. Comparison of MVs isolated by both methods showed that in
UC-MVs the particle size pattern was typical of large protein aggregates that
co-sediment with MVs during ultracentrifugation. In SC-MVs, the major
particles had the typical MV size range (90 to 300 nm). Presence of MVs in
SC-MVs was corroborated by detection of the typical MV markers CD9,
CD63 and CD81. In our hands, immunosuppressive potential of BM-MSCsderived MVs as well as AT-MSCs-derived MVs was not observed at any of
the tested doses.
Conclusions: Our findings show that SC-MVs are purer than UC-MVs,
but fail to exhibit an immunosuppressive potential. Our data suggest that the
isolation method may impact the potency of MVs and that this has to be
carefully taken into account before clinical application.
310
LYOPHILISED PLATELET LYSATE AS CELL CULTURE
SUPPLEMENT
C Kreissig1, I Rehfeld1, E Scheel2
1
ZBST, DRK-Blutspendedienst West, Ratingen, Germany, 2ZB Plasma,
DRK-Blutspendedienst West, Hagen, Germany
It is well known, that platelets play an important role in regulation of cell
division and differentiation. So use of platelet lysate in mesenchymal stem
cell culture became a frequently used method. Unfortunately there is no
standardised platelet product for use as cytokine source available. Because of
short half life of cytokines and limited stability preparations mostly are
produced for every single experiment. These platelet lysates do not contain a
standardized content of cytokines. So reproducibility of different experiments in a row is restricted. We developed a lyophilisation technique for
human platelet products. Many platelet preparations can be pooled, lyophilized and stored for a long period of time. So an experimental series of MSC
cultures can be performed using the same cell culture supplement. As previously published we analyzed influence of lyophilized platelet preparations
(LPL) on MSC cultures. After two years of storage of LPL we tested stability
of the preparation in a functional assay. Therefore we used human mesenchymal stroma cells, which were grown in MSC Growth Medium. For differentiation assays we used Mesenchymal Stem Cell Adipogenic and
Chondrogenic Differentiation Medium. We observed, that cytokines contained in LPL are functionally active even after two years of storage. We
compared adipogenic and chondrogenic differentiation in MSC cultures.
While adding LPL instead off specific cytokine cocktails we could observe
comparable numbers and structure of adipocytes and chondrocytes. We
could prove, that LPL can serve as MSC supplement. It promotes cell division and adipogenic and chondrogenic differentiation. Lyophilisation of
platelet lysates lead to a stable supplement with defined cytokine concentrations. So an experimental series of MSC cultures can be performed using
the same cell culture supplement. This contributes to a reproducibility of
different experiments in a row.
311
312
DECELLULARIZED LIVER SCAFFOLD AS A POTENTIAL
RESOURCE FOR THE DEVELOPMENT OF FUNCTIONAL
HUMANIZED LIVER
AA Khan, SK Vishwakarma, G Bhavani, S WilayathHussain, MA Habeeb
Centre for Liver Research & Diagnostics, Deccan College of Medical Science,
Hyderabad, Andhra Pradesh, India
Background& Aim: Liver transplantation is the only established treatment
forend stage liver diseases. One of the major challenges for tissue engineering
is to produce large volume tissues and organs for clinical applications. Whole
organ decellularizationand recellularization approach represents an ideal
choice for development of new organs. The present study was designed to
develop a technology that can provide more reliable liver microarchitecture
and extracellular matrix.
Methods: Decellularization of xenogenic liver was performed using perfusion through portal vein. Liver was perfused using detergents to ensure
S89
complete removal of the cells.Vascular tree was visualized using contrast imaging. Decellularized liver bioscaffold was characterized to ensure complete
removal of cells retaining thevascular architecture and connective tissues as
scaffold. FDA labeled EpCAM+ve human hepatic progenitor cellswere infused
in decellularized liver through portal vein. Repopulation of cells within the
liver scaffoldwas determined by various markers. Functional assays were performed to identify the cell survival, engraftment and migration within the liver
scaffold.
Results: Hematoxylene and Eosinstains revealed multiple collagen layers
with vascularchannels.PCR analysis of scaffold did not show any residual
nuclei or cell in the decellularized Liver. Tracking of the FDA labeled cells
throughout the networkshowed a defined vascular tree with multiple
branching and residual niches of the cells.RT-PCR revealed the presence of
transplanted cells.
Conclusion: The study has demonstrated a potential technology in the
fabrication of liver tissue that can be readily transplanted into host animals and to study of liver cell biology and drug discovery with further
development.
313
MESENCHYMAL STEM CELLS TRANSPLANTS AFTER PELVIC
RADIOTHERAPY
LIMITS
THE
DEVELOPMENT
OF
RADIATION-INDUCED FIBROSIS, WITHOUT PROMOTING
THE RESIDUAL TUMOR GROWTH
S Francois1,2,3, B L’homme1, MM Benderitter1, L Douay2, AA Chapel1,2
1
LRTE, IRSN, Fontenay-Aux-Roses, France, 2UMR_S 938, UPMC Cdr
Hôpital saint Antoine, Paris, France, 3IRBA/EBR, Biomedical Research
Institute of the Armed, Bretigny sur Orge, France
Radiation therapy is a key component of the management of various pelvic
tumors. Unfortunately, normal tissues located in the vicinity of target organs
are radiosensitive, and long-term cancer survivors may develop late treatment-related injury, most notably radiation-induced fibrosis (RIF). This
process is considered irreversible, and there is currently no effective treatment for preventing or reducing the development of RIF. The objective of
this study is to investigate the anti-fibrotic effect of mesenchymal stem cells
(MSC) on prostatic fibrosis. For this study, we have developed a model of
fractionated irradiation in the pelvic area in Sprague Dawley rats after
chemical induced colonic tumors. The anti-fibrotic effect of MSC in prostate was evaluated by histology study. Expression of fibrosis biomarkers was
studied after radiotherapy alone and radiotherapy associated with MSC
therapy. Our study was conducted from 24 hours to one year after the last
radiation exposure. Over a period of 12 months the variation of fibrosis
biomarkers expression has highlighted that the process of prostatic fibrosis
evolves step by step with reaction peak at 2 months after radiotherapy.
These preliminary results suggest that MSC must be performed during the
first months after radiotherapy for an optimal efficiency of MSC. In the
prostates of rats treated with radiotherapy + MSC transplants, the stoichiometric ratio of MMP / TIMP seems to be respected suggesting tissue
homeostasis and lack of progression of a RIF. In this study we found that the
lifetime of the animals receiving MSC grafts was significantly greater. Pelvic
radiotherapy combined with MSCs has reduced the number and size of
colonic tumors as well as protection of the prostate tissue in long term
against the RIF.
314
ADIPOSE STEM CELLS: EFFECTS OF CRYOPRESERVATION
AND DONOR AGE ON UTILITY IN REGENERATIVE MEDICINE
DT Harris1, A Muise1, M Badowski1, J Pierce2
1
Immunobiology, Univ. of Arizona, Tucson, Arizona, United States, 2Aesthetic
Surgery of Tucson, Tucson, Arizona, United States
Introduction: Adipose stem cells are being increasing used in regenerative
medicine, often at the point of care at the time of treatment. If adipose tissues
and stem cells could be cryopreserved without loss of utility it would broaden
the uses of these cells and tissues, as well as avoiding addition collection procedures for the donors. In addition, many individuals that could benefit from
regenerative medicine are older but the effect of donor age on adipose stem cell
utility is unknown and could impact efficacy.
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Poster Abstracts
Method: Adipose tissue was collected by liposuction from donors ranging
from 20-80 years of age (N¼29). Whole tissue was cryopreserved using a
DMSO cryoprotectant, controlled rate frozen, and stored in liquid nitrogen
dewers. Samples were thawed and evaluated at time points of 1-24 months for
viability, cell recovery, cell phenotype, proliferation, CFU-F and differentiation capacity.
Results: Cryopreservation of adipose tissue had no effect on any variable
measured, with viability routinely exceeding 80% even at 2 years post-storage.
In addition, thawed adipose tissue was capable of differentiating into adipose,
cartilage, bone and neurons. Clinical use of frozen and thawed adipose tissue
was successful for scar revisions, breast and buttocks augmentation, and
treatment of Dupytren’s contractures. However, increasing donor age had a
negative effect on the ability of adipose stem cells to form cartilage and bone,
but not neurons and adipose tissue.
Conclusions: Adipose tissue may be banked at the time of harvest for later
use without loss of cells or function. Adipose tissue harvested from donors of
any age may be used for cosmetic applications as well as potentially for
neurological indications. However, older stem cells may have limited use for
orthopedic applications.
(TRA-1-60+ or CD326+) and untouched IPSC culture. The protocol of differentiation included a mesodermal derivation step followed by an expansion
step on gelatin coated dishes. Routine IPSC and umbilical cord MSC were
included as controls. All the lineages successfully finished the mesoderm
derivation step and showed a loss of pluripotency markers TRA-1-60, TRA-181 and ALP and an increase of the early differentiation marker SSEA-1. At this
stage, none of the lineages expressed MSC markers. The cells rapidly switched
on positive CD44 and CD105 MSC-like cells when placed in expansion medium, but with different efficiency and viability depending of the IPSC lineages
used as starting material. Only the MSC-like coming from purified TRA-1-60
IPSC showed high expression of MSC markers and homogenous MSC-like cell
morphology through passaging. Impurities generated from MSC-like cultures
originated from IPSC purifed CD326 and untouched IPSC were used to test
complementary markers of endodermal and ectodermal differentiation. The
capability of the different MSC-like populations to continue the differentiation
to adipocytes, chondrocytes and osteoblasts was also analysed. In conclusion,
phenotyping assays were successfully developed and validated following biopharmaceutical requirements for in process controls of multipotent and
pluripotent cells.
315
EARLY UC-MSC INJECTION DAMPENS THE SYNOVIAL
CATABOLIC ACTIVITY IN EARLY-STAGE OF EXPERIMENTAL
OSTEOARTHRITIS
N Saulnier1, C Boulocher2, S Maddens1, E Pillet2, T Roger2, E Viguier2
1
Vetbiobank, Marcy l’Etoile, France, 2VetAgro Sup, Marcy l’Etoile, France
317
NOT ALL THE STEM CELLS MEET ALL THE CLINICAL NEEDS:
MESENCHYMAL STEM CELLS IN REGENERATIVE MEDICINE
T Montemurro, M Vigano, V Parazzi, B Baluce, C Lavazza, S Budelli,
V Boldrin, M Barilani, E Ragni, L Marino, E Montelatici, L Lazzari,
R Giordano
Cell Factory, Fondazione IRCCS Cà Granda, Milan, Italy
Introduction: Osteoarthritis (OA) is a chronic degenerative joint disorder
associated with synovitis, exacerbating the catabolism process via a positive
feed-back loop. Mesenchymal Stromal Cells (MSC) injections have been
recently proposed as a therapeutic alternative to counteract the local inflammation. Nonetheless, contrasting results have been published in preclinical
models of OA. In this study, we compared the ability of early or delayed single
injections of Umbilical Cord (UC)-derived MSC to prevent the disease
process.
Material & Methods: Early OA was induced by Medial Meniscal Release
(MMR) in rabbit knee joint (d0). On day 3 (early group) or 15 (delayed group),
intra-articular injection of 3.106 UC-MSC was performed. Two separate
control groups were injected with PBS at the same time points. All animals
were euthanized 8 weeks after. Cartilage gross evaluation and EPIC-mCT
thickness measurements were performed to confirm early grade of OA. Synovial tissue was harvested and submitted for histological examination and
molecular analysis of inflammatory and matrix turn-over associated genes.
Results: MMR induced cartilage fibrillation and discrete osteophytes,
mimicking the early events of OA. Articular lesions were reduced in the early
cell-treated group. Histological evaluation of synovial tissue showed lymphoplasmacytic infiltrates in both cell-treated groups. In the early cell-treated
group, synovial gene expression of IL1-b and metalloproteinases MMP-1, -3,
-13 was significantly reduced compared to the matching control group. Similar
data were observed in the delayed group, but not statistically significant.
Conclusion: Early injection of UC-MSC dampens the catabolic process in
OA joint more efficiently that delayed injection. Our data suggest the synovium
as a major responder of MSC therapy, modulating the expression of matrixdegrading enzymes. Further investigations are required to decipher the interactions between MSC and the synovium.
316
FROM IPSC TO MSC-LIKE : CELL LINEAGES SUCCESS AND
THEIR IN-PROCESS MONITORING BY SPECIFIC FLOW
CYTOMETRY MARKERS
N Theys, V Deffontaine, F Vandermeers, S Thys
Strategy & Innovation, Quality Assistance, Donstiennes, Belgium
Induced pluripotent stem cells (IPSC) can be directed to generate cells for
regenerative medicine. The heterogeneicity of IPSC culture represents a
challenge to initiate process of cell differentiation. In addition to target
markers, in-process characterisation should include general markers of early
and definitive mesodermal, ectodermal and endodermal differentiation. We
have designed multicolored panels for flow cytometry to offer an in-process
characterisation of IPSC differentiate to MSC-like cells. These markers were
used to monitor the differentiation of three IPSC lineages: preselected IPSC
Mesenchymal stem cells(MSCs)are multipotent cells that can be isolated
from many sources, including bone marrow(BM), adipose tissue, umbilical
cord blood, Wharton’s Jelly and amniotic fluid. The use of these cells in a
clinical trial requires that their production complies with Good
Manufacturing Practice(GMP).The translation step is even more challenging
when MSCs are obtained from an old and or diseased patient such as in the
case of autologous approaches for degenerative disorders.Besides the patient/
donorerelated issues, critical factors in the expansion procedures are starting
density, doubling rate, cell confluence, culture duration and use of FBS or
other substitutes which could potentially influence the in vitro and in vivo
cell properties. With regards to all these parameters, here we describe the
GMP procedures and the extensive validation process to obtain BMMSCs
suitable for different clinical applications. In particular, two GMP MSC
manufacturing processes were validated:the production of BMMSC from
healthy donors as off-the-shelf products for allogeneic use and from patients
affected by a rare form of parkinsonism (Progressive Supranuclear Palsy)
within a specific clinical protocol (NCT01824121).With each donor type,
two different culture conditions with either bovine serum or platelet lysate
were tested to choice the best culture conditions in consideration of the
expected therapeutic effect. With this aim, we established protocols, standard
operating procedures, production and quality control processes, we validated
specific reagents suitable for clinical applications and clearly defined release
specifications and final quality control tests. The evidences from this validation study support the concept that each clinical approach in the context of
regenerative medicine needs a keen knowledge of the results that can be
obtained by applying different procedures and a critical decision based on the
specific clinical needs.
318
EFFECTS OF A NOVEL CERAMIC BIOMATERIAL ON IMMUNE
MODULATORY
PROPERTIES
AND
DIFFERENTIATION
POTENTIAL OF MESENCHYMAL STROMAL CELLS
G Bassi1, F Guilloton2, C Menard3, M Di Trapani1, L Pacelli1, R Carusone1,
M Midolo1, E Amati1, I Bezier3, F Deschaseaux2, L Sensebe2, S Baroth4,
H Schrezenmeier5, M Rojewski5, P Layrolle6, R Giordano7, C Lavazza7,
L Lazzari7, P Bourin2,8, M Dominici9, K Tarte3, M Krampera1
1
Laboratory, University of Verona, Verona, Veneto, Italy, 2EFS Pyrenees
Mediterranee, Universite Paul Sabatier UMR5273 - Inserm U1031, Toulouse,
France, 3INSERM U917, Universite Rennes 1, Rennes, France, 4Biomatlante
SAS, Vigneux de Bretagne, France, 5Institute of Transfusion Medicine,
University of Ulm and German Red Cross Blood Donor Service BadenWürttemberg, Hessen, Ulm, Germany, 6Inserm U957, Faculte de Medecine,
LPRO, Nantes Cedex, France, 7Cell Factory, Fondazione IRCCS Ca, Milan,
Italy, 8CSA21, Toulouse, France, 9Medical and surgical sciences for children
and adults, University Hospital of Modena and Reggio Emilia, Modena, Italy
The aim of this study was to assess the immune modulatory properties of human
mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs)
and cord blood (CB-MSCs) in the presence of a novel hydroxyapatite and
tricalcium-phosphate (HA/TCP) biomaterial as scaffold for MSC delivery. In
resting conditions, a short-term culture with HA/TCP did not modulate the antiapoptotic and suppressive features of the various MSC types towards T, B and
NK cells; in addition, when primed with inflammatory cytokines, MSC maintained or not on HA/TCP similarlyincreased their suppressive capacities. The
long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like
phenotype with up-regulation of OSTERIX and OSTEOCALCIN, similarly to
what obtained with dexamethasone and, to a higher extent, BMP-4 treatment.
MSC-derived osteoblasts did not trigger immune cell activation, but were less
efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells.
Interestingly, their suppressive machinery included not only the activation of
IDO, which plays a central role in T-cell inhibition, but also COX-2 that was not
significantly involved in immune modulatory effect of human undifferentiated
MSCs. COX-2 is significantly involved in bone healing, suggesting that its induction by HA/TCP could also contribute to the therapeutic activity of MSC for
bone tissue engineering.
319
QNPQ: A COMPUTER AIDED MULTI-TARGET STRUCTURAL
RADIOPROTECTIVE PEPTIDE CRITICAL FOR THE COINDUCTION OF THE SLIT2, SPONDIN1 PROTEIN LIGANDINDUCED FUNCTION AND HOMOLOGY MODELING OF THE
ROBO-1 RECEPTOR REVERSE TRANSCRIPTION SITES: A
COMBINATORIAL DIRECTED CONSERVATION MOTIF BASED
ANALYSIS
I Grigoriadis, N Grigoriadis
biogenea pharmaceuticals, Thessaloniki, Greece
An increasing amount of evidence from experimental and computational
bioinformatic analysis suggests that there are many domains in DNA sequences that remain evolutionarily conserved. In some cases, these
conserved patterns in a collection of unaligned DNA and protein sequences
present the same functional and regulatory properties and are significant for
the molecular role of these sequences. Discriminative motif finding algorithms aim to increase the sensitivity and selectivity of conserved motif
discovery by utilizing a specific set of DNA and protein sequences, and
searching for binding sites and homolog repeats among the sets of the
selected sequences. In the present study we introduce a combined bioinformatic software-based discriminative methodology to detect short,
highly and most conserved motifs between the DNA sequences within the
proteins Slit-2 and Spondin-1 and their receptor Robo-1 and then, on
finding out more motif conserved features about them including their
physical regulatory properties, as well as their function as therapeutic adjuvants for the enhancement of host tolerance to aggressive chemoradiotherapy for eradicating metastatic cancers.
320
CELL THERAPY FOR TREATMENT OF STRESS URINARY
INCONTINENCE IN WOMEN: POTENTIAL DOSE EFFECT OF
AUTOLOGOUS MUSCLE-DERIVED CELLS FOR URINARY
SPHINCTER REPAIR (AMDC-USR)
R Jankowski1, S Werner2, S Snyder3, M Chancellor4, P Kultgen3, R Pruchnic1
1
Cook MyoSite, Inc., Pittsburgh, Pennsylvania, United States, 2Cook, Inc.,
Bloomington, Indiana, United States, 3MED Institute, Inc., West Lafayette,
Indiana, United States, 4Beaumont Health System, Royal Oak, Michigan,
United States
Background: Stress urinary incontinence (SUI) is the involuntary leakage of
urine upon effort or exertion. In animal studies, muscle-derived cells have
successfully integrated within tissue to improve sphincter function. To examine
the clinical potential of AMDC-USR, Cook MyoSite, Inc. has completed 4
phase I/II clinical trials examining the safety and efficacy of AMDC-USR for
treatment of SUI in women with a focus on the potential effect of cell dose on
safety and efficacy.
S91
Methods and Results: In phase I/II of the clinical development program,
126 women with SUI have received intrasphincteric injection of AMDC-USR.
Administered AMDC-USR doses ranged from 1 e 200 million cells (Table).
Safety was assessed by the incidence and severity of adverse events. Secondarily,
efficacy was assessed via changes in diary-reported stress leaks and pad tests.
Patients were followed for 12 months. No adverse events have been attributed
to AMDC-USR product. Procedure-related adverse events are consistent with
the known risks of needle muscle biopsy and intrasphincteric injection and selfresolve or are easily treated. Efficacy data from a dose escalation study (IND1/
CTHM) suggest that more patients are responsive to doses of 100 and 200
million AMDC-USR at 12-month follow-up than to lower doses.
Phase I/II
Trial
AMDC-USR
Dose(s)
N (million cells)
MCMT
8
Efficacy Conclusions
20 2
Five patients experienced reduction
in stress leak frequency and pad
weight.
MCDR
38 1, 2, 4, 8, 16, Compared to lower doses, a higher
32, 64, 128
percentage of patients receiving
doses of 32 million AMDCUSR experienced reduction in
stress leak frequency and/or pad
weight.
IND1 and 80 10, 50, 100, Compared to lower doses, a higher
CTHM
200
percentage of patients receiving
doses of 100 million AMDCUSR experienced reductions in
stress leak frequency and/or pad
weight.
Conclusions: AMDC-USR appears safe at doses ranging from 1-200
million cells. Efficacy data suggest that more patients are responsive to doses of
100 and 200 million AMDC-USR than to lower doses, providing critical data
for phase III trials.
321
HIGHLY CELLULARIZABLE THREE-DIMENSIONAL HUMANHEART DERIVED SCAFFOLDS
D Holt, J Theisen, F Silva, D Grainger, D Bull, AN Patel
Surgery, University of Utah, Salt Lake City, Utah, United States
Background: Heart failure results from damaged myocardium that can
severely reduce function and lead to death with very few options for treatment.
Recent studies have injected stem cells into the coronary arteries to improve
function. Though the potential of stem cell therapy is great, only limited efficacy has been attained, Extracellular matrix (ECM) proteins derived ECM
from bowel or bladder and stem cells in concert may have superior benefits to
either alone but the homing cues are still limited for engraftment due to lack of
organ specific signalling and xenogenic issues. Our goal was to derive a novel
three-dimensional human heart extracellular matrix (ECM)-derived scaffold
that could serve as a vehicle to deliver cardiac or stem cells directly to the
damaged tissue of the heart and remain at the treatment site.
Methods: Scaffolds were created from purified human heart ECM proteins
to expedite clinical translation and actively promote and preserve heart cell
phenotype. Scaffolds were imparted with pores 250 um in size to enhance
diffusion and cell penetration and distribution. Human cardiomyocytes and
cardiac derived-iPSCs were seeded into scaffolds 3 mm in diameter and 0.3 mm
thick, cultured and subsequently implanted into NOD-SCID mice.
Results: The human cardiomyocytes and cardiac derived iPSCs readily
adhered to human cardiac-derived ECM protein scaffolds. Cardiomoycytes
within the scaffold maintained representative phenotypes including expression
of cardiomyocyte-specific markers and remained electrically active, even
beating the scaffold in unison in vitro. In vivo, cardiomyocyte-seeded scaffolds
spontaneously adhered to and incorporated with murine infarcted tissue.
S92
Poster Abstracts
Conclusions: These results indicate that a novel 3D scaffold made of purified human cardiac ECM can be used as a delivery vehicle for human cardiac
cells to directly target damaged heart tissue. Optimization of scaffolds and cell
interactions need to studied further in vivo.
322
INJECTABLE BONE VERSUS BONE CONSTRUCT FOR
CRITICAL SEGMENTAL BONE DEFECT REPAIR
E Ferdhany1, R Erfani1, A Sadeghilar1, A Adraii1, M Osman1, AM Kassim1,
NM Haflah1, AA Rashid1, RB Haji Idrus2,3, A Ng2
1
Department of Orthopaedics and Traumatology, Universiti Kebangsaan
Malaysia Medical Centre , Kuala Lumpur, Federal Territory, Malaysia, 2Tissue
Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, Kuala
Lumpur, Federal Territory, Malaysia, 3Department of Physiology, Faculty of
Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Federal Territory,
Malaysia
The combination of osteogenic cells, platelet-rich plasma (PRP) and tricalcium phosphate/ hydroxyapatite (TCP/HA) have been prepared either in
an injectable form or as a cylindrical solid bone construct. Three-centimeter
length segmental gap were made on the left sheep tibiae and the tibiae were
stabilized with external fixators. In the same sitting, autologous mesenchymal stem cells were derived from sheep bone marrow via iliac crest aspirations. Cells were expanded and osteogenically induced in vitro.
Autologous PRP was derived from sheep whole blood. Sintered TCP/HA
granules were mixed with osteogenic cells and PRP to form an injectable gel
mixture. To form the bone constructs, osteogenic cells were mixed with
PRP and seeded on a three centimeter-cylindrical TCP/HA (sintered) block.
Segmental defects were repaired three weeks later after the creation of the
defect. In the injectable bone group, twenty millimeters of gel mixture was
injected percutaneously into the bone gap. In the bone constructs group,
bone construct was implanted directly into the bone gap. Sheep were
monitored for the next 3 months. Serial radiological assessment showed
faster and greater bone regeneration in the bone construct group. e bone
regeneration. Bone union was achieved by three months post-implantation
in the bone construct group while little and randomized bone spicule
regeneration were seen in the injectable group. Multiple bone injections
increased amount of bone regenerated but did not result in bone union. Our
study to date concludes that solid 3D scaffolds are required for critical
segmental bone repair.
Radiographs taken at week 12 post-injection / post-implantation.
323
LIMBAL STEM CELL PHENOTYPE IS SUPPORTED BY A NEW
CULTURE MEDIUM
A Popova1, A Ulyanov1, O Fechin1, I Valamina2, O Shilovskih1
1
Eye microsurgery clinic, Ekaterinburg, Russian Federation, 2Ural State
Medical University, Ekaterinburg, Russian Federation
The aim of present study was to compare morphological and immunophenotypic features of limbal epithelium cultivated with the application of two culture
mediumseDMEM/F12 and EpiCult-C(for human mammary epithelial cell
culture).
Methods: Limbal rims were harvested from 5 cadaver donors and cultivated on
dAM in two different culture mediums e DMEM/F12(Culture I) and EpiCultC(Culture II). Epithelial morphology was studied by histology and phase-contrast
microscopy while phenotype was defined by immunostaining with monoclonal
antibodies to stem cell marker p63, proliferation marker Ki-67 and differentiation
marker CK3. As a control group 2 cadaver donor corneas were studied.
Results: Basal cells in Culture II were significantly smaller than in Culture I:
median size 11.7mm(range 7.2-37.7 mm) and 20.4 mm(range 11.0-60.0 mm)
respectively, p¼0.02. The corresponding values for superficial layers in Culture I
and Culture II were: median size 45.1 mm(range 23.8-73.0 mm) and 70.5
mm(range 45.8-76.4 mm) respectively, p¼0.09. Cultivated corneal epithelium on
dAM in both culture mediums formed from 3 to 6 layers. Cuboidal basal cells and
the nuclei-cytoplasmic ratio 1:1 in Culture II morphologically were more similar
to control samples of donor corneas. There were no significant differences between Culture I and Culture II in an amount of p63 positive cells: median percentage 2.75%(range 1.9-3.1) and 3.5%(range 2.9-4.0%) respectively, p¼0.47.
The proliferative index didn’t differ significantly: for Culture I median was
13.0%(range 9.0-17.0%), for Culture II - 15.8%(range 6.0-36.0%), p¼0.36. CK3
expression was detected only in superficial layers in both Culture I and Culture II.
Conclusions: Our data demonstrates that EpiCult-C is comparable with
DMEM/F12 in supporting of limbal stem cell phenotype and cell differentiation.
Moreover epithelium cultivated in EpiCult-C has better morphological features
and may be applicated in long-term restoration of the damaged ocular surface.
324
COMPARATIVE STUDY OF THE BIOLOGICAL CHARACTERISTICS OF SERUM-FREE AND FETAL BOVINE SERUMCONTAINED MEDIUM CULTURED UMBILICAL CORD-DERIVED
G Chen, M Qiao, H Tian, D Wu
the First Affiliated Hospital of Soochow University, Suzhou, China
To compare the difference of biological characteristics between human umbilical
cord-derived mesenchymal stem cells (UC-MSC) cultured by serum free medium and fetal bovine serum-contained complete medium and to create a
xenogeneic protein-free UC-MSC culture system. Healthy human umbilical
cord segments were digested with collagenase. Umbilical cord-derived mesenchymal stem cells were cultured by serum free MesenCult-XF medium and
FBS-based aMEM complete medium. We analysed the morphology, immunophenotype, expansion potential, trilineage differentiation potential, karyotype
and immunosuppression of early passage of UC-MSC. The average cell diameters of UC-MSC in suspension cultured by serum free medium and FBSbased medium are 26 mm and 35 mm, respectively. Cell expansion folds with
serum free medium and FBS-based medium were (5.20.2) and (3.50.1) in the
first five passage, respectively. The expansion potential of MSCs was significantly
higher with serum free medium compared to FBS-based medium (P<0.05). The
cpm were (4.570.14)104, (2.040.16)104 and(0.420.04)104 when
serum free medium cultured MSCs were added to the cultures at ratios MSCs/T
cell of 1:100, 1:10 and 1:5. While the cpm were (4.570.14)104, (2.040.16)
104 and (0.420.04)104 when serum free medium cultured UC-MSCs were
added to the cultures. The immunosuppressive potential of serum free mediumcultured UC-MSC was higher than that of serum-contained medium cultured
UC-MSC at three different ratios MSC/T cell (P<0.05). Compare with serumcontained medium cultured early passage of UC-MSC, the cell diameter of
serum free medium cultured MSCs was smaller and the expansion potential was
higher. No xenogeneic proteins were presented in UC-MSC preparation when
UC-MSC was cultured with serum free medium. Human UC-MSC suppresses
T-cell proliferation in a dose-dependent manner. The immunosuppressive potential of UC-MSC was higher when cultured in serum free medium compared
with FBS-based medium.
325
ROLE OF BONE MARROW-DERIVED STEM CELL THERAPY IN
TREATMENT OF EXPERIMENTALLY INDUCED TYPE 1
DIABETES MELLITUS IN CANINE MODEL
H Gabr1, WA Elkheir2
1
Clinical pathology, Faculty of medicine-Cairo university, CAIRO,
Egypt, 2Immunology, Medical academy, cairo, Egypt
Background: Type 1 diabetes mellitus (DM) results from a cell-mediated
autoimmune attack against pancreatic beta cells and characterized by severe
damage to many tissues. Several in vitro studies have shown that bone marrow
derived stem cells could be reprogrammed to become functionally insulin
producing cells under certain culture conditions.
Purpose: The aim of this study is to demonstrate that bone marrow derived
mesenchymal stem cells (MSCs) can be induced to differentiate into insulin
producing cells (IPCs) under suitable culture condition and to assess the ability
of these differentiated cells to correct the functional defect in insulin secretion.
Materials and methods: This study was done on 9 dogs, one as a negative
control and 8 were induced to be diabetic. MSCs were isolated from bone
marrow and induced to differentiate into IPCs. Two diabetic dogs received
undifferentiated MSCs transplant, while another 2 diabetic dogs received
differentiated MSCs and 1 diabetic dog remained as a positive control.
Results: The diabetic dogs that received undifferentiated MSCs showed no
improvement in FBG level during the observation period while the diabetic
dogs that received differentiated MSCs showed improvement in FBG on day 6
after transplantation.
Conclusion: This study suggests that bone marrow MSCs can be differentiate into IPCs that can improve FBG levels in diabetics.
326
INVESTIGATION OF ASC-MEDIATED WOUND HEALING IN IN
VITRO SKIN INJURY MODELS
SE Riis1, R Newman2, D Kuninger2, S Boucher2, M Vermuri2, V Zachar1,
T Fink1
1
Department of Health Science and Technology, Aalborg University, Aalborg,
Denmark, 2Cell Biology and Stem Cell Systems, Life Technologies, Frederick,
Maryland, United States
Normal wound healing is characterized by a sequence of partially overlapping
stages including inflammation, proliferation, and remodeling. When this
sequence of events is perturbed, for instance by hypoxia, neuropathy or
dysfunctional immune response, there is a risk of the wounds becoming chronic.
Since chronic wounds represent a significant burden to both patients and health
systems worldwide, there is significant interest in developing new treatment
modalities. The ideal treatment would facilitate a nomalization of the wound
healing process. As adipose-derived stem cells (ASCs) have pro-angiogenic,
immunomodulatory and anti-apoptotic properties, they are proposed as candidates for regenerative therapies of chronic wounds. In order for successful
treatments to be developed, it would be beneficial to have simple in vitro assays
for each of the stages of wound healing. In particular, for the study of the proliferation stage of wound healing, the so-called scratch assay is often employed, as
it mimics fibroblast and/or keratinocyte behavior. Here we describe modifications of this scratch assay in order to assess the effects of ASC secretome on
fibroblast and keratinocyte migration and proliferation. Because optimal culture
conditions for ASCs are different than for skin cells, particularly keratinocytes,
assay modifications were required in order to evaluate stem cell derived factors in
the scratch wounds. These included concentration of soluble factors by filtrations, media exchange by dialysis as well as evaluating culture parameters
including seeding densities, feed schedules and surface coatings. Here we
describe and quantify how these changes impact cell behavior in scratch wound
assays using time-lapse imaging and analysis of scratch wound closure. We
describe some of the pitfalls we experienced during this study and suggest possible
solutions to make the fibroblast- or keratinocyte-based scratch assays suitable
models to study ASC effects on wound healing.
327
VALIDATION OF ANALYTICAL METHODS FOR IN-PROCESS
CHARACTERIZATION OF HUMAN MESENCHYMAL STEM
CELLS USED FOR CLINICAL STUDIES
N Theys, S Thys, F Vandermeers
Strategy &Innovation, Quality Assistance, Donstiennes, Belgium
S93
Introduction: Cell therapy products emerge from a long process of cell culture, differentiation and purification steps where appropriate cell identity and
purity monitoring are mandatory. The characterization of human MSCs used
for manufacturing of cell therapy products requires the development and the
validation of analytical methods to encounter biopharmaceutical requirements.
Material and Methods: Multicolor stainings were designed to deeply
characterized MSC using flow cytometry. These panels contain viability,
positive and negative markers for MSC. These assays were qualified for precision, specificity, limit of detection and quantification. After culture under
specific conditions, specific colorations were performed in order to evaluate
MSC differentiation to adipocytes (Oil Red staining), osteoblasts (Alizarin Red
staining), chondrocytes (Alcian Blue staining) and hepatocytes (Periodic AcidSchiff staining). Moreover, CYP3A4 activity measurement was performed to
show hepatocytes activity.
Results and conclusions: The validated flow cytometry analysis showed
that more than 95 % of MSC are positive for CD105, CD90, CD73 and CD44
while less than 2 % of these cells express CD34, CD45, CD11b, CD19 and
HLA-DR. Differentiation assays showed that MSC were able to differentiate to
adipocytes, osteoblasts and chondrocytes. MSC were also able to differentiate
to hepatocyte-like cells showing intracellular glycogen and CYP3A4 activity.
Ongoing works consist in designing complementary assays to quantify MSC
(absolute count) and differentiated cells using flow cytometry and quantitative
PCR. In conclusion, assays that we developed allow characterization and precise follow-up of MSC during the course of cell expansion and meet pharmaceutical requirements for product of quality control.
328
CULTURING OF HUMAN ADIPOSE DERIVED MESENCHYMAL
STEM CELLS (ADSCS) UNDER HYPOXIC CONDITIONS
AFFECTS PRODUCTION OF WOUND HEALING CYTOKINES
AND GROWTH FACTORS
M Mirlashari1, D Josefsen1, K Landsverk2, G Hasvold2, H Gullestad3,
G Kvalheim1
1
Department of Cellular Therapy, Oslo university Hospital, Radiumhospitalet,
Oslo, Norway, 2Department of radiation Biology, Oslo, Norway, 3Department
of Surgery, Oslo, Norway
ADSCs are easily accessible in large quantities with a minimal invasive, safe and
well-established surgical procedure. There are more than 300-500 times more
stem cells in 1 gram of fat when compared to 1 gram of aspirated bone marrow.
ADSCs is therefore an attractive cell source for tissue repair. We have recently
initiated a phaseI/II study in patients with chronic wounds following curative
radiotherapy by injecting adipose stem cells into the wound. Following liposuction cells from the Stromal Vascular Fraction (SVF) were isolated using the
CelutionTM system and administered fresh. Preliminary data from this study
show that injection of SVF cells give complete healing of the lesions after 8
weeks. However, the number of SVF cells selected from 200ml adipose tissue
might become too little when larger chronic wounds needs to be treated.
Therefore, we are currently investigating the properties of ex vivo expanded
ADSCs from SVF and their potential use in the treatment of chronic wounds.
We like others have shown that ADSCs do not propagate in vivo and the
positive effect of ADSCs in tissue repair is through paracrine mechanisms. In
this study we want to investigate the effects of hypoxia on the production of
wound healing cytokines (IL-6, IL-8, and IL-10) and growth factors (VEGF
and bFGF) by ADSCs. ADSCs were cultured under normoxia 21% O2 and
hypoxia 1% O2 for 48h. Hypoxia induced stabilization of hypoxia inducible
factor 1 alpha (Hif-1 alpha) and affected secretion of wound healing growth
factors and cytokines. But hypoxia did not affect the phenotype and
morphology of ADSCs .Our finding support the strategy of incubation of
ADSCs in hypoxia in order to induce increased secretion of wound healing
cytokines and growth factors.
329
SUCCESSFUL CULTURE AND CHARACTERISATION OF EX
VIVO EXPANDED HUMAN AUTOLOGOUS ORAL MUCOSA
EPITHELIUM USING A FEEDER- AND ANIMAL PRODUCTFREE METHOD FOR THE TREATMENT OF TOTAL
BILATERAL LIMBAL STEM CELL DEFICIENCY
M Lako1, S Kolli1,2, S Ahmad1,2, HS Mudhar3, A Meeny3, F Figueiredo1,2
1
Institute of genetic medicine, newcastle university, Newcastle, United
Kingdom, 2Ophthalmology department, newcastle univeristy, newcastle,
S94
Poster Abstracts
United Kingdom, 3Dept of Histopathology, Royal Hallamshire Hospital,
Sheffield, United Kingdom
Ocular surface reconstruction with ex vivo expanded limbal stem cells (LSCs)
is a widely used clinical treatment for patients with limbal stem cell deficiency (LSCD). This is not applicable to patients with bilateral LSCD where
there are no remaining LSCs. Cultivated oral mucosa epithelium (OME) has
been used as an alternative source of autologous epithelial stem cells for
ocular reconstruction in few clinical trials. However, successful generation of
stratified OME epithelium has only been achieved in the presence of animal
feeder cells and/or animal derived products in the culture media, likely to
contribute to increased risk of pathogen transmission and graft rejection. In
this study we report generation of multi-layered OME epithelium that shares
many of the characteristics of corneal epithelium using a fully compliant
Good Manufacturing Practice, feeder- and animal product-free method.
Proof of concept was achieved by transplantation of autologous ex vivo
expanded OME in two patients with histologically confirmed bilateral total
LSCD that resulted in successful reversal of LSCD in the treated eye up to
24 months.
330
CELL SURVIVAL AND SEEDING EFFICIENCY FOR SEVERE
CRANIOFACIAL RECONSTRUCTION USING STEM CELLS: A
PROOF-OF-CONCEPT CLINICAL STUDY
A Rajan, E Eubanks, S Edwards, S Aronovich, I Rudek, F Wang, A Lanis,
D Kaigler
University of Michigan, Ann Arbor, Michigan, United States
Traumatic injuries involving the face are very common, yet, the clinical
management of the resulting craniofacial deficiencies is very challenging.
These injuries are commonly associated with missing teeth, for which
replacement is often compromised due to damaged or deficient jawbone
support. Using a novel cell therapy, we report the reconstruction of the upper
jaw of a patient who lost teeth and supporting bone following a traumatic
injury. A 44 year old female with a history of a traumatic injury to the face
presented with four front teeth missing and an associated severe jawbone
deficiency. Replacing the teeth required significant bone regeneration of the
anterior segment of the upper jaw using a novel cell therapy. Cell survival and
attachment conditions were optimized with a biodegradable polymer
comprised of beta-tricalcium phosphate, which was used to delivery ex vivo
expanded stem cells directly to the jawbone defect. Following 4 months of
healing, cone beam computed tomography (CBCT) and a bone biopsy were
performed to evaluate the bone regenerated within the previous defect area
and oral implants were placed and functionally loaded. Cell seeding efficiency
of the polymer was highest following incubation of the cells for one hour, at
either room temperature or on ice. However, at one hour, cell survival was
highest when the cells remained on ice whereas at 30 minutes, the incubation
temperature did not effect cell survival. Volumetric CBCT and histological
analyses revealed that the cell therapy resulted in significant regeneration of
vascularized and mineralized bone tissue sufficient to stably place oral implants
4 months following cell transplantation. Implants were biomechanically
restored with tooth prostheses 6 months following implant placement. In
conclusion, stem cell therapy using the appropriate transplantation conditions
can be considered for reconstruction of severe craniofacial defects resulting
from traumatic injury.
331
ENCAPSULATION OF HUMAN MESENCHYMAL STEM CELLS IN
SODIUM CELLULOSE SULFATE-BASED MICROCAPSULES
REQUIRES IMMORTALIZATION
C Sanz-Nogu
es1, J Horan2, G Ryan2, M Kassem3, T O’Brien1
1
Regenerative Medicine Institute (REMEDI), National University of Ireland,
Galway, Galway, Ireland, 2Ziel Biopharma Ltd., Limerick,
Ireland, 3Department of Endocrinology, Odense Universitetshospital, Odense,
Denmark
Peripheral vascular diseases represent a major global health problem. Stem
cells such as human mesenchymal stem cells (hMSC) may promote vascular
regeneration. However, current limitations of stem cell therapies include poor
rate of engraftment and limited cell survival after transplantation. This might
be due to their susceptibility to be attacked by host’s immune cells, mainly
through antibodies and complement proteins. The use of biomaterials to
encapsulate cells overcomes these problems, and is likely to improve their
therapeutic outcome. Microcapsules made of sodium cellulose sulfate (SCS)
and poly-diallyl-dimethyl-ammonium chloride (pDADMAC) are very stable
and biocompatible and have been previously used in the clinic. Our aim is to
encapsulate primary and immortalized hMSCs using these materials and
characterize their behavior and angiogenic potential. Ultrastructure of
microcapsule membrane determined by SEM shows a very dense structure
with no visible pores. Determination of molecular weight cut-off of microcapsules shows that small molecules (i.e. angiogenic factors) are able to cross
the microcapsule membrane. Angiogenic potential is confirmed by ELISA and
HUVECs cord formation in a matrigel tubule assay. Furthermore, microcapsules provide immunoprotection to encapsulated cells since large molecules like antibodies (160kDa) are not able to permeate. Finally, encapsulated
cells are viable and metabolically active for the culture time tested (14 days),
although it is much poorer in the case of the primary cells. We hypothesize
that this result is not due to direct cytotoxic effects from microcapsule
components but due to cell attachment or nutrient diffusion problems that
may affect primary more than immortalized cells. These results show that
SCS-pDADMAC microcapsules can be used as a device for delivery of
angiogenic factors secreted from MSCs. However, viability of primary
hMSCs is limited and needs further improvement to enable successful
translation.
332
CLINICAL
AND
IMMUNOLOGICAL
RESPONSE
TO
MESENCHYMAL STEM CELL (MSC) THERAPY FIRST
EXPERIENCES IN IRRADIATION INDUCED COLITIS
J Voswinkel1, J Lataillade2, M Mothy1, N Gorin1, J Simon3, S Francois4,
MM Benderitter4, J Perrot1, A Chapel4,1
1
Department of Haematology, APHP, Paris, France, 2Burn Treatment Center,
Sevice de sante des Armees, Clamart, France, 3Department of Radiation
Oncology, AP-HP, Paris, France, 4PRP-HOM, IRSN, Fontenay, France
Background: Therapy by Mesenchymal Stromal Cells (MSC) enable functional recovery of the intestine and dampen the systemic inflammatory
response in radiation-induced colitis. Allogeneic bone marrow-derived MSC
from family donors were intravenously infused to four patients with refractory
irradiation-induced colitis.
Patients and Methods: MSC were obtained by culture from bone marrow
aspirates of the patient children. Two patients were subjected to two MSC
infusions and two patients to one infusion, respectively. Pain, hemorrhage,
frequency of diarrheas and fistulisation as well as the lymphocyte subsets in
peripheral blood served as parameters evaluated before MSC therapy and
during the follow-up.
Results: Two patients revealed a substantiated clinical response for pain and
hemorrhage after MSC therapy. In one patient pain reappeared after 6 months
and again substantially responded on a second MSC infusion. A beginning
fistulisation process could be stopped in one patient resulting in a stable
remission for more than 3 years of follow-up. The frequency of painful diarrhea diminished from an average of 6/d to 3/d after the first and 2/d after the
2nd MSC injection in one patient. A decline of CD4+ and CD8+ T lymphocytes and an increase of potentially regulatory CD25+ T cells accompanied the
clinical response in this patient after the MSC injections. In all patients prostate
cancer remained in stable complete remission. No toxicity occurred.
Conclusion: A modulation of the lymphocyte subsets towards a regulatory
pattern and diminution of activated T cells accompanies the clinical response in
refractory irradiation-induced colitis. MSC therapy was safe and effective on
pain, diarrhea, haemorrhage, inflammation and inhibited fistulisation. For
patients with refractory chronic inflammatory and fistulising bowel diseases,
systemic MSC injections represent a safe option for salvage therapy. A clinical
phase I/II trial is actually initiated.
333
TREATMENT OF VENOUS LEG ULCERS WITH BONE MARROW
DERIVED STEM CELLS: NEED TO RE- INJECTION?
G Otero1, C Agorio1, L Diaz2, A Tchekmedyian5, A Sujanov4, D Leal2,
L Echarte4, M Montelongo3, I Rodriguez3, A Rodriguez3, C Touriño4
1
Dermatology, Hospital de Clinicas, Montevideo, Montevideo,
Uruguay, 2Hematology, HOSPITAL DE CLINICAS, MONTEVIDEO,
Uruguay, 3Transfusional Medicine, HOSPITAL DE CLINICAS,
MONTEVIDEO, Uruguay, 4Basic Medicine, HOSPITAL DE CLINICAS,
montevideo, Uruguay, 5CETEP, HOSPITAL PEREIRA ROSSELL,
MONTEVIDEO, Uruguay
Leg ulceration is a chronic recurrent condition with considerable cost, both to
patient and health services with a prevalence of 1-3% in European countries.
Venous insufficiency is the most common cause of this disease. In recent years,
different growth factors and cell types (autologous epidermal cell, blood
mononuclear cells, bone marrow derived stem cells (BMDSC), mesenchymal
stem cells) have been employed to treat ulcers of different etiologies with
improvement in healing rates. In addition, some authors repeated cell implantation to ameliorate ulcers outcomes.
Objective: To evaluate BMDSC re-injection in the treatment of venous leg
ulcers (VLU).
Methods: Female, 70 years old with a VLU area reduction of 73% in 12
months following the first BMDSC injection. Nevertheless, VLU still persist as
two small ulcers on her left leg with an area of 37 and 13 cm2 each. The new
procedure consisted in the extraction of 179 ml of bone marrow (BM) by
puncture of posterior iliac crest under anesthesia. The BM was centrifuged and
processed under laminar flow, obtaining 24 ml of final cell product at a concentration of 25 x 106 nucleated cells / ml. Multiple injections of 0.2 ml each
were made, separated by a distance of 2 cm from each other covering the edge
and bed ulcer. The total volume injected was 4, 2 ml. After ten month of follow
up only one ulcer persists with and area of 13 cm2 and the other healed
completely. The area reduction was 65 and 100% respectively.
Conclusion: Autologous BMDSC transplantation can lead to an improvement in wound healing rates and seem to be a safe complementary treatment.
However, additional injections or combination of treatments will be required in
great area wounds and proper control trials are needed. We are starting a pilot
study to evaluate this condition.
334
FLAGELLIN PRETREATMENT ENHANCES THE IMMUNOSUPPRESSIVE CAPACITY OF MESENCHYMAL STEM CELLS IN
ANIMAL MODEL OF PROCTITIS
C Linard, J Lacave-Lapalun, MM Benderitter
IRSN, Fontenay aux Roses, France
Enhancing tissue repair by mesenchymal stem cells (MSCs) may be an important
advance in treating radiation-induced proctitis. TLR ligands can improve MSCs
capacity to suppress immune responses and repair. In a colorectal model of 27Gy
radiation in rats, we investigated the effects of i) systemic MSCs (5.106) administration (MSCs) ii) TLR5 ligand flagellin administered (3 days post-radiation)
prior the systemic MSCs administration (flagellin pre-treatment) or iii) MSCs
preconditioned 24h in culture with flagellin (Flag-MSC). MSCs or Flag-MSCs
were administered 7 days post-radiation. One month post-radiation, lesions are
similar to those seen clinically (acute mucosal ulceration, large immune cell
infiltration). Flow cytometry of isolated colonic immune cells population showed
that MSCs, Flag-MSC and flagellin pre-treatment reduced the innate immunity
response (neutrophil and macrophage infiltration) as compared to irradiated rats.
Although the among of NK cells was not decrease by MSCs, Flag-MSC and
flagellin pre-treatment, INOS, crucial to achieve immunosuppression was
overexpressed by Flag-MSC and flagellin pre-treatment. The CD4/CD8 ratio
decreased with MSCs, increased with flagellin pre-treatment and was normalized
with Flag-MSCs compared to irradiated rats. Surprisingly, in the irradiated colon
no modification of the suppressive T-cell population Treg (CD4+CD25+) was
induced by MSCs alone as compared to irradiated rats, but Flag-MSC and
flagellin pre-treatment overexpressed FOXP3 and IL-2Ra characterizing an
enhanced functional Treg (Flag-MSCs overexpressed IL-10). In addition, MSCs,
Flag-MSCs and flagellin pretreatment restored the epithelial cell proliferation
and the antimicrobial Reg3g protein implicated in host defense. In conclusion, on
a proctitis model, MSCs exposed to activation through TLR5 ligand (preactivation of the host or pre-treated-MSCs) have an enhanced immunosuppressive capacity.
335
EFFECT OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS
(ADMSCS) THERAPY IN CALCANEAL TENDON HEALING
AA Aro, GD Carneiro, LF Teodoro, GF Simões, ALR Oliveira, CP Vicente,
ER Pimentel
Structural and Functional Biology, UNICAMP, Campinas, Sao Paulo, Brazil
S95
The tendon is composed of highly organized collagen fibers that form a
complex supramolecular structure. After lesions, the functional capacity of the
tendon is not completely restored. Thus, tendon lesions are still a serious
clinical problem because the site of the injury becomes a region with high
incidence of recurrent rupture. ADMSCs represent an alternative therapy for
tendons lesion, due their multipotential properties and high responsiveness to
different stimuli and the possibility of helping reorganizing tendon matrix.
Therefore, we hypothesized that ADMSCs can migrate to the injured region,
improving the tendon healing. ADMSCs were obtained from the inguinal
region of the Lewis rats and the cells were maintained at a subconfluent level
until their use (5p). Flow cytometry analysis showed the expression of the
main positive markers for ADMSCs, CD90 and CD105, as well as negative
marker CD34. For the analyses of the partially transected calcaneal tendons
on the 14th day after surgery, Lewis rats (120-day-old) were separated in:
Normal (N) - tendons without transection; Transected (T) - transected
tendons and treated with DMPBS; Transected+ADMSCs (T+ADMSCs) transected tendons treated with a unique injection of ADMSC (1.5x106)
around the lesion. Rats were analyzed using the CatWalk system (max
contact intensity of the paw), and the treatment with ADMSCs improved the
gait of rats with significant difference on the 3rd day of the healing process
when compared to the group T. Western blotting analysis of collagen type I
demonstrated increased amount in the T+ADMSCs group when compared
to T. Total collagen concentration determined by the hydroxyproline dosage
presented no significant difference between T and T+ADMSCs. In
conclusion, the use of ADMSCs as a therapy may have an important role in
tendon repair, improving animal gait response and collagen amount after
lesion. Financial support: FAPESP (2012/14973-8).
336
APPLICATIONS OF COOK HUMAN PLATELET LYSATE IN CELL
THERAPY
Cook General BioTechnology, Indianapolis, Indiana, United States
Human platelet lysate (hPL) has emerged as a viable human-derived alternative
to fetal bovine serum (FBS) for cell manufacturing for cell-based therapeutics.
Derived from human platelets, hPL contains similar growth factors and cytokines found in FBS at comparative levels. COOK HPLÔ is provided in two
different versions, a heparin-requiring hPL (PL-H) and heparin-free hPL (PLNH) and is produced at an industrial scale (minimum lot size of 20L), in
accordance to GMP standards and proper documentation, from large number
of pooled platelets resulting in high lot-to-lot consistency and purity. COOK
HPL provides researchers with the ability to incorporate hPL in their early
research and development phase through a high quality research grade
including both versions with the ability for a smooth transition into clinical
manufacturing using the GMP clinical grade. Preliminary results of both
versions of hPL at different concentrations (2.5, 5, and 10%) supported cell
proliferation of primary cells such as dermal fibroblasts, endothelial, keratinocytes, and mesenchymal stem/progenitor cells such as amniotic-, bone
marrow-, adipose-, and cord blood-derived MSCs to comparable levels of FBS
at all concentrations, as well as keratinocytes serum free media (SFM). COOK
HPL was also shown to support large-scale expansion of MSCs using xeno-free
microcarriers in 3D cultures. COOK HPL-based freezing media maintained
high cell viability and normal biological properties of neonatal and adult MSCs
from various tissues post-thaw, and was comparable to serum-free freezing
media and FBS containing freezing media. These studies show the ability of
using COOK HPL as an alternative to FBS for the growth of primary and
various neonatal and adult stem cells derived from various tissues and for largescale expansion in 2D and 3D cultures for cell-based therapeutics, as well as a
freezing and recovery solution.
337
HOMOLOGOUS DEMINERALISED BONE MATRIX (DBM)
ASSOCIATED TO AUTOLOGOUS ADIPOSE MESENCHYMAL
STROMAL CELLS (AMSC) AND EFEFCTER CELLS (EC)
AGAINST DBM INDUCE BONE TISSUE FORMATION AT TWO
WEEK POST TREATMENT OF EXPERIMENTAL JAW LESIONS
IN PIGS
B Han, N Blasetti, LA Ugartemendia, D Vincens, S Ferraris, M Nimni,
GA Moviglia
S96
Poster Abstracts
Introduction: In order to improve the existent biologic methods to promote
bone formation in extreme conditions the addition of EC against DBM to the
matrix and stem cells added was tested.
Methods: DBM was prepared using fresh piece of bone. Process was
conducted in a GMP laboratory. Bone was fragmented in small pieces,
demineralized in a solution of Nitric acid 0.6 Molar at 4C, Frozen to -160C
and reduced to powder. Swine aMSC were obtained isolaited and cultured.
EC against DBM were obtained from circulating peripheral blood mononuclear cells activating and expanding them culturing in DEMEM + Insulin
(Humalin), and 1% (W/V) of DBM. After 96 hours of culture, cells were
harvested and named EC. The two square holds on each jaw’s branch of 3
pigs (15 to 20 kg body weight) were performed by surgery under anesthesia
and in a clean animal operating room. The first hold was left without any
refill; the second was filled with DBM alone, the third with DBM + aMSC
and the fourth with DBM + aMSC and EC. The four holds were covered with
steel meshes which were fixed to both sides. Two week later animal were
euthanized and the jaw was histological processed. Obtained slides of each
treated piece of jaw were stained with H&E, PAS, Collagen I, Alkaline
Phosphatase and Calcium.
Results: the lesions left without any treatment were filled of fibrotic tissue
and small new bone tissue was observed at the borders of the lesion. The cavity
filled with DBM only showed well expansion of the implanted scaffold with
small invasion of cells and no new bone tissue formation. Similar results but
with more cellularity was observed in the hold filled with DBM + aMSC.
Opposite the hold filled with DBM, aMSC and EC had approximately 40% of
their volume filled with new bone tissue.
Conclusions: Biological active DBM may be produced in our laboratory
under GMP rules. The addition of EC improves the quality and speed of bone
formation.
338
LIP AND PALATINE CLEFT SURGERY OUTCOME IS IMPROVED
BY THE ADJUNVANT USE OF ACELLULAR SWINE DERMIS AND
UMBILICAL CORD BLOOD STEM CELLS (UCB SC)
GC Trigo, MC Ortega, MI Vilacha, H Drago, N Blasetti, GA Moviglia
Introduction: To prove safety and efficacy of reconstructive surgery following
a modified Malek’s technique and supported with the use of UCB SC and
decellularized swine dermis we conducted the following controlled open label
prospective clinical trial.
Methods: After University bio ethic comity approval 9 patients with complete lip and palatine cleft congenital dysmorphia after prenatal diagnosis saved
their own UCB SC (experimental group). A Group of 9 patients with similar
lesions that did not save the UCB SC were considered as a control group. All at
the age of 3-5 month old, underwent to a first surgery that sutured the soft
plate muscles, nasal mucosa, oral mucosa and dental arcade. During a second
surgery was sutured the anterior muscle circle, skin and mucosa of the lip. Only
in experimental group, a scaffold of swine decellularized dermis seeded with
own UCB SC had placed at the space between the two borders of the bone
cleft. Rest of UCB SC was injected in all the borders of mucous membranes,
muscles and bone pieces.
Results: No mayor side effects were observed in any of the treated patients.
The scar formation of muscles and mucous membranes of lips was faster and
stronger and smother in the experimental group (less than 7 days against 10
days or more). The oral mucosa on the bone gap had a dehiscence larger than 4
mm in all the control group patients. Opposite 3 patients for the treated group
had any dehiscence and the rest the dehiscence was smaller than 4 mm. Three
treated patients developed bone tissue and two of them developed also a dent
piece in the place of the closed gap.
Conclusion: The use of UCB SC associated with decellularized swine
dermis seems to be safe and produce improvements in the evolution of the
treated lip and palatine cleft carrier patients. New cell therapies are under
development to improve the reported results.
339
SYNERGISTIC EFFECT OF BMP-2 AND MESENCHYMAL
STROMAL CELLS ON THE HEALING OF RADIODERMATITIS
THROUGH HIF-1A INDUCTION
S Francois1,3, V Eder2, N Gorin3, L Douay3, MM Benderitter1, A Chapel1,3
1
PRP-HOM, IRSN, Fontenay aux roses, France, 2UMR_S938, INSERM
UMRS_938, Paris, France, 3LAB.P.ART.-EA3852, University of Tours,
Tours, France
Mesenchymal Stromal Cells (MSCs) are effective to treat burns. We
investigated whether Bone Morphogenic Protein-2 (BMP-2) known to
enhance angiogenesis, potentiates MSCs. Posterior limbs of rats were irradiated. 140 rats were divided into 14 groups. Six groups received 25, 35, 45,
55, 65, and 75 Gray (Gy). 55 Gy induced ulceration . Five groups then
received 55 Gy, and either PBS or 2104, 2105, 2106, and 2107 MSCs
from eGFP transgenic rats. MSCs were deposited by 17 intramuscular infusions into the ulcerated area. Three groups irradiated at 55 Gy were used
to study co-infusion of MSCs with BMP-2. Results were evaluated by
clinical examination, histology, confocal and electron microscopy and In
Vitro Endothelial Cell Capillary Tube Formation for vasculogenesis. Hypoxia inducible factor-1 (HIF-1a) was measured by Western blotting and
ELISA and its role confirmed by siRNA blocking. A minimum of 2105
MSCs was required to attain significant improvement. Co-injection of BMP2 and MSCs stopped progression and accelerated repair. BMP-2 and MSCs
synergistically enhanced angiogenesis. Skin repair was mediated through
HIF-1a. These results suggest that new strategies adding cytokines to MSCs
should be evaluated for treating radiodermatitis, burns, and chronic ulcers in
man.
340
LONG-TERM QUANTITATIVE BIO-DISTRIBUTION AND
SIDE EFFECTS OF HUMAN MESENCHYMAL STEM CELLS
(HMSCS) ENGRAFTMENT IN NOD/SCID MICE FOLLOWING
IRRADIATION
S Francois1,2, B Usunier1, L Douay2, MM Benderitter1, A Chapel1,2
1
Radiological Protection and Human Health Division, Institute of Radiological
Protection and Nuclear Safety, Fontenay, France, 2UMR_S938, INSERM,
Fontenay aux roses, France
There is little information on the fate of infused Mesenchymal Stem Cells
(MSCs) and long-term side effects after irradiation exposure. We addressed
these questions using human MSCs (hMSCs) intravenously infused to nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice submitted to total body irradiation (TBI) or local irradiation (abdominal or leg
irradiation). The animals were sacrificed 3 to 120 days after irradiation and
the quantitative and spatial distribution of hMSCs were studied by Polymerase Chain Reaction (PCR). Following their infusion into non-irradiated
animals, hMSCs homed to various tissues. Engraftment depended on the dose
of irradiation and the area exposed. Total body irradiation induced an
increased hMSC engraftment level compared to non-irradiated mice, while
local irradiations increased hMSC engraftment locally in the area of irradiation. Long-term engraftment of systemically administered hMSCs in NOD/
SCID mice increased significantly in response to tissue injuries produced by
local or total body irradiation until 2 weeks, then slowly decreased depending
on organs and the configuration of irradiation. In all cases, no tissue abnormality or abnormal hMSCs proliferation were observed at 120 days after
irradiation. This work supports the safe and efficient use of MSCs by injection
as an alternative approach in the short- and long-term treatment of severe
complications after radiotherapy for patients refractory to conventional
treatments.
341
CORRELATING EX VIVO AND IN VIVO OSTEOGENIC ASSAYS
FOR QUALITY CONTROL OF CLINICALLY DESTINED CGMP
GRADE BM-MSC
A Murgia1, E Veronesi1, V Rasini1, O Candini1, L Sensebe2, P Layrolle3,
H Schrezenmeier4, P Paolucci1, J Burns1, M Dominici1
1
Department of Medical and Surgical Sciences for Children & Adults,
University of Modena and Reggio Emilia, Modena, Modena, Italy, 2Inserm
U1031, EFS, Toulouse, France, 3Inserm U957 - Laboratoire Physiopathologie
de la Resorption Osseuse, Faculte de Medecine de Nantes, Nantes,
France, 4Institute of General Zoology and Endocrinology, University of Ulm,
Ulm, Germany
Among diverse approaches in translational medicine, there is growing interest
in use of mesenchymal stem cells (MSC) to enhance bone regeneration. We
aimed to evaluate the osteogenic potency of human bone marrow
(BM)-derived MSC isolated and expanded according to good manufacturing
practice (GMP) . Recently, numerous genes have been associated with the
osteogenic differentiation, however their expression in differentiated hMSC
ex vivo rarely predict in vivo bone formation . We wished to explore whether
such biomarkers changes were applicable using primary hMSC grown in
medium supplemented by platelet lysate at 1 week instead of 2 weeks time
points. Testing 6 donor BM-hMSCs for osteogenesis using ex vivo matrix
mineralization stainings revealed 4 were positive for Alizarin Red (ALZ) and 3
for Von Kossa (VK). In vivo bone formation assays, implanting hMSC with
osteoconductive scaffold in NOD/SCID mice for 6 weeks, revealed that
hMSC from 4 donors generated new bone ( 15% total area). Notably, the ex
vivo phenotype of matrix mineralization staining at 2 weeks did not necessarily correlate with in vivo bone formation. To address the problem of
donor-specific heterogeneity we selected hMSC from osteogenically functional donors when analyzing 1 week gene expression. Using hMSC from
donors that were positive for ex vivo mineralization, 7 genes showed statistically significant changes using ALZ+ donors and 6 genes using VK+ donors.
Using hMSC from bone-forming donors, 7 genes showed statistically significant changes and 5 of these were also relevant to ex vivo mineralization.
Using these 5 genes for cluster analysis, a strong correlation with boneformation was identified; distinctly clustering hMSC from bone-forming
versus non-bone forming donors. We provide proof of principle that genetic
analysis of hBM-MSC early responses to osteogenic factors ex vivo can
indicate bone forming potential, suggesting that this approach may prove
helpful for clinical trials.
342
QUANTITATIVE
ANALYSIS
OF
BONE
MARROW
CONCENTRATE (BMC) PRODUCED USING SYNGENXÔ-2000
SYSTEM- A POINT OF CARE MEDICAL DEVICE
V Kumar1, JV Perea1, J Walker2, J Sheu2, G Bauer2
1
Scientific Affairs, SynGen, Inc., Sacramento, California, United States, 2Davis
Medical Center, University of California, Sacramento, California, United
States
Introduction: There is growing interest in the use of bone marrow-derived
stem cells to treat a variety of acute and chronic conditions, including cardiovascular and orthopedic indications. The SynGenXÔ-2000 system is a
novel point of care device for efficient and reproducible preparation of bone
marrow concentrate (BMC) from bone marrow aspirate (BMA). The objective
of this study was to characterize the cell counts of BMA from normal healthy
donors and of BMC following processing with the SynGenXÔ-2000 using
complete blood counts (CBC) and flow cytometer-based analysis, and to
determine the viability of the CD34+ cells in the BMC.
Methods: Bone marrow aspirate (108 +/- 9 mL) was concentrated into a
final volume of 20 +/- 0.2 mL in a 12 minute process using the SynGenXÔ2000 system. All 10 bone marrow aspirates used in this study were processed
and sample aliquots were analyzed within 8 hours of collection. For colony
forming unit (CFU-H) assays, cells were plated at a density of 1 and 2x104/
plate. Flow cytometric analysis was performed using the Becton Dickinson
FACS Calibur machine.
Results: The cell counts, cell enrichment factor and cell recoveries of the
10 samples are reported in the table. The SynGenXÔ-2000 system removed
greater than 95% of the RBCs, resulting in a mean Hct of 6.5 2.1% in the
final 20 mL BMC product. The CD 34+ cell viability of the BMC was
S97
98.8 1.1% and microscopic evaluation of the distribution of red, white and
mixed colonies demonstrated no change in the BMC as compared to the
BMA.
Conclusion: The results demonstrated an average recovery of 90 % and 5
times enrichment for mononucleated cells (MNC), CD34+ and CD45+ cells in
the final BMC product. The SynGenXÔ-2000 system demonstrated that it is
an efficient, rapid and easy method for preparing bone marrow concentrate
from human bone marrow aspirate at the point of care.
343
AUTOMATION
OF
UPSTREAM
AND
DOWNSTREAM
MANUFACTURING PROCESS OF CELL THERAPEUTIC
PRODUCT: GAIN IN QUALITY, YIELD AND CONSISTENCY
WITH REDUCED MANPOWER
P Willemsen1, S Snykers1, V Codutti1, C Gumy1, F Collignon2, J Goffinet2,
M Egloff2, J Drugmand2, B De Vos1, E Sokal1, C Dedry1, J Castillo2,
E Halioua1
1
Promethera Biosciences, Mont-Saint-Guibert, Belgium, 2ATMI Life Science,
Brussels, Belgium
Promethera BiosciencesÒ produces the cell-therapy product, HepaStem, to
treat serious metabolic liver disorders. Today, 20 patients have been treated
during a European phase I/II clinical trial. Promethera is currently preparing
its next clinical phases in US and Europe. To upscale the process, minimize
manual operations & related-risks, and reduce costs, Promethera has developed a fully-closed semi-automated system from expansion to final filling.
This next-generation process is based on ATMI’s Xpansion bioreactor
technology followed by in-line centrifugation and filling in Aseptic Technologies closed vials. HepaStem cells were successively expanded in mid-scale
XP100 (61200cm2) and large-scale bioreactors XP200 (122400cm2) without
change in growth-rate, population doubling-time, in-process impurities and
quality. A 400-fold concentration of the harvested product (150000 cells/ml
upon harvest to 60.10E6 cells/ml after centrifugation) with satisfactory
preservation of viability and quantity was accomplished via ATMI’s in-line
centrifuge. Sterility was preserved at all-time. In-line filling in closed vials
substantially reduced batch-to-batch variation in terms of content uniformity
with less than 15% variation in terms of cell quantity/vial and less than 2% in
terms of volume To date, this combined closed process has passed all qualification steps with increased sterility risk mitigation. The first full-scale GMPbatches are currently being manufactured. In conclusion, the stream-lined
expansion-centrifugation-filling process via combination of ATMI’s closed
XpansionÔ bioreactor/centrifugation with in-line filling in Aseptic Technologies closed vials offers a valuable technology for large-scale commercial
production. This technology results in reduced costs, increased batch-tobatch consistency and is able to increase the yield of Promethera’s production
process by 10-fold while reducing manpower and global operational time by
50-to-60%.
344
MESENCHYMAL STROMAL CELLS FOR OSTEONECROSIS OF
THE FEMORAL HEAD (ONFH). DATA FROM ONGOIN
CLINICA TRIAL
R Coll1, M Aguirre2, A Hernandez2, M Caminal3, J Vives3, I Oliver-Vila3,
M Codinach3, M Blanco3, A Pla3, J Garcia3
1
XCELIA-Clinical Development, Banc de Sang i Teixits, Barcelona,
Barcelona, Spain, 2Orthopedic Surgery and Traumatology, Hospital Vall
Hebron, Barcelona, Barcelona, Spain, 3XCELIA, Banc de Sang i Teixits,
Barcelona, Spain
Background: ONFH is an ischemic event that eventually progresses to
collapse, total articular degeneration and arthroplasty. It affects mainly young
adults and it’s associated to pain, functional limitation and a great socio-economic impact. Current treatments lack efficacy limiting its progression. The
working hypothesis proposes that XCEL-MT-OSTEO-ALPHA (ex-vivo
expanded autologous bone marrow mesenchymal stromal cells fixed in allogeneic bone tissue, produced at Xcelia- Advanced Therapy Division of the
Blood and Tissue Bank of Catalonia, under GMP conditions) is a useful
product to achieve bone regeneration and avoid progression to collapse. This
study has been funded by the Spanish government (MSSSI and MICINN) and
the EU (ERDF).
S98
Poster Abstracts
Methods: Prospective, randomized, two-arms, parallel, single-dose, openlabel (blinded assessor), phase I-II study in which 24 patients affected with
ONFH (ARCO I/II) will be randomized to core decompression/XCEL-MTOSTEO-ALPHA (test arm) or isolated core decompression (standard treatment) and followed up for 12 months. Primary objective is to assess the
feasibility and safety of XCEL-MT-OSTEO-ALPHA. Secondary objectives
are to assess the efficacy by MRI and clinical questionnaires (VAS for pain,
QoL SF-36 and WOMAC Index). Patient’s asigned to the test arm will undergo previous surgery for bone marrow extraction. After the expansion, the
product is implanted surgically by means of a throcar (cylinder shape), through
the bone decompression tunnel.
Results: 13 patients have been randomized (6 to test arm). Four (2 in each
arm) have finished the 12-month follow-up while 6 have reach the 6-month
follow-up. There has been no hip collapse. Reported AEs are negligible and
not related to the test product. No SAEs have been reported so far.
Conclusions: Present data suggest that the use of XCEL-MT-OSTEOALPHA for the surgical treatment of ONFH is feasible and safe, possibly
providing a new treatment option for this pathology.
345
MESENCHYMAL STROMAL CELLS FOR SPINAL FUSION. DATA
FROM ONGOING CLINICAL TRIAL
R Coll1, E Caceres2,6, A Garcia de Frutos2, M Ubierna3, A del Arco4, G Salo5,
M Codinach7, M Blanco7, A Pla7, J Garcia7
1
XCELIA-Clinical Development, Banc de Sang i Teixits, Barcelona, Spain
2
Orthopedic Surgery and Traumatology, Hospital Vall Hebron, Barcelona,
Barcelona, Spain, 3Orthopedic Surgery and Traumatology, Hospital
Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain, 4Orthopedic
Surgery and Traumatology, Hospital de la Santa Creu i Sant Pau, Barcelona,
Barcelona, Spain, 5Orthopedic Surgery and Traumatology, Parc de Salut Mar,
Barcelona, Barcelona, Spain, 6ICATME, Hospital Universitari Quiron Dexeus,
Barcelona, Barcelona, Spain, 7XCELIA, Banc de Sang i Teixits, Barcelona,
Barcelona, Spain
Background: Spinal fusion is a surgical technique for the treatment of a great
variety of pathologies affecting the spine. Spinal coalition using patient’s iliac
crest is the standard surgical technique, although it is associated with a relevant
percentage of failures and local morbidity of the bone donor area. The working
hypothesis proposes that XCEL-MT-OSTEO-ALPHA (ex-vivo expanded
autologous bone marrow mesenchymal stromal cells fixed in allogeneic bone
tissue, produced at Xcelia- Advanced Therapy Division of the Blood and
Tissue Bank of Catalonia, under GMP conditions) is useful to achieve bone
generation for spinal fusion. This study is funded by the Spanish government
(MSSSI and MICINN) and the EU (ERDF).
Methods: Prospective, multicenter, randomized, two-arms, parallel, singledose, open-label (centralized blinded assessor), phase I-II clinical trial in which
62 patients affected with L4-L5 degenerative spondylolisthesis (Meyerding
grade I-II) and/or degenerative discopathy will be randomized to XCEL-MTOSTEO-ALPHA or the standard treatment, with a 12-months follow-up.
Primary objective is to assess the feasibility and safety. Secondary objectives are
to assess the efficacy by imaging procedures (helicoidal tomography and x-Ray)
and clinical questionnaires (VAS for pain, QoL SF-36 and Oswestry Disability
Index). Patient’s asigned to the test treatment will undergo previous surgery for
bone marrow extraction. After the expansion, the product is implanted
surgically.
Results: 19 patients have been randomized (9 to experimental arm). Two
patients (experimental arm) have finished the 12-month follow-up and 10 have
a follow-up of 6 month. Imaging data suggest that the test treatment produces
complete fusion at month 12, with no associated AEs. No SAEs have been
reported.
Conclusions: Present data suggest that the use of XCEL-MT-OSTEOALPHA for surgical treatment in spinal fusion is feasible and safe, reaching
consolidation of treated patients at month 12.
346
EFFICIENT CRYOPRESERVATION OF HUMAN ES AND IPS
CELLS IN A CHEMICALLY DEFINED, CGMP PRODUCED,
SERUM-, XENO-, AND DMSO-FREE FREEZING MEDIUM
L Hagbard1, J Ericsson1, C Karlsson2, T Kallur1
1
BioLamina AB, Stockholm, Sweden, 2Cellectis stem cells, Gothenburg,
Sweden
Human pluripotent stem cells (hPSCs) show great promise in regenerative
medicine, drug discovery, as well as importance for basic research. It is
therefore a priority to establish efficient, user-friendly, and robust methods
for bulk hPSCS cryopreservation. Thus far, obtaining good survival after
thawing is problematic and, furthermore, current slow-freezing protocols
result in hPSCs with a tendency to spontaneously differentiate. We have
developed a novel, current good manufacturing practice (cGMP) produced,
chemically defined, xeno- and dimethyl sulfoxide (DMSO)-free cryopreservation medium denoted FREEZEstem for cryostorage and banking of human
embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs).
The hPSCs were frozen in FREEZEstem and compared to a commercial
cryomedium containing 10% DMSO and a standard cryomedium with 10%
fetal calf serum and 10% DMSO. Viability after thawing, toxicity of the
freezing media as well as the impact of different thawing solutions was
assessed. The cellular phenotype was evaluated using immunocytochemistry
or flow cytometry analysis and the proliferation and differentiation potential
were investigated. We found that directly after thawing, the same or a higher
number of hESC and iPSC colonies were detected for cells frozen in
FREEZEstem and these cells were less sensitive to the thawing solution.
Abundant expression of stem cell markers, high proliferation rate and
extensive differentiation potential were detected for hPSCs frozen in all three
cryomedia. Importantly, we have optimized the protocol for single-cell
freezing and, thus, is ideal for use in a hPSC culture system which supports
single-cell passaging such as the LN-521 matrix. FREEZEstem not only has
the advantage of being cGMP manufactured and DMSO-free, it is also a userfriendly, robust freezing medium with very low toxicity enabling total control
over hPSC cultures, thus offering an excellent, simple option for banking
human ES and iPS cells.
347
EFFECT OF AUTOLOGOUS MESENCHYMAL STEM CELL(MSC)
INJECTION ON HEALING OF CARTILAGE DEFECTS IN A
CANINE MODEL OF OSTEOARTHRITIS
H Gabr2, WA Elkheir1
1
Clinical Pathology, Cairo University, cairo, Egypt, 2Clinical pathology,
Faculty of medicine-Cairo university, CAIRO, Egypt
Chondrogenesis is a well-orchestrated process derived by chondroprogenitors that undergo to condensation , proliferation and chondrocyte differentiation. Because cartilage lacks blood supply, it lacks
regenerative power and subsequent wound healing. The end stage of cartilage damage frequently leads to O.A. resulting in a significant decrease in the
quality of life of millions of people. In vitro, MSCs showed the potential to
differentiate and can be multiplied without losing their multilineage capacity
of differentiation. This made the MSCs the cell of choice in tissue engineering. MSCs are multilineage progenitor cells and responsible for the
turnover and repair of mesenchymal tissues, such as bone, cartilage, ligament, muscle, and fat. The objective of this work was to confirm the fitness
of the dog as a good model of OA; effect of cell therapy in cases of acute and
chronic,compared to control group surgically induced partial thickness
chondral defects through the injection of autologus bone marrow derived
MSCs in dogs. This work was done on 24 knees of male domestic mongrel
dogs by doing surgical chondral defects then injected intra-articular with
MSCs according to classified groups: acute (injected after 1 day),chronic(after 1 month) and control group not injected. The dogs sacrificed after
1,2,6,8 weeks of injection. Assessment by histological scoring of cartilage
repair (Os Score) for blind randomized samples and by clinical examination
for lameness degree score.
Results: Our results showed that dogs possess characteristics that are not found
in traditional rodent models and confirmed the efficacy of direct intraarticular
injection of MSCs to home and function in cartilage defects both in acute and
chronic lesions.
Conclusion: The local delivery of MSC is a good therapeutic option for O.A.
348
STELLATE CELLS AND LIVER PARENCHYMA GENE
TRANSCRIPTION CHANGES AFTER STEM CELLS THERAPY
IN EXPERIMENTAL LIVER FIBROSIS AND CIRRHOSIS
TP de Paula1, C Resende4, MD Rossi2, M Pavão3, S Rehen2, J Lapa e Silva1,
GF Rezende1
S99
1
Clínica Medica - Faculdade de Medicina, Federal University of Rio de Janeiro,
Rio de Janeiro, RJ, Brazil, 2Instituto de Ciências Biomedicas, Federal
University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil, 3Instituto de
Bioquímica Medica, Federal University of Rio de Janeiro, Rio de Janeiro, RJ,
Brazil, 4Radiodiagnóstico - Hospital Universitário Clementino Fraga Filho,
Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil
Introduction: The regulation of hepatic stellate cells (HSCs) genes expression
in stem cell therapy may be a useful strategy for reversal of fibrosis in chronic
liver disease.
Aim: To evaluate the effect of two types of stem cells (SC) in HSCs and liver
parenchyma (LP) genes transcription in an experimental model of liver fibrosis
or cirrhosis.
Methods: Balb/C mice received CCl4 peritoneally triweekly and alcoholic
liquid diet ad-lib for 12 or 33 weeks. First, 3-5 x105 bone marrow mesenchymal
stem cells (MSCs; n¼6) or embryonic stem cells (ESCs; n¼6) were injected
weekly in the LP of animals presenting liver fibrosis (12w induction), guided by
ultrasound imaging, and the animals were sacrificed one week after the third
cell infusion. As controls, other groups received the respective cell vehicles
(n¼3, each) or no intervention (n¼3). After, mice presenting liver fibrosis (12w
induction) or cirrhosis (33w) were submitted to the same protocol, with ESCs,
vehicle or no intervention. TGF-b, MMP-9, TIMP-1 and type I collagen (ColI) gene transcription of HSC and LP was analized by real-time PCR and
compared to pretreatment groups.
Results: In liver fibrosis mice, a decreased expression of TGF-b and Col-I
in HSCs and LP was observed only after MSC injections . The expression of
TIMP-1 was not influenced by SC. The expression of MMP-9 in LP was
increased after both types of SC, but little has changed in HSCs. The stage of
liver fibrosis did not interfere in TIMP-1 expression after SC, however influenced TGF-b in LP and MMP-9 or Col-I in HSCs.
Conclusions: SC modulate gene transcription in LP and HSC and the
response appear to be better with MSCs. Transcript changes similarity in
HSCs and LP suggests the former are responsible for the modifications. The
stage of fibrosis seems to influence gene expression of HSCs.
349
ATTACHING
CELLS
TO
NATURALLY
OCCURRING
EXTRACELLULAR MATRICES PRIOR TO CELL DELIVERY
RDR Ritchie1, S Charlebois2, MC Hiles1
1
Cook Biotech, Inc., West Lafayette, Indiana, United States, 2Med Institute,
Inc., West Lafayette, Indiana, United States
Cell therapy is showing promise for efficacy in the treatment of a variety of
conditions. Yet the almost immediate decline in the number of viable cells after
implantation is troublesome. The pursuit of a delivery mechanism that increases the survival of cells after implantation and sequestration of cells at the
site of delivery has led to investigations utilizing naturally occurring extracellular matrices, such as small intestinal submucosa (SIS). Adherent-dependent
cells, such as mesenchymal stem cells (MSC), undergo anoikis when no
attachment sites are available. Combining such cells with an ECM containing
native cell attachment binding sites should enhance the survival of implanted
cells. Cell/ECM combinations have been implanted before, but an injectable
version of an ECM would create a less invasive treatment. The data presented
here utilizes an intact, solid-phase configuration of SIS that can be delivered via
injection through a needle. Therapeutic cell types, such as placenta-derived
MSCs, readily attach to this configuration of SIS, which maintains its native
three-dimensional structure and composition (Figure 1). With an eye on
clinical use and suitability for therapy, we investigated the combination of cells
and SIS in a quick assay to simulate bedside usage and to avoid the need for
prolonged co-culturing. We demonstrated that 500,000 skeletal musclederived cells will attach in 1 hour to SIS in a delivery volume of 0.25 ml. The
attachment rates and the number of cells that attach vary for each cell type. The
attachment of cells to SIS prior to delivery will provide the cells with a healthy
microenvironment within a damaged or ischemic tissue. Studies to determine if
the combination of the SIS and therapeutic cells enhances cell survival after
implantation are ongoing. Fewer cells may be required for clinical efficacy if
the number of cells that survive increases by co-delivering cells and SIS.
Placenta-derived MSC on injectable SIS.
350
CRYOPRESERVATION OF STEM CELLS FOR THE FUTURE:
LOOKING BEYOND DMSO
S Matosevic, C Zylberberg
Akron Biotechnology, Boca Raton, Florida, United States
Cryopreservation is important for the storage and banking of cells that would
otherwise be prone to damage over time. Improved strategies for cryopreservation that advance our understanding of the effects of preservation media
and freezing conditions on the viability, funcionality and differentiation
potential of stem cells are of growing interest. Recently, cryoprotectant
formulations have emerged that aim to reduce toxicity associated with
traditional media, the use of which calls for the rapid removal of DMSO
following thawing. One such DMSO-free formulation involves the polyampholyte poly-l-lysine (PLL), a homopolymer of the essential amino acid
lysine. Like DMSO, PLL media has been applied to vitrification and slow
cooling mechanisms. But unlike DMSO e which penetrates the cell membrane and prevents intracellular ice crystal formation e poly-l-lysine cannot
cross the cell membrane and works by acting as an antifreeze protein. The
active, carboxylated form of PLL is obtained after blocking reactive amino
groups, with an optimal conversion of amino-to-carboxyl groups of 50-65%.
Work in our laboratories has focused on developing both PLL-based,
DMSO-free as well as traditional DMSO-rich cryoprotectant formulations.
Validation studies have shown distinct cryoprotective behavior that is
dependent on the composition of the medium. While cryopreservation of
stem cells is not defined by any solid standards, novel formulations must not
only ensure that cell functionality and viability is maintained after thawing,
but must provide a methodology that can be routinely implemented by
different end users. Cell toxicity and post-thaw viability and functionality
needs to be optimized for each cell type. Any procedures must also comply
with appropriate international laws and guidelines. We are showing data for
several cell types, namely mesenchymal stem cells and induced pluripotent
stem cells, together with an indication to pursue this product for future logistic purposes.
S100
Poster Abstracts
351
HUMAN PLATELET LYSATE SUPPORTS GROWTH OF HUMAN
SKIN AND VASCULAR eDERIVED CELLS SIMILAR TO FETAL
BOVINE SERUM AND SERUM FREE MEDIA
Acellular extracellular matrices (ECM) such as dermis and blood vessels have
advanced the field of regenerative medicine and became life-saving clinical
practices. However, the prolonged angiogenesis and homing of endogenous
cells impede tissue remodeling and may lead to graft failure. Dermal fibroblasts, keratinocytes, and vascular endothelial cells are commonly used for
research. However, these cells are often grown in FBS supplemented media
which hinders their rapid transition into clinical application in the field of
skin and vascular tissue engineering due to regulatory challenges and risk of
disease transmittance. Thus, there is a need to find a human-based media
additive. hPL is derived from human platelets and contains similar growth
factors and cytokines found in FBS at comparable levels. It has been
demonstrated that hPL supports the growth of various cells. The focus of this
study was to evaluate the ability of heparin-free hPL (PL-NH) and hPL
requiring heparin (PL-H) to support proliferation and maintenance of multipotency properties of primary human cells from different tissues at different
concentrations of media. COOK HPLÔ is produced at an industrial large
scale (minimum20 L/ lot) with high lot-to-lot consistency and purity. Preliminary results of both versions of hPL at different concentrations (2.5, 5,
and 10%) supported cell proliferation of fibroblasts, endothelial, and keratinocytes to comparable levels of FBS at all concentrations, as well as keratinocytes SFM. Morphology of all cells expanded in hPL did not change over
several passages and were similar to cells grown in FBS. Also, flow cytometry
analysis indicated that cells maintained their respective expression of surface
markers throughout several passages. This study shows the ability of using
hPL as an alternative to keratinocytes SFM and FBS for the growth of dermal
fibroblasts, keratinocytes, and endothelial cells for skin and vascular cellbased therapeutics in xeno-free cultures.
352
FREEZING AND RECOVERY OF MESENCHYMAL STEM CELLS
IN HUMAN PLATELET LYSATE
Fetal bovine serum (FBS) is still used as a standard media supplement for cell
expansion and freezing along with dimethyl sulfoxide (DMSO). FBS poses several
regulatory and potential species cross-contamination challenges hindering its
potential use for clinical application of cell expansion and freezing. In an attempt
to limit these challenges, there is a need for a human-derived alternative. Human
platelet lysate (hPL) is derived from human platelets and contains similar growth
factors and cytokines found in FBS at comparable levels. The focus of this study is
to evaluate heparin-free hPL (PL-NH) as a freezing media, in conjunction with
DMSO, and a recovery media post thaw of neonatal and adult MSCs from
various tissues. In this study, different freezing media formulations including 90%
hPL/10% DMSO, 95% hPL/5% DMSO, and 90% culture media containing
10% hPL/10% DMSO were tested and compared to similar formulations
substituted with FBS instead of hPL and commercially available serum-free
freezing solutions. MSCs were assessed for viability and proliferation in cultures
post-thaw. Preliminary results of COOK HPLÔ PL-NH with different concentrations showed high cell viability (>90%) and normal proliferation post-thaw
and were comparable to serum-free freezing media and FBS containing freezing
media of amniotic-, bone marrow-, and adipose-derived MSCs, as well as human
dermal fibroblasts. This study demonstrates the ability of using hPL to substitute
FBS in freezing solutions and serum-free freezing solution.
353
STEM CELLS RECOVERED FROM ADIPOSE TISSUE FORM NEW
CARTILAGE IN OSTEOARTHRITIS OF THE HUMAN KNEE
R Krebs, H Pototschnig, P Schöttle, E Alt
Isar Medical Center, Munich, Germany
Osteoarthritis (OA) is the most frequent joint disease causing pain, functional
loss and disability. Cell-based treatments have shown encouraging results in
animal studies and in initial human case reports. Mesenchymal stromal cells
provide analgesic, anti-inflammatory and immune-modulatory effects. In
addition, cartilage repair has been shown. With a novel point-of-care-system
(POC) a high number of adipose-tissue derived regenerative cells (ADRC)
(1 +- 0.25 mio cells per g adipose tissue) can be obtained without culturing.
These fresh autologous ADRC contain a significantly higher amount of
pluripotent cells (10%) compared to bone marrow derived cells (0,1%). 13
male and 9 female patients (PX) suffering from OA of the knee grade 3-4
were operated by the same orthopedic surgeon between 6/12 and 4/13. All
PX were treated by ADRC, arthroscopic abrasion combined with Pridie
drillings and osteotomy for malalignment correction. Before arthroscopy,
60 ml abdominal adipose tissue was harvested by liposuction under local
anaesthesia. The CE-marked and by EMA as “Non-ATMP” regulated
Transpose RTÔ System (InGeneron Inc. Houston TX) was used as POC.
Within 60 minutes, an ADRC cell suspension (5-7 ml) was obtained and
applied to the knee within the same operative procedure. At arthroscopic 1year follow-up (FU) (17/22 PX studied so far), coverage of the cartilage
defect of more than 95% was found in all patients. The quality of coverage
was rated 4,5/5 points. All PX reported significant improvement concerning
pain and function, 90 % of the PX were completely restored and pain free at
FU. ADRC are an efficient, cost effective complementary treatment for
cartilage repair avoiding any artificial treatment such as endoprosthetics.
The InGeneron POC is easy to use, effective and safe. The encouraging
results are superior to results of patients treated by conventional methods
only. Further studies with a longer FU time will be performed to validate
those preliminary results.
354
SINGLE-USE CENTRIFUGATION SOLUTION FOR VOLUME
REDUCTION AND CELL WASHING PROCESS IN CELL
THERAPY MANUFACTURING
S Mehta
Operations, kSep Systems, LLC, Durham, North Carolina, United States
Current bioprocessing technologies have limited applications in cell therapy
manufacturing as they are optimized for the manufacture of recombinant
proteins. For example, most cell harvest technologies are efficient in
removing cells but not in maximizing viable cell density, viability, washing,
and recovery; parameters that are most important for cell therapy
manufacturing. Most commonly cells are retained by either centrifugation
or filtration based devices. These approaches are often problematic. Conventional centrifugation based devices cause stress and nutrient deprivation
to the cells in the pellet which results in low viability, while filtration based
devices often suffer from issues such as clogging, retention of cell debris,
and shear stress on cells. We have developed the first single-use fluidizedbed centrifugation systems that are completely closed. Our results show that
kSep systems can concentrate diverse types of cells with high recovery while
maintaining high viability. Additionally, kSep systems can remove cell
debris, light particulate impurities, all while significantly reducing aggregation of cells. We have designed these systems without any rotary seals
(providing reduced sterility risk) or filters (for reduced issues from clogging). Once captured and concentrated, the cells can efficiently be washed,
manipulated, and harvested. This technology is a breakthrough for applications requiring maintenance of cellular integrity during processing.
Results show >90% recovery of cells with unchanged viability and 99.9%
removal of contaminants. Our automated systems are currently being
used for cell therapy, recombinant proteins, and vaccine manufacturing
processes.
355
S101
being she the first case of use of bone marrow autologous Mesenchymal stem
cells expanded in vitro during 1 month with a posterior supra selective infusion
in the colonic area by endovascular catheterism in superior and inferior
mesenteric artery to accurate the arrival of the cells to the tissue. After one
month of the treatment several changes in the patients was documented like
low of the number of diarrhea episodes , bloodiness , pain and down regulation
in the number of CDAI score. Several important changes in the cytokines
patterns are fund, like upregulation of IL-10, TGF-B, and IL-6 and dowregulation of IFN-gamma and IL-2. The autoantibodies like ANAS and ASCAS
was negative three month after the therapy with complete remission after 3
months maintained until one year then. We conclude that autologous expanded
and endovascular infused Mesenchymal Stem Cells offer an safety opportunity
of immune regulation and tissue regeneration in Chron’s Disease.
357
PLX-PAD
CELL
TREATMENT
MITIGATE
TOLL-LIKE
RECEPTOR INDUCED PREECLAMPSIA SYMPTOMOLOGY IN
MICE
L Pinzur1, V Chiasson2, P Chatterjee2, M Hatahet2, E Abraham1, A Chajut1,
R Ofir1, B Mitchell2
1
Pluristem Therapeutics Inc., Haifa, Israel, 2Division of Nephrology &
Hypertension, Texas A&M Health Science Center/Scott & White Healthcare,
Temple, Texas, United States
Preeclampsia is a human pregnancy-specific disease, defined as the occurrence
of hypertension and significant proteinuria after the 20th week of gestation,
and affects 2-6% of previously healthy women. Preeclampsia is characterized
by a generalized systemic maternal inflammatory response, placental dysfunction and has no effective treatment except abortion or delivery. PLacenta
eXpanded (PLX)-PAD are placenta derived mesenchymal-like adherent stromal cells expanded in Pluristem’s proprietary bioreactor system using a threedimensional culture method. PLX-PAD possess immunomodulatory properties as well as pro-angiogenic and anti-fibrotic properties and have demonstrated a therapeutic potential in various animal models. PLX-PAD are suitable
for allogeneic administration without HLA-matching due to their low immunogenicity. . The therapeutic effect of PLX-PAD was tested in a mouse model
of preeclampsia. Preeclampsia was generated by intra-peritoneal introduction
of either TLR3 agonist (poly I:C) or TLR7 agonist (R837) on days 13, 15, and
17 of gestation in C57BL/6 mice followed by PLX-PAD administration on
day 14 of gestation. PLX-PAD treatment resulted in progressive reduction of
SBP over a 3 day period and normalization of urinary protein/creatinine ratio
and aortic endothelium-dependent relaxation responses within 4 days after
treatment. PLX-PAD had no significant effects on the number of fetuses or
incidence of fetal demise. PLX-PAD also reduced spleen weight/body weight
ratios, normalized splenic levels of gamma-delta T cells, decreased plasma IL6 levels, and restored plasma IL-4 levels in TLR agonist treated mice.
Additionally, PLX-PAD treatment decreased fibrin deposition in the
placental vasculature and significantly reduced placental HIF-1alpha protein
levels. These data suggest that PLX-PAD treatment is a potential therapy for
preeclampsia.
356
IMMUNOMODULATION AND INDUCTION OF REMISSION
IN CROHN’S DISEASE WITH AUTOLOGOUS EXPANDED
MESENCHYMAL STEM CELLS. CASE REPORT
J Arturo1,3, C Perez1,3, L Larios3, O Segura2, Y Bastidas1,3, O Segura2,
C Lucena4, E Lucena4, C Ruiz2
1
molecular immunology, inmugen corporation, BOGOTA,
Colombia, 2Haemodinamia, Diagnosticos e Imagenes, Bogota,
Colombia, 3Molecular and Regenerative Medicine, Inmugen Corporation,
Bogota, Colombia, 4Cecolfes, Bogota, Colombia
358
SCALABLE PRODUCTION OF HUMAN MESENCHYMAL STEM/
STROMAL CELLS IN MICROCARRIER-BASED CULTURE
SYSTEMS
CL da Silva1,2, JG Carmelo2, A Fernandes-Platzgummer2, JL Weber3,
M Bear4, M Hervy4, MM Diogo1,2, JS Cabral1,2
1
Department of Bioengineering, Instituto Superior Tecnico, University of
Lisbon, Lisbon, Portugal, 2Stem Cell Bioengineering and Regenerative
Medicine Laboratory, Instituto Superior Tecnico, University of Lisbon,
Lisbon, Portugal, 3Corning Life Sciences, Corning Incorporated, Corning,
New York, New York, United States, 4Corning SAS, CETC, Avon, France
Chron’s disease is a severe and progressive autoinflammatory and autoimmune
disease with major complications like fistula, ulcers and cancer. Today the
current treatments include immunesuppressors and anti cytokine therapies like
anti TNF-blockers, whereas in the world several patients who use this medications, can present chronic imflammatory activations of the disease and serious
complications like adverse effects. Here we present a case of a patients with
resistance and not response to the current treatments like anti TNF-blockers,
The clinical application of human mesenchymal stem/stromal cells (MSC)
for a wide range of diseases is at the front line of stem cell-based cellular
therapies. Nevertheless, successful implementation of these cell-based
therapies requires the ability to generate large numbers of cells with welldefined characteristics in a cost-effective way. To this end, significant
research has been done on the development of microcarrier-based cultures
in scalable stirred bioreactors alongside with the development and evaluation
S102
Poster Abstracts
of well-defined serum-free and xeno-free media formulations which represent important milestones towards the production of MSC for cellular
therapies. In this work, a microcarrier-based suspension culture was explored
for the scale-up of MSC expansion in xeno-free medium using synthetic
peptide acrylate surface beads. Cells were maintained on CorningÒ SynthemaxÒ II polystyrene and CELLstartÔ-coated Solohill plastic microcarriers (dos Santos et al, 2011) for 14 days in xeno-free medium. Bone
marrow (BM) derived- and adipose tissue (AT) derived-MSC were seeded at
50,000 cells/mL with 4.5 cm2/mL of microcarriers in spinner flasks (80 mL
volume). To maximize cell seeding, the adhesion step was performed in 50%
final volume during the first 24 hours. The efficiency of initial cell adhesion
to microcarriers was similar for both cell types; slightly better cell adhesion
was observed on Synthemax II compared to CELLstart-coated microcarriers: 48% and 43% for AT MSC and 42% to 37% for BM MSC. The
homogenous cell distribution on the first days of culture resulted in a higher
expansion rate with exponential growth from day 4 to day 6 for AT MSC
and day 4 to day 9 for BM MSC. The longer growth phase observed for BM
MSC resulted in higher cell densities of 350,000 cells/mL compared to
250,000 cell/mL for AT MSC cultures. Expanded cells maintained their
characteristic immunophenotype and multilineage differentiation potential.
Ongoing work includes the translation of this culture system to a fully
controlled stirred-tank bioreactor (1 L) and the study of the impact of
different culture parameters namely feeding regime and dissolved oxygen
concentration on cell productivity. This scalable xeno-free, microcarrierbased culture platform is anticipated to enable cost effective and robust
production of MSC meeting the needs of the allogeneic “off-the-shelf”
MSC therapy sector.
360
CONTROLLED CULTURE OF ADHERENT CELLS IN A NOVEL,
CLOSED BIOREACTOR SYSTEM FOR CELL THERAPY
PRODUCTION
R Das1, R Roosloot1, W Tra1, H Roelofs2, P van Santen1, J de Bruijn1,3
1
Xpand Biotechnology BV, Bilthoven, Netherlands, 2Hematology and Blood
Bank, Leiden University Medical Centre, Leiden, Netherlands, 3Twente
University, Enschede, Netherlands
There is an increasing need to make adherent cell expansion cheaper, safer and
easier. Standard culture practice should be translated to closed, controlled systems
to increase safety and quality. Current closed systems are limited in their culture
range, only limited maximal cell numbers can be obtained, or the minimal volume
is too large to support growth of small cell numbers. Therefore, a bioreactor was
designed that supports growth of limited cell numbers to therapeutic quantities.
BM-MSCs (P2) were expanded under controlled conditions (DO 25%, pH 7.3) in
a closed bioreactor system (Fig 1). MSCs were grown for 17 days on microcarriers
in an expandable culture bag and compared to stirred vessel culture. Flow
cytometry was performed and differentiation capability was assessed. Therapeutic
potential will be assessed through stimulation assays using various cytokines (e.g.
IDO production upon IFN-g stimulation). Using closed procedures, culture was
maintained for 17 days (Fig 2) and volume was expanded twice, resulting in a final
volume of 850 ml and 5.0E5 cells/ml (400E6 cells, Fig 3), whereas stirred vessels
yielded 60*106 cells (PDL 6.8 vs 4.1). Cells were positive (>95%) for CD73,
CD90 and CD105 and negative (<2%) for CD43, CD11b, CD19, CD45 and
HLA-DR. All cells could be differentiated along the osteo- and adipogenic lineage. However, immunesuppressive potential needs to be assessed. A novel
359
DEVELOPMENT OF CELL-ENHANCED CHITOSAN SCAFFOLDS
TO OVERCOME LONG GAPS AFTER PERIPHERAL NERVE
INJURY
S Wrobel1,2, M Morano3, C Meyer1,2, A Shahar4, O Ziv4,
K Haastert-Talini1,2, S Geuna3, C Grothe1,2
1
Institute of Neuroanatomy, Hannover Medical School, Hannover,
Germany, 2Center for Systems Neurosciences (ZSN) Hannover, Hannover
Graduate School for Veterinary Pathobiology, Neuroinfectiology, and
Translational Medicine (HGNI), Hannover, Germany, 3Department of
Clinical and Biological Sciences, Neuroscience Institute of the Cavalieri
Ottolenghi Foundation (NICO), University of Turin, Turin, Italy, 4NVR
Laboratories LTD., Neural and Vascular Reconstruction, Ness Ziona,
Israel
Chitosan-based artificial nerve guidance conduits are developed in the
BIOHYBRID project. Composite nerve grafts existing of a chitosan outer
shell and a core scaffold made of three-dimensionally structured NVR
hydrogel are enriched with genetically engineered rat Schwann cells (SCs).
Cell survival, growth factor expression pattern and cellular bioactivity and
potency to support functional peripheral nerve regeneration were investigated in vitro and in vivo. In vitro studies demonstrated that selected
growth factors (fibroblast growth factor- 2, FGF-2 18kD, and glia-derived
neurotrophic factor, GDNF) were over-expressed by genetically engineered
SCs seeded into the core scaffold (0.5% NVR gel) in comparison to emptyvector expressing and naïve SCs. Neurite outgrowth of sensory DRG
neurons was supported by SCs-FGF-2 and SCs GDNF in co-culture bioassays. The support of neurite outgrowth was comparable to that produced
by the same growth factor coupled to ferro-nanoparticles. We further
demonstrated that SC viability was preserved when seeded into NVR-Gel/
chitosan scaffolds over up to 9 days in vitro (WST-1 assay). During this
period, the cells formed aggregates during the first 24hrs but then grew out
of these to populate the NVR-Gel scaffold. Encouraged by the in vitro
results cell seeded composite nerve guides (naïve SCs, SCs-FGF-2, SCsGDNF) were transplanted into sciatic nerves of adult rats to bridge 15 mm
nerve gaps. Autologous nerve transplantation served as the clinical standard
control condition. Over 16 weeks in vivo functional recovery is currently
tested by electrophysiology, static sciatic function index motor function
evaluation and von-Frey mechanosensitivity evaluation. First in vivo results
will be available till April 2014. Our results give a promising perspective of
an efficient composite nerve guide for long gap nerve repair. This work has
received funding from the European Community’s Seventh Frame work
Programme (FP7-HEALTH-2011) under grant agreement n 278612
(BIOHYBRID).
Xpand’s bioreactor system.
MSCs were successfully cultured inside a bioreactor bag for 17 days.
Therapeutic cell numbers were obtained at harvest.
S103
D Giroux1, P van der Aar2, M Serra3
PBS Biotech Inc., Camarillo, California, United States, 2DYNC, Breda,
Netherlands, 3IBET, Lisbon, Portugal
1
Cell count of bioreactor culture versus three stirred vessels. Cells in the
bioreactor doubled 6.8 times, while the stirred vessel controls resulted in
only 4 population doublings. A total of 400 million cells were obtained.
bioreactor system with sampling possibilities is introduced for GMP-grade MSC
production. The system enables culture from minimal numbers to therapeutic
quantities in a controlled environment. The closed design reduces contamination
risk and enables operation outside expensive cleanroom facilities. Operator
involvement is minimized, limiting labor and production costs. The switch from
open, uncontrolled procedures to closed systems for production of cell therapy
products presents a major step forward to improved quality, reproducibility and
affordability of these therapies.
361
HUMAN-DERIVED
RAW
MATERIALS:
CONTROLLED,
CONSISTENT COLLECTION AND CRYOPRESERVATION
ENABLE SUCCESSFUL MANUFACTURING OF CELL-BASED
PRODUCTS
TV Ramos1, W Wang1, J Okhovat1, G McPherson1, S Burger2
1
HemaCare Corporation, Van Nuys, California, United States, 2Advanced Cell
& Gene Therapy, LLC, Chapel Hill, North Carolina, United States
Daniel Giroux, PBS Biotech, Inc. USA, [email protected] Margarida
Serra, Instituto de Biologia Experimental e Tecnologica (iBET) Marcos Sousa,
Instituto de Biologia Experimental e Tecnologica (iBET) Bone-Marrow
derived Mesenchymal Stem Cells (BM-MSCs) are multipotent non-hematopoietic cells. They are easy to isolate, grow well in vitro, and possess both
immunomodulatory and low immune reactivity properties. MSCs are an ideal
cell for the development of “off the shelf” allogeneic cellular therapeutics, To
move from these small trials to commercialization, cells must be manufactured
at large scale while remaining potent and safe. To address this manufacturing
bottleneck, we have developed a unique MSC manufacturing process where
BM-MSCs are cultured on microcarriers with low shear mixing using the AirWheel Bioreactor. BM-MSCs (StemCell Technologies) were cultured in xenofree Mesencult medium (StemCell Technologies) on Synthemax II microcarriers (Corning) in a PBS 3 Air-Wheel Bioreactor. Processes for microcarrier
seeding, expansion via bead-to-bead transfer, and harvest of MSCs all took
place within the Bioreactor. Cell growth and metabolic rates were monitored,
and a traditional stirred-tank bioreactor was run in parallel as control. In the
Air-Wheel Bioreactor, the cells attached to the microcarriers faster and reached
confluence more quickly than a stirred-tank bioreactor. MSCs cultured as
described met the International Society of Cellular Therapy (ISCT) minimum
criteria for BM-MSC (> 95% CD73, CD105, CD90 and < 2% CD34, CD45,
CD14, CD19, HLA-DR) and tri-lineage differentiation. In this study we show
that BM-MSCs can be expanded on microcarriers using a low-shear Air-Wheel
Bioreactor with bead-to-bead transfer that is amenable to scale up to commercial manufacturing. When BM-MSCs are cultured using this novel
expansion system, microcarrier loading is fast and uniform, the MSCs are
highly proliferative, and they retain the characteristic markers of BM-MSC.
363
DEVELOPMENT OF A NEW ADVANCED THERAPHY MEDICINAL
PRODUCT FOR BONE REGENERATION TREATMENT; FROM
BENCH TO BEDSIDE
M Caminal, N Pujals-Fonts, J Vives, M Codinach, I Oliver-Vila,
A Casamayor-Genesca, M Blanco, R Coll, A Pla, J Garcia
XCELIA, Banc de Sang i Teixits, Barcelona, Barcelona, Spain
Human cells are critical raw materials for manufacturing cell therapy products,
but often introduce significant variability. Rigorous operational controls and
quality systems, however, enable optimal collection of high-quality, consistent
cellular material. HemaCare, a long-standing supplier of human-derived blood
components, controls apheresis procedures and collection sites under a formal
quality system, with GMP-compliant, validated procedures and equipment, and
GTP-compliant donor screening and tracking. HemaCare performed 86,799
cellular apheresis collections in the last seven years (year ending July 31, 2013),
including patient and normal-donor peripheral blood mononuclear cells
(MNCs), and plateletpheresis products for research, clinical trials, and commercial products. HemaCare’s unmobilized apheresis products showed
consistently high MNC purity, with 93.8% of products containing 75%
MNC, and an average of 85.66% MNC 7.1% (mean 1 SD). Red blood cell
contamination was low, with hematocrit averaging 1.78% 0.7%. Approximately 85% of HemaCare donors have donated apheresis products 5 or more
times; this repeat-donor pool also contributes to product consistency. HemaCare’s laboratory is equipped with Miltenyi Biotec technology for isolation of
cellular raw material into purified cellular subpopulations. The consistency and
viability of the purified products are measured with flow cytometry. Using
BioLife Solutions’ serum-free and protein-free fully-defined cGMP CryoStorÔ cryopreservation medium with purified cells, post-thaw recovery rates
of cell fractions have been above 95%, based on 7AAD staining. Dendritic cells
and macrophages have demonstrated post-thaw recovery rates of 90%.
CryoStorÔ cryopreservation medium, in combination with freezing in the
BioCision CoolCellÔ freezing container, has enabled HemaCare to standardize the cryopreservation process, reducing variability while optimizing
post-thaw viable cell recovery of its research products.
Purpose: In the bone regeneration context, tissue engineered products provide
a significant advance in new emerging therapeutic options for the treatment of
bone injuries as an alternative to the classical techniques (autologous bone
graft, distraction osteogenesis or allogenic bone). Here we present the development of a new advanced therapy medicinal product (ATMP) composed of ex
vivo expanded mesenchymal stromal cells (MSC), loaded onto bone scaffolds
and shaped as the lesion to treat using fibrin sealant, from preclinical studies to
phase I/IIa clinical trials.
Materials and methods: Three preclinical studies were performed: - Two
large animal studies were conducted in order to evaluate safety and efficacy of
the ATMP. The sheep models chosen were 1) osteonecrosis of the femoral
head and 2) Critical size segmental bone defect. - A small animal model was
carried out to evaluate tumorigenicity after subdermal implantation of the
ATMP. Large number of MSC (15-30x106) were expanded from bone marrow
mononuclear cells and immobilized in a bonny scaffold. This process was
developed and patented by XCELIA (Procedure for obtaining a tissue engineering product for the regeneration of bone tissue, EP 2361971 A1).
Currently, two clinical trials are undergoing: 1) Mesenchymal Stem Cells in
Osteonecrosis of the Femoral Head (NCT01605383) and 2) Safety Study of
Mesenchymal Stem Cells and Spinal Fusion (NCT01552707). Some patients
have already been treated and the recruitment process is still open.
Results and conclusions: Preclinical studies in both large and small animal
models confirmed the safety of the ATMP and evidenced its efficacy. A total
number of eleven batches were GMP manufactured to date, and no adverse
effects have been found in treated patients. Developed ATMP has promising
clinical results treating both osteonecrosis and spinal fusion bone regeneration.
362
DEVELOPMENT OF A SCALABLE MANUFACTURING PROCESS
FOR BONE-MARROW DERIVED HMSC’S IN A LOW-SHEAR
SINGLE-USE BIOREACTOR SYSTEM
364
ADVANCED CELL THERAPY INDICATED FOR GONARTHROSIS
TREATMENT: THE WAY TO THE COMPLETED CLINICAL
TRIAL
S104
Poster Abstracts
N Pujals-Fonts, M Caminal, J Vives, M Codinach, I Oliver-Vila,
A Casamayor-Genesca, M Blanco, R Coll, A Pla, J Garcia
XCELIA, Banc de Sang i Teixits, Barcelona, Barcelona, Spain
Purpose: Osteoarthritis (gonarthrosis is the most frequent) is one of the most
common causes of pain and disability in middle-aged and older people. The
successful repair of degenerated articular cartilage is still a major clinical
challenge. An advanced cell therapy product has been developed in the context
of a phase I/IIa clinical trial (NCT01227694) with the aim to improve the
outcome of the patient’s articular cartilage after intra-articular injection of
autologous mesenchymal stromal cells (MSC). M & MPreclinical studies
Three studies have been performed in order to assess pharmacodynamics/
pharmacokinetics, subchronic toxicology and dose response. The osteoarthritis-induced animal models used were sheep in the first case and rat for the
rest. Manufacturing An optimized close-system process has been developed and
patented by XCELIA. Process manufacturing has been taking place in GMP
facilities and under approval by the Spanish Regulatory Authorities. The cell
expansion methodology allows to obtain high doses of MSC in a relative short
period of time. Quality assurance tests are exhaustive in order to confirm a
compliant product releasing. The cell-based product oriented for the clinical
trial is composed by 40x106 MSC in a saline solution. To date, we have
experience in producing more than 50 cell batches. Clinical trial The clinical
trial was designed for the treatment of gonarthrosis grade II-III (Kellgren &
Lawrence). Fifteen patients were included in this single-arm study. Currently,
the clinical trial has been finished and compassion treatments are being
administered at present.
Results and conclusions: Preclinical results demonstrated the safety and
efficacy of the cell-based product application. The manufacturing experience has demonstrated the robustness and reproducibility of the process
design. As promising results in the clinical trial have been obtained after
one year of follow-up post-treatment, a phase II/III is currently under
design.
365
GENERATION OF ASPERGILLUS FUMIGATUS-SPECIFIC TH1
CELLS AGAINST INVASIVE ASPERGILLOSIS
A Jochheim-Richter1, P Bacher2, A Ortigao1, N Mockel-Tenbrinck3,
E Wingenfeld1, D Karpova1, M Assenmacher3, R Alex3, A Scheffold2,
H Bönig1
1
Institute for Transfusion Medicine and Immunohematology, German Red
Cross Blood Service Baden-Württemberg-Hessen, Frankfurt,
Germany, 2Department of Cellular Immunology, Clinic for Rheumatology and
Clinical Immunology, Charite - University Medicine, Berlin,
Germany, 3Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
Invasive fungal infections are a common cause of morbidity and mortality in
patients undergoing allogeneic hematopoietic stem cell transplantation
(HSCT). Importantly, these infections are predominantly observed after
neutropenia has abated, during the prolonged period of immunosuppression. TH1 mediated responses have been shown to be involved in the
control of Aspergillus infection, but the number of anti-Aspergillus TH1
cells remains low after HSCT. Selective restoration of antifungal cellular
immunity with antigen-specific T-cells therefore represents an attractive
strategy to protect against or to treat invasive aspergillosis. As the frequency
of Aspergillus-specific T cells is low, technical limitations and poor
compatibility of proposed protocols with current GMP standards have
hampered the establishment of this approach so far. Here we report a novel
strategy which allows direct enrichment of naturally occurring anti-Aspergillus T cells under GMP-conditions. Steady-state apheresis of the original
stem cell donor is used as starting material and depleted of potentially
harmful CD8+ and CD25+ cells in the first step. Antigen-specific T-helper
cells are then incubated with Aspergillus fumigatus lysate, which induces
expression of the activation marker CD137. The CD137+ target cells are
subsequently isolated with the CliniMACSÒ Cell Separation System. The
purity of the enriched CD137+ Aspergillus-specific T cells among total T
cells is 75% and higher. Contaminants include residual B-cells, NK-cells,
monocytes and granulocytes. The isolated cell population proliferates in
response to Aspergillus fumigatus lysate in vitro and can be induced to
produce the cytokines IFN-gamma and TNF-alpha by antigen-specific restimulation. A multicentre clinical trial using cells isolated as described has
been submitted to the authorities and will provide evidence of the therapeutic potential of Aspergillus-specific T-cells in patients with invasive
aspergillosis.
366
FAST PRODUCTION OF HUMAN PLATELET LYSATE BY
PLATELET RICH PLASMA SONICATION FOR THE EX-VIVO
EXPANSION OF BONE MARROW-DERIVED MESENCHYMAL
STROMAL CELLS
M Bernardi1,2, E Albiero1,2, A Alghisi3, K Chieregato1,2, C Lievore3,
D Madeo2, F Rodeghiero1,2, G Astori1
1
Advanced Cellular Therapy Laboratory, Department of cellular therapy and
hematology, San Bortolo Hospital, Vicenza, Italy, 2Research Laboratories,
Hematology Project foundation, Vicenza, Italy, 3Immune Transfusion Service,
department of cell therapy and hematology, San Bortolo Hospital, Vicenza,
Italy
Introduction: In addition to their use in regenerative medicine, mesenchymal stromal cells (MSC) are used in the prophylaxis and treatment of
Graft Versus Host Disease due to their strong immune-modulatory
properties. For obtaining a sufficient number of cells for clinical application, MSCs need to be ex-vivo expanded. Expansion is possible when
fetal bovine serum (FBS) is added to basal media but its use is discouraged by regulatory authorities, due the risk of zoonoses and immune
reactions. Human platelet lysate (Hu-PL) has been proposed as a valid
substitute of FBS. Platelet growth factors release is commonly achieved
after overnight freezing/thawing cycles of platelet rich plasma (PRP);
however the process is time consuming and difficult to standardize. We
have developed a fast method to obtain PL from PRP by using ultrasound. Efficiency was tested by expanding bone marrow (BM)-MSC in
the obtained Hu-PL.
Materials and methods: sonication was performed by treating PRP bags
in an ultrasonic bath at a frequency of 20 kHz for 30 minutes at room
temperature. To evaluate platelet lysis we measured PDGF-AB release by
ELISA. Efficiency was tested by measuring cumulative population
doubling time (cPD), differentiation capacity and immunogenic properties
of BM-MSC expanded in D-MEM+10%Hu-PL and in D-MEM+10%
FBS.
Results: 74% of PDGF-AB was released after treatment. Hu-PL significantly enhanced BM-MSC proliferation rate compared to FBS. The cPD of
cells growth in Hu-PL at 10%, 7.5% and 5% was significantly better when
compared to cells expanded in 10% FBS. BM-MSC expressed MSC markers
and were able to differentiate into adipogenic, osteogenic and chondrogenic
lineages. Immunosuppressive activity of BM-MSC expanded in Hu-PL was
maintained when co-coltured with T-cells.
Discussion: Hu-PL can be quickly produced by sonication; moreover, HuPL reduce the cell PD time compared to FBS maintaining both cell differentiation potential and immunomodulatory capacities.
367
ABSENCE OF MICRONUCLEUS FORMATION IN CHO-K1 CELLS
CULTIVATED IN PLATELET LYSATE ENRICHED MEDIUM
PRODUCED BY SONICATION OF HUMAN PLATELET RICH
PLASMA
M Bernardi1,2, V Adami3, E Albiero1,2, D Madeo1, F Rodeghiero1,2, G Astori2
1
Research Laboratoties, Hematology Project Foundation, Vicenza,
Italy, 2Advanced Cellular Therapy Laboratory, Department of cellular
therapy and hematology, San Bortolo Hospital, Vicenza, Italy, 3High
Throughput Screening Core facility (CIBIO), University of Trento,
Trento, Italy
Introduction: Human platelet lysate (Hu-PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cells (MSC)
expansion. Compared to FBS, Hu-PL favors MSC proliferation avoiding
the risks related to animal derivatives. Growth factors in the platelet
granules are released upon disruption following freezing/thawing (F/T)
cycles. Alternatively we described the use of ultrasounds for the production
of Hu-PL starting from platelet rich plasma (PRP). Since Hu-PL
significantly increases the growth rate of MSC compared to FBS and given
the possibility of free radicals formation during PRP sonication, we
investigated both the MSC chromosomal stability during expansion by
karyotype analysis and Hu-PL safety applying the cytokinesis-block
micronucleus assay (CBMA).
Materials and methods: Hu-PL was produced by sonication of the
PRP bags for 30 minutes at 20 kHz. MSCs were isolated from bone
marrow (BM) samples and expanded in D-MEM+10% Hu-PL. Karyotyping was carried out at the end of passage 6. For CBMA, the CHOk1 cell line lacking DNA repair mechanism was exposed to increasing
concentrations of Hu-PL from 0.1% to 30% in three independent experiments and using different Hu-PL batches. For positive control,
micronucleation was induced by exposing the CHO-k1 to Mitomycin-C.
After 24h, cytokinesis was blocked by Cytochalasin B. Cells were fluorescent stained with Hoechst and micronuclei were automatically detected
using an high content imaging system (Operetta) analyzing at least 2000
binuclear cells/well.
Results: Growth proliferation induced by the use of Hu-PL did not lead to
micronuclei formation on CHO-K1 cells compared to negative control
(p<0.01). Karyotype did not reveal genomic alterations on BM-MSC expanded
in 10% Hu-PL.
Discussion: micronuclei formation is considered a biomarker of chromosomal damage, genome instability and cancer risk. Our results suggest that
MSC can be safely expanded in Hu-PL produced by sonication.
368
NEXT
GENERATION
XENOGENIC-FREE
BIOLOGICS/
REAGENTS
1
1
1
2
1
C Johnston , F Silva , R Walker , T Warbington , AN Patel
1
United States, 2Ibiologics LLC, Phoenix, Arizona, United States
Background: Cell and tissue therapy has emerged as a growing application in
regenerative medicine. The use of xenogeneic components to manufacture or
derive cells or scaffolds for regenerative medicine has been an industry standard
for decades with many risks, such as prion, viral transmission or adverse
immunological reactions against xenogeneic components. Most manufacturing
protocols use fetal bovine serum (FBS) as a cell culture supplement to expand
cells used for hybridoma production and cell-based therapies. Regulatory
agencies have developed strict guidelines for translating preclinical into
clinical-grade large-scale cell expansion to minimize risk. Although these
animal-free alternatives have been proposed, the lack of a scaleable supply
for large-scale manufacturing have prevented these reagents to be widely
used by industry. Our objective was to create a xeno-free program for human biologics.
Methods: Human platelet lysate (hPL) was produced using a scalable
system (>1000 liters) without the use of heparin and tested on different
human cell populations. Stem cells derived in our xeno-free platelet lysate
were tested for multipotency. Human biological scaffolds were made of
purified extracellular matrix from various organs (heart, adipose, liver and
lung).
Results: hPL can be produced in a scaleable manner without the use of
heparin, and successfully used as an FBS replacement. Cells cultured in our
lysate had shorter doubling times compared to other tissue culture supplements
and maintained there cell potency and function. Human biological scaffolds
were developed from various tissues and demonstrated efficient cell penetration
and distribution.
Conclusions: Here we report the development of industry scaleable (greater
than 1000 liters) xenogenic-free hPL and the development of human tissue
specific scaffolds. These xenogenic-free biological reagents provide tools for a
wide range of regenerative medicine applications.
369
370
ALLOGENEIC MESENCHYMAL STEM CELL THERAPY FOR
KNEE OSTEOARTHRITIS: SAFETY AND EFFICACY RESULTS
OF A RANDOMIZED, DOUBLE BLIND, PHASE 2, PLACEBO
CONTROLLED, DOSE FINDING STUDY
S105
A Majumdar1, S Balasubramanian1, C Thej1, M Zagan1, S SundarRaj1,
U Kumar1, U Baikunje1, N Anthony1, N Shetty2, V Pandey3, V Agarwal4,
B Dasgupta5, S Wagh6, A Das1, A Chullikana1, PK Gupta1
1
Stempeutics Research, Bangalore, Karnataka, India, 2Department of
Orthopaedics, M.S Ramaiah Medical College & Hospitals, Bangalore,
India, 3Department of Orthopaedics, Kasturba Medical College and Hospital,
Manipal, Karnataka, India, 4Department of Rheumatology, Sanjay Gandhi Post
Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh,
India, 5Department of Orthopaedics, Seth G. S. Medical College and KEM
Hospital, Mumbai, Maharashtra, India, 6Department of Rheumatology,
Jehangir Clinical Development Center, Jehangir Hospital, Pune, Maharashtra,
India
Osteoarthritis (OA) is a degenerative disease of the connective tissue and
progresses with age. Owing to the lack of vasculature within the articular
tissue, the cartilage is especially vulnerable to damage and has poor potential for regeneration. Conventional therapies using pharmacologic agents
are beneficial in reducing pain and inflammation during the early stages of
the disease, additional treatments are required as the disease progresses
ultimately TKR remains the only viable option. MSCs are increasingly
being tested in clinical trials of many degenerative diseases including OA,
since these cells possess potent anti-inflammatory, immunosuppressive
properties and potential of differentiating into cartilage. Using BMMSC
obtained from multiple volunteers, we have developed a pooled allogeneic
cell therapy product called Stempeucel and extensively characterized it both
in vitro and in vivo. Preclinical safety and toxicity studies clearly demonstrated that stempeucel is nontoxic, non-teratogenic and non-tumorigenic.
Moreover, stempeucel is also found to promote cartilage regeneration in a
rat model of OA. A single intra articular injection of stempeucel in combination with HA into the knee joints of patients showed considerable
reduction in pain compared to HA alone. The pain reduction was measured
using three different methods, namely VAS, WOMAC and ICOAP, all of
which have been widely used to monitor the pain score and knee functions
in these patients. Four different doses of stempeucel have been used, 25
million cell dose showed maximum pain reduction in patients by all three
methods and, the effect has lasted for 12 months and the final follow-up will
be done at the end of the trial period at 24 months. This study demonstrates that stempeucel administration in OA patients is safe and well
tolerated (primary end point) and also efficacious in reducing the pain for at
least a year and may have the potential to halt disease progression and
cartilage regeneration.
371
CONCENTRATION AND HARVEST OF HEPATIC PROGENITOR
CELLS USING CONTINUOUS CENTRIFUGE SYSTEM
M Egloff1, J Goffinet1, M Pasquiou1, P Willemsen2, S Snykers2, V Codutti2
1
ATMI LifeSciences, Brussels, Belgium, 2Promethera, Mont Saint Guibert,
Belgium
The implementation of bioreactor systems for large scale cell therapy
industrialization is crucial but challenging. As the production scale increases, the volumes to be processed become substantial and require an
adaptation of the downstream strategy. To answer this new challenge, as
well as to meet the needs of customers, ATMI LifeSciences has developed
a single-use centrifuge device able to concentrate, wash and resuspend cells
after culture. The system respects constraints from the cell therapy
application: low-shear stress necessary to process stem cells, capture very
low cell suspension concentration and high concentration factor, while also
filling a gap in the centrifuge market by processing intermediate volumes
(5L to 60L) in short times. The centrifuge, which relies on a continuous
centrifugation technology, is composed of a hardware controller and a
disposable module. It combines a centrifugation bowl and a 1L-harvest bag
that are plugged on the hardware part. It features a flexible and compact
design including a peristaltic pump (0-500 ml/min) and a rotating bowl
support (550-3800 G). The centrifuge is fully flexible and can process
different types of cells from stem cells to Vero cells. This presentation
illustrates an application of ATMI centrifuge with a case study. The
IntegrityÒ Cell Recovery System is already used by a Belgian cell therapy
company and has achieved efficient and reliable results. Cell viability was
maintained after the harvest of several XpansionÔ 200 bioreactors with a
recovery yield between 85% and 95% (equivalent to a yield between 90%100% of a conventional discontinuous centrifugation process used as
S106
Poster Abstracts
control) and a concentration factor of 400 (150,000 cells/ml to 60.106 cells/
ml after centrifugation).
medium is a good alternative for MSC isolation and expansion in Good
Manufacturing Practices conditions.
372
SCALING-UP CELL MANUFACTURING TO A CLOSED AND
CONTROLLED SYSTEM: USING A MULTIPLATE BIOREACTOR
COMBINED TO A CONTINUOUS CENTRIFUGATION
M Egloff, J Castillo, J Drugmand
ATMI LifeSciences, Brussels, Belgium
374
PRINCIPLES OF PLURIPOTENT STEM CELL BIOPROCESSING:
MICROCARRIER-CELLS AGGREGATE DIMENSIONS DICTATE
EFFICIENT
AND
RELIABLE
EXPANSION
AND
CARDIOMYOCYTE DIFFERENTIATION IN BIOREACTORS
S Oh1, A Lam1, A Chen1, J Li2, W Birch2, S Reuveny1
1
Bioprocessing Technology Institute, Singapore , Singapore, 2Institute of
Materials Research and Engineering, Singapore, Singapore
A successful transition from laboratory scale, to an efficient and robust good
manufacturing practice (GMP) process is key and represents a major challenge for cell based products manufacturing. Implementing innovative technologies is essential to support such industrialization. Single-use bioreactors
represent a solution by minimizing manual handling, realizing economies of
scale and delivering the benefits of a closed system. However the implementation of bioreactor systems for large scale cell therapy industrialization is
challenging. Scale-up is not as simple as providing a larger surface area.
Changing the niche environment has a big impact on cell behaviour as cells
are highly sensitive to the microenvironment. Also as the production scale
increases, the volumes to be processed become substantial and require an
adaptation of the downstream strategy. The combination of the XpansionÔ
multiplate bioreactors and a continuous centrifuge enables an easy transfer
from existing multitray stack process to a large scale and closed production
system. The bioreactor offers the same cell growth environment (polystyrene
multiplate) plus the regulation of the critical cell culture parameters as pH,
dissolved oxygen and cell density. Single-use centrifuge device enable
continuous concentration, wash and resuspend cells after culture. The system
respects constraints from the cell therapy application: low-shear stress
necessary to process stem cells, capture very low cell suspension concentration
and high concentration factor. This presentation illustrates with different
cases studies the transition from traditional open R&D systems to a closed
manufacturing platform. Trends analysis of pH and DO for several cell cultures, as well as cell confluence monitoring, highlight efficiency of controls
and monitoring to develop a robust scalable process. Cell quality was assayed
and showed equivalence between all scales. Value and benefits are also
examined through COGs analysis.
373
CULTURE AND CHARACTERIZATION OF MESENCHYMAL
STEM CELLS IN A TOTALLY DEFINED MEDIUM
M Gadelorge1, VV Mariono1, J Dulong2, J Descamps1, L Caillot3,
C Conway3, K Tarte2, L Sensebe1
1
STROMAlab, UMR CNRS 5273, U1031 Inserm, EFS-PM, Univ. P.
Sabatier, EFS-Pyrenees-Mediterranee, Toulouse, France, 2EFS Bretagne,
U912, Rennes, France, 3ABCell-Bio, Montpellier, France
Mesenchymal Stem Cells (MSC), which can be from different origins, have
many properties that make it an ideal tool for cell therapy and regenerative
medicine. The growing number of clinical trials using these cells requires to
secure their production. In this context, the use of standardized and animalfree conditions represents a major issue for their clinical uses. For this
purpose, the ABCellbio company has developed a fully defined medium for
MSC: the SPE-IV medium. The SPE-IV medium was tested for AT-MSC
(Adipose Tissue- MSC) and BM-MSC (Bone Marrow- MSC) isolation from
fresh tissue and for their expansion (n ¼ 3 for AT, n¼3 for BM). Several
controls were performed: clonogenicity, phenotypic analysis, differentiation
potential, stemness and genetic stability, immunosuppression. The medium
currently used for clinical trials (aMEM + Platelet Lysate) was used as
internal control. The AT-MSC and BM-MSC grew better and faster in
SPE-IV medium compared to control medium (ASC: population doublings
time ¼ 23.4 hours vs 38.1 hours; BM-MSC: population doublings time ¼
29.8 hours vs 37.6 hours). Clonogenic potential and immunosuppression
capacities of cells were maintained in SPE-IV medium. Moreover, the
expression of genetic stability markers, stemness markers and senescence
marker were not modified. Although phenotypic profile of MSC was
maintained with only few changes in adherence molecules expression, differentiation capacities of MSC appeared slightly decreased for adipocyte
lineage in AT-MSC (ns), for chondrocyte lineage (p < 0.05) and for
osteoblast lineage (ns) in BM-MSC. Our results showed that SPE-IV
The expansion of human pluripotent stem cells for biomedical applications
compels a defined, reliable and scalable platform in suspension bioreactors.
In previous work, we have demonstrated successful suspension cultures with
undefined, Matrigel coated microcarriers or uncoated microcarriers with
cultures treated with the ROCK inhibitor for cell production. This study
describes a special technology whereby 100 micron diameter polystyrene
microcarriers are layered with a cationic charge followed by either defined
Vitronectin or Laminin coatings to provide cell attachment, spreading and
growth. This coating combination critically enables formation and evolution
of microcarrier-cell aggregates which generate high cell yields reaching over
3.5 million cells/ml in 7 days. The numbers of these microcarrier-cell aggregates increase from 20/ml to w80/ml at a quazi-constant size of w300
micron indicating that growth occurs within a self-regulating microenvironment. Importantly, this paired coating enables single cell seeding and cell
growth under continuous agitation. Different cell lines (HES-3, H7, IMR90)
show highly reproducible bioresponses to these surface properties. Post
expansion, these microcarrier-cell aggregates were further directed towards
the cardiomyocyte lineage with Wnt signalling modulators (CHIR 99021
and IWP2) in 4 different conditions. The continuous agitation bioreactor
process yielded the highest numbers of cardiomyocytes of 1.9 million cells/
ml compared to the conventional embryoid bodies (EB) method which
generated 0.1 million cells/ml e a significant 19 fold improvement. Cardiomyocytes expressed high levels of cardiac troponin (>50%) compared
to the EB method (13%). Cardiomyocytes exhibited normal responses to
drug einduced QT prolongation assays against E-4031 and Verapamil. This
superior technology is ready to be translated to larger bioreactor scales
for integrated expansion and differentiation of pluripotent stem cells to
cardiomyocytes.
375
NOVEL PROCESS CONTROL IN A CLOSED SYSTEM
BIOREACTOR FOR CULTURE OF ADHERENT CELLS
R Das1, R Roosloot1, P van Santen1, J de Bruijn1,2
1
Xpand Biotechnology BV, Bilthoven, Netherlands, 2Department of
Biomaterials and Technology, Twente University, Enschede, Netherlands
As cell therapies move to clinical practise, there is a need to simplify and
optimize production protocols and move away from traditional culture
techniques in flasks. Reducing handling lowers production costs and risk
for contamination, while process optimization using multi-parameter
control increases quality, reproducibility and traceability. We designed a
bioreactor (Fig 1A) for GMP-grade culture of adherent cells (e.g. MSCs).
Cells are cultured from biopsy to harvest on microcarriers in a single use
bioreactor bag (Fig 1B). The volume is minimized to support growth of
minimal cell amounts and can be expanded as more culture surface is
required. The cabinet and bag are fitted with sensors, allowing control of
critical parameters at the site of culture. A unique oxygenator was
designed for the tight control of DO and pH, while biomass was determined using radio frequency impedance (RFI). To validate these process
controls, human MSCs were cultured on microcarriers inside the bioreactor bag for up to 26 days. DO and pH were controlled throughout the
culture and the ability of RFI to measure viable biomass was determined.
The new oxygenator was able to control critical culture parameters more
accurately than traditional systems (e.g. head space aeration). DO was
controlled around setpoint within 3% and pH variations were <0.05 (Fig
2). RFI measurements of live biomass (attached cells) inside the bag
correlated highly (R2 ¼ 0.998) with offline cell counts (Fig 3). Production
of cell therapy can benefit from culture procedures that minimize
S107
1
Voisin Consulting Life Sciences, London, United Kingdom, 2Voisin
Consulting Life Sciences, Rennes, France
Xpand’s bioreactor system (A) and the single-use bioreactor bag (B) with
sensor placement.
The new oxygenation system resulted in very accurate control of DO and
pH. Variation of pH was minimal (<0.05) and DO was controlled within
3%. Setpoints were restored quickly after medium refreshment (peak).
Measurements of viable biomass inside the bioreactor bag correlate highly
with offline measurements using a standard cell counter(Coulter Counter
Z1). Coeffecient of correlation (R 2) is 0.998.
ˇ
operator involvement and reduce cost. The presented bioreactor enables
one operator to handle one entire culture with minimal labour. Precise
monitoring and control increases reproducibility and quality of the final
cell product. The newly developed oxygenator resulted in a precise
regulation of critical parameters, without introducing unwanted factors.
Moreover, biomass can be monitored without the need for destructive
sampling.
376
HOW TO EFFICIENTLY APPLY A RISK BASED APPROACH
TO CELL BASED MEDICINAL PRODUCT DEVELOPMENT.
REGULATORY CHALLENGES AND RECOMMENDATIONS TO
ACHIEVE A STREAMLINED DEVELOPMENT PROGRAM
N Thomas1, AD Poiseau2
Similarly to all products, the risk profile of Advanced Therapy Medicinal
Products (ATMPs) must consider the nature of the product, its mechanism
of action, impurity profile, manufacturing process, as well as clinical aspects,
such as bio-distribution and the target indication. The risk profile of a cell
based ATMP is further complicated by risks specific to the nature of the
therapy including cell origin, the degree of cellular differentiation, level of
cell manipulation and the risk of oncogenicity or immunogenicity. The
adoption of the European Medicines Agency (EMA) guideline on the riskbased approach (RBA) to the development of ATMPs demonstrates the
EMA’s acknowledgement of the differences in risk profiles of various
ATMPs, and the inability to standardize the development program to
support marketing authorisation of these complex products. There is little
doubt that the risk assessment for an ATMP and resulting adaptation of the
development program needs to be an iterative process. However, while the
aforementioned guideline provides worked examples of key considerations
for ATMP development once risks have been identified, the implementation of the RBA in a product development plan, and the planning of key
studies to conduct prior to milestones such as first in man studies, or
submission of a marketing authorisation application (MAA), taking the RBA
in to account remains purposefully vague. This presentation will examine
the key studies and regulatory considerations in the development of
ATMPs, and highlight the common barriers to regulatory approval of both
clinical trial applications and MAAs in the EU. Furthermore, the presentation will discuss approaches for justification of a streamlined product
development plan, and key interactions with regulators to obtain support
for this approach.
377
SUCCESSFUL TECHNOLOGY TRANSFER FOR CELL THERAPY
PRODUCTS
R Dennett1, A Gibbons2, V Pimpaneau1
1
Voisin Consulting Life Sciences, Rennes, France, 2Voisin Consulting Life
Sciences, Paris , France
One of the innate challenges in spring boarding a new cell therapy candidate
out of the laboratory and into first in man studies is the suitable development and alignment of the process for cGMP clinical manufacture. This
often presents a new encounter to many cell therapy companies. The careful
management of this pivotal stage is critical and can easily make the difference between a successful or a failed project. In this presentation we explore
how an objective technology transfer approach should be applied for cell
therapy products and provide detail of the mechanisms and tools which
should be used in order to make sure that the transition from development
to cGMP is correctly achieved. Effective technology transfer is based on a
logistical series of events involving change management processes that
encompass a clear understanding of the process and product and critical
quality attributes, planning and a systematic approach, risk-management,
change-control and comparability. This tried and tested approach has been
adapted to the discipline of cell therapy, and aims to accommodate the
complexity and heterogeneity of cell based products. The understanding of
the difficulty in producing cell therapies at different sites, including minimal
source tissue, quality of raw materials, issues associated with the heterogeneity of products from batch to batch, difficulty to characterize the products
and different operational scientists makes methodology for technology
transfer for this product sector all the more important and challenging. We
will draw upon real life case studies of a failed and successful technology
transfer and provide usable guidance that can be taken away and put into
actual practice.
378
ESTABLISHMENT OF A LICENSED CORNEAL EPITHELIAL
STEM CELL CLINICAL PRODUCT FOR USE IN THE
TREATMENT OF PATIENT WITH SEVERE OCULAR SURFACE
DISEASE
C Bienek1, A Atkinson1, L Bailey1, J Barry1, J Drain1, B Cuthbertson1,
J Pelly1, T McQuillan1, G Galea1, N McGowan1, J Campbell1,
M MacDonald2, A Agrawal2, K Ramaesh3, S Mantry3, S Ahmad4, S Kaye4,
B Dhillon2, M Turner1
S108
Poster Abstracts
1
Scottish National Blood Transfusion Service, National Services Scotland,
Edinburgh, United Kingdom, 2Princess Alexandra Eye Pavilion, NHS
Lothian, Edinburgh, United Kingdom, 3Tennent Institute of Ophthalmology,
NHS Greater Glasgow and Clyde, Glasgow, United Kingdom, 4St Paul’s Eye
Unit, Royal Liverpool University Hospital, Liverpool, United Kingdom
The aim of this study was to establish a corneal epithelial stem cell product
for use in a pilot clinical trial for the treatment of patients with severe ocular
surface disease (OSD) (the clinical trial is funded jointly by the UK Stem
Cell Foundation, Scottish Enterprise and the Chief Scientist Office). This
clinical trial will treat 20 patients from 3 centres (NHS Lothian, NHS
Greater Glasgow and Clyde, and Liverpool). Ten patients will receive
allogeneic ex vivo expanded limbal stem cells on human amniotic membrane
(AM), and 10 will receive cultured AM control. This is the first prospective
trial to compare these two products for the treatment of OSD. The primary
outcome for the trial is best corrected visual acuity. The culture process for
this cell therapy product is based on the method of Kolli et al, with explants
from the corneal limbal region, positioned on human AM, kept flat by fitting
the AM over glass coverslips, then cultured for up to 21 days. The established process generates a product with cellular outgrowth from allogeneic
limbal tissue of 200mm2 and minimum diameter of 15mm on AM. The
facility and equipment used in the manufacture of this product were qualified to Good Manufacturing Practice (GMP) requirements, and the production method, testing, and shelf life were qualified and final product
specifications set. Following inspection of SNBTS by the MHRA in April
2011, an MIA (IMP) licence to manufacture was granted in August 2011. A
CTA for the pilot study as well as a licence to use the product on a Named
Patient Basis, was granted in September 2011. To date sixteen patients have
been recruited to the study and at varying stages of treatment. In conclusion,
SNBTS has established a corneal epithelial stem cell clinical product that
has been licensed for use by the MHRA and is being used to treat patients
with OSD as part of a pilot clinical trial.
379
IMPROVED EXPLANTS METHOD TO ISOLATE UMBILICAL
CORD-DERIVED MESENCHYMAL STEM CELLS AND THEIR
IMUUNOSUPPRESIVE PROPERTIES
Y Mori1, T Nagamura-Inoue1, J Ohshimo2, T Shimazu1, A Takahashi1,
H Tsunoda3, A Tojo1
1
Dep. of Cell Processing and Transfusion, Institute of Medical Science, Univ.
of Tokyo, Tokyo, Japan, 2R&D Department, Tsubakimoto Chain Co., Tokyo,
357-8510, Japan, 3Dep. of Obsterics, NTT Medical Center Tokyo, Tokyo,
141-8625, Japan
Umbilical cord (UC) have become one of the major sources of mesenchymal
stem cells (MSCs). Different from bone marrow-derived MSC (BM-MSCs),
to isolate UC-derived MSCs (UC-MSCs), we need to digest the UC with
enzyme such as collagenase, or explants method. To avoid the non-human
enzyme, we took explant method, although the segmented tissues were often
floating during culture, resulting in the loss of cell recovery. To overcome
this, we here show the improved explants method using the stainless mesh
(CellamigoÔ) to cover the tissues and the immunosuppressive effect of the
isolated UC-MSCs in vitro. UC were minced into small fragments and the
fragments were aligned at regular intervals in culture dishes and semi-dried
for 30 min to attach to the bottom in the conventional explant method.
On the other hand, in the improved explants method, we covered with
CellamigoÔ (Tsubakimoto Chain Co., Japan) on he minced fragments and
immediately poured the medium into the dishes. The culture medium was
refreshed in every 3 days until the adherent cells reached 80-90% confluence.
In 9 to 12 days, the adherent cells and tissue fragments were harvested using
trypsin. The number of UC-MSCs isolated by explants method with or
without Cellamigo was 3.071.36x106g (fUC1g) and 0.860.54x106/g,
respectively (n¼4, P<0.05). Processing time and incubation time were
shortened in the improved explants method compared with the conventional
method. It took three weeks to get the cells 2 106/g(n ¼ 23) in the conventional explants method. The isolated UC-MSCs in the improved methos
showed CD105+CD73+ CD90+, CD45-. In MLR, UC-MSCs efficiently
inhibited the responder T cells derived from the same donor of UC-MSCs,
triggered by allogeneic dendritic cells (DC). With Cellamigo, we shorten the
processing and incubation time to isolate UC-MSCs by improved explant
method. Conclusively, UC-MSCs may be a feasible for banking for clinical
uses, such as treatment of GVHD.
380
IMPORTANCE OF IDENTIFYING THE MECHANISM OF
ACTION TO SUPPORT ATMP DEVELOPMENT
A Gibbons1, R Dennett2, V Pimpaneau2
1
Voisin Consulting Life Sciences, Paris, France, 2Voisin Consulting Life
Sciences, Rennes, France
ATMPs are often composite, heterogeneous mixtures. Their biological function encompasses complex interactions ranging from biochemical and immunological activity to structural replacement of damaged tissue. Although
difficult to elucidate, it is necessary to understand the Mechanism of Action
(MoA) as early as possible in development in order to define the scientific and
regulatory pathways. We will discuss how it helps guide the different stages of
development leading to the implementation of adapted manufacturing processes, appropriate analytical tools and non-clinical models. Understanding the
MoA is useful to allow for ATMP Classification by the Committee for
Advanced Therapies (CAT) and to confirm the status of the product as an
ATMP as this will influence the regulatory requirements. The MoA is also
essential to define name and description of the Drug Substance at time of
submissions. The MoA is at the core of the Chemistry, Manufacturing and
Control strategy to define the identity of the drug substance and the design
of an adapted manufacturing process leading to desired Quality Target
Product Profile. In this regard, product characterization including detailed
understanding of the moieties inducing clinical effects together with the
definition of the impurity profile will drive the overall testing scheme. The
MoA is also critical to the development of the potency assay(s) indicative of
the product’s intended biological effect and forms the basis for establishing
and controlling the dose necessary to obtain the expected therapeutic activity.
The MoA may not be fully elucidated but even only partial understanding will
help forge a strong basis to define the quality attributes to monitor
throughout process, release and stability and develop the appropriate
analytical tools. In turn, a solid testing strategy will reinforce the comparability exercises supporting changes occurring from clinical development to
commercialization.
381
CLINICAL-GRADE DEPLETION OF NAÏVE T CELLS USING
IMMUNOMAGNETIC CD62L BEADS: APPLICATION FOR
ADOPTIVE IMMUNOTHERAPY AND ALLODEPLETION WITH
MAINTENANCE OF CENTRAL AND EFFECTOR MEMORY T
CELLS
ER Samuel1, L Beloki2, J Hood1, M Murray1, R Chakraverty3,4, M Lowdell1,4
1
Haematology, University College London, London, United
Kingdom, 2Oncohematology, Navarrabiomed-Miguel Servet Foundation,
Pamplona, Spain, 3Institute of Immunity and Transplantation, University
College London, London, United Kingdom, 4Haematology, Royal Free
London NHS Trust, London, United Kingdom
The depletion of naïve T cells from donor leukapheresis collections (LCs) to
reduce alloreactivity and the harmful effects of graft-versus-host disease whilst
preserving memory T cell reactivity and the graft-versus-leukaemia response
holds great promise for adoptive immunotherapy post allogeneic stem cell
transplantation. This study established a clinical-grade protocol under clean
room GMP conditions for purging naïve T cells through CD62L depletion
using immunomagnetic beads. The efficacy of CD62L depletion was assessed
in three steady-state donor LCs and analysed for cell composition and
functional immune responses. CD62L depletion eliminated >99.9% of
CD62L+ T cells with a mean CD62L- T cell yield of 47.1% ( 9.4). Depleted
cells comprised an equal mix of CD4+ (45.4% 19.7) and CD8+ T cells
(45.3% 21.3) with elimination of B cells but maintenance of the NK cell
population. CD62L depleted cells were predominantly effector and central
memory (>90%) in phenotype with a reduction in the mean naïve T cell
population from 42.1% ( 10.6) in unmanipulated LCs to 9.7% ( 4.3)
following depletion. Assessment of pathogen-specific responses by IFN-g
secretion following direct in vitro stimulation with CMVpp65 and EBV
EBNA-1 peptide pools, demonstrated persistence of anti-viral T cell reactivity in CD62L depleted cells with enhancement of T cell responses
observed in two donors. Alloreactivity was measured in MLRs between donors with complete HLA-mismatch and revealed a reduction in the alloreactive immune response in CD62L depleted cells compared with
unmanipulated LCs. Clinical-grade depletion of naïve T cells using immunomagnetic CD62L beads from steady-state LCs is feasible and highly efficient, with sterility maintained throughout processing. Therapeutic doses of
CD3+ T cells, comprising CD4+ and CD8+ T cell subsets, for adoptive
transfer can be achieved, with the maintenance of reactivity to common viral
pathogens and a reduced risk of inducing GvHD.
382
MICROCARRIER CULTURE OF MESENCHYMAL STEM CELLS
WITH PLATELET LYSATE AS A SERUM SUBSTITUTE
W Tra1, T Beuving1, R Das1, R Roosloot1, P van Santen1, J de Bruijn1,2
1
Xpand Biotechnology, Bilthoven, Netherlands, 2Biomaterials Science and
Technology, Twente University, Enschede, Netherlands
Concerns about the use of animal derived products for the production of
therapies in humans has resulted in the search for alternatives that can replace
fetal bovine serum (FBS). One of these possible substitutes is platelet lysate
(PL). For the production of clinically relevant numbers of mesenchymal
stromal cells (MSCs) for cell therapy in humans, an upgrade to large-scale
culture is necessary to acquire therapeutic cell quantities. Large-scale culture
with microcarriers is effective in the presence of FBS, whereas large microcarrier aggregates are formed in the presence of PL. Therefore, a pre-treatment of PL to stop the aggregates from forming is essential. Here, we present
two methods to use PL in microcarrier culture. Human MSCs were isolated
from bone marrow aspirates and cultured in aMEM with 5% platelet lysate or
15% FBS. Two types of PL culture medium was prepared: 1) heat-inactivation
of PL (HIPL); and 2) pre-gelling of PL (PGPL). MSCs were seeded at 400
cells/cm2 and cultured for 6 passages, or cultured for 21 days in spinner flasks
(Cytodex1, 2 g/L) Growth kinetics were determined for both types of culture.
Cells were characterized using FACS. HIPL cultured MSCs were smaller and
more elongated and PGPL cultured MSCs were smaller, more condensed with
multiple dendrites per cell when compared with the FBS cultured MSCs (Fig
1). Analysis of cell growth showed that PGPL resulted in the highest cell
numbers for both monolayer and microcarrier culture (Fig.2). FACS analysis
showed comparable expression of the typical MSC surface markers (Fig.3).
Alizarin Red, Safranin O and Oil Red O staining showed that all MSCs could
be induced into the osteo-, chondro- and adipogenic lineage, respectively,
regardless the serum supplement. Based on the results we conclude that with
the correct pre-treatment, it is possible to use PL in a microcarrier culture.
Therefore, PL could be used for large-scale cultivation of MSCs for cell
therapy.
383
NOVEL PLATFORM TECHNOLOGY FOR SCALE DOWN AND
PROCESS OPTIMISATION FOR REGENERATIVE MEDICINE
R Drake, B Zoro, K Bure
TAP Biosystems, Royston, Herts, United Kingdom
Clinical and commercial success of allogeneic cellular therapies will depend on
robust, well defined and cost effective manufacturing processes. The bioprocessing industry employs Quality by Design (QbD) to understand the
manufacturing process, how a process affects product critical quality attributes
(CQAs) and relationships between CQAs and a product’s clinical properties. In
Regenerative Medicine such studies are extremely labour intensive and costly,
and conventional cell culture vessels often lack suitable analytical technology
for effective monitoring and control. We describe an advanced automated
system, ambrTM, based on a novel, scaled-down 10-15ml culture vessel. The
vessel mimics features of a full scale bioreactor; 24 or 48 bioreactors can be
processed in parallel, sensors provide continuous monitoring and control of
culture pH, dissolved oxygen, temperature and stirring rate, there is sampling
for off-line analysis (e.g. metabolites) and integrated cell count and viability
measurement. This innovative platform supports investigation and analysis of
multiple conditions in a single run; media, growth factors, cell density and O2
can be systematically varied and optimised, greatly improving efficiency.
Increasingly suspension cell culture (on microcarriers) is being explored for
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large scale production. We show ambr provides controlled conditions for
mammalian cells growing in suspension, as cell aggregates (e.g. embryoid
bodies) or on a range of microcarriers. The availability of consistent, reliable
tools that increase the numbers of parallel experiments at a micro scale will
significantly reduce cost per experiment, and precise control will allow QbD
approaches to be adopted at an early stage. Reducing the time and labour
required to develop, optimise and characterise the complex processes involved
in making novel Regenerative Medicine therapies has the potential to reduce
development timescales, and helps address a major biomanufacturing
bottleneck.
384
QUANTIFICATION OF VARIATION IN BIOLOGICAL INPUT
MATERIALS AND ITS EFFECT ON CLINICAL OUTCOME AND
MANUFACTURE
JA Thurman-Newell, J Petzing, DJ Williams
Centre for Biological Engineering, University of Loughborough,
Loughborough, Leicestershire, United Kingdom
Regenerative medicines (RMs) will revolutionise healthcare by improving patient quality of life and reducing costs due to their substantial long term health
benefit. One particular RM is stem cell therapy, which contains a self-renewing
progenitor cell with the capability to differentiate into any cell type of the body.
This has exciting possibilities for treatment of chronic disease and injury.
Haematopoietic stem cells (HPCs) are one type, derived from the bone
marrow, and released into the peripheral blood. These are used in the treatment of blood based cancers and immunological disorders where they can reestablish haemalogical function or allow higher doses of chemo/radiotherapy
than the bone marrow would usually tolerate. HPCs are derived from human
blood or bone marrow; a raw material that is inherently variable due to geography, ethnicity, lifestyle and quality of life of the donor. This biological
variation can affect the safety and efficacy of any cell derived product, and
therefore the cell therapy industry will need to design a process around this
variation, or control it. A greater understanding of biological variation and its
causes will allow a greater understanding of HPCs’ mode of action, of the
relationship between mode-of-action and dose/response, and in design of RM
clinical trials. Blood and bone marrow derived HPC therapies were used as a
case example to study the effects of variation. The literature was systematically
searched, using the PRISMA guidelines, for data related to donor characteristics to establish a database on baseline biological variation. Bearing in mind a
number of noted assumptions, variation in the collected and transplanted
material can be as high as 3.5 orders of magnitude of the mean, with a suggestion that the processing step adds up to 2 orders of magnitude of this
variation. Additionally a number of observations about the available data were
made.
385
MICRORNA PROFILING AS A QUALITY SIGNATURE FOR
CELLULAR THERAPIES
D Olijnyk, D Mallison, S Ridha, S Paterson, D Dunbar, V O’Brien
Sistemic Ltd, Glasgow, United Kingdom
Background: Cell therapies have significant promise for regenerative medicine. However, there is a requirement for reliable tools to monitor cell
populations during bioprocess development and scale-out/scale-up to safe,
therapeutically-useful cell populations of suitable quality and consistency.
Given that microRNAs (miRNAs) are important determinants of cell
phenotype and, unlike other RNA molecules, highly stable ex vivo, miRNA
profiling is proving to be highly informative for product characterization.
Measurement of miRNA expression in cell populations provides a relatively
straightforward and generally-applicable way to characterise and compare cell
populations.
Methods: Total RNA was prepared using a column-based kit (Exiqon) and
miRNA expression profiles were analysed using microarray slides (Agilent
human miRNA 8*60K V16.0 of miRBase).
Results: Data will be presented to support the use of miRNA profiling for
the selection of donor starting material, comparability of bioprocessing steps in
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Poster Abstracts
the development of a cell therapy and to illustrate of the utility of the miRNA
technology as a surrogate potency test.
Conclusion: MiRNA profiling serves as a generic tool which can be
applied to a number of the activities associated with the development of a
cell therapy including donor selection, comparability assessments and potency testing.
386
FROM ACADEMIC IP TO AN INDUSTRIAL PROCESS UNDER
GMP
G Franco, N Sadr
PharmaCell B.V., Maastricht, Netherlands
In recent years, many cell-based therapies have originated in academic laboratories. However, only a few have been successfully translated from the bench
into clinical trials. Given the stringent regulatory framework in force, the
translation of a scientifically sound cell-based approach into a candidate ATMP
is a complex process for which the initial scientific promoters have often no
experience. Production of a cell-based therapeutic for clinical trials requires
thorough compliance to the GMP guidelines and deep understanding
of Technology Transfer processes, Quality Control, clean-room-based
manufacturing and the overall encompassing Quality Assurance system. The
familiarity of the scientific community with these concepts is critical. Meaningful examples are allogeneic therapies relying on cell lines generated within
research laboratories. Complete traceability of the cell history, required for its
use in humans, is often lacking. The reagents involved in cell manipulation
can also represent a hurdle, especially if they cannot be purchased within a
GMP-compliant system. Methods and techniques can also be source of delays.
Open-handling cell manipulation protocols constitute a major hurdle for
economically sustainable scale-up of production processes. Another obstacle is
the use of qualitative, labor-intensive, highly variable, operator-dependant assays. Quantitative methods, which can be validated and are compatible with the
needs of the manufacturing process, are critical as well. While solutions to
these problems are available, the time and costs involved could imply severe
setbacks. Familiarity of initial scientific players with the later stages of the
product cycle must go along a comprehensive strategy involving, not only the
innovators, but also CMOs and technology providers, who can play a key role
in increasing the success rate of the translation process from the bench to the
bedside.
387
STORAGE OF CD34-POSITIVE SELECTED HPC
S Bleau1, J Tonon1, J Tassy1, A Jakubowski2, G Koehne2, K Smith1,
P Maslak1, S Giralt2, RJ O’Reilly3, M Pessin1, RC Meagher1
1
Laboratory Medicine, [email protected], New York, New York, United
States, 2Medicine, Memorial Sloan Kettering Cancer Center, New York, New
York, United States, 3Pediatrics, Memorial Sloan Kettering Cancer Center,
New York, New York, United States
Use of T-cell reduced (TCD) grafts at MSKCC to prevent the incidence
and severity of graft-versus-host disease (GVHD) is a major theme of the
program’s treatment strategy. A recent multicenter study has confirmed that
TCD results in durable engraftment with a low incidence of GVHD.
Enriched CD34+ TCD grafts can be successfully frozen following routine
cryopreservation methods. However, scare information exists about viability
and functional capacity of CD34+ cells held at refrigerated temperature.
Such information is especially important as newer treatment strategies utilize
these TCD grafts in complex fractionated infusion schedules over multiple
days and in combination with other stem cell products. Based on busy
collection schedules and high throughput cell processing laboratory, we reexamined cell storage methodology. In preliminary studies, we examined
short-term refrigerated storage for maintenance of enriched CD34+ cells
over time to determine the feasibility of providing TCD products when
needed without cryopreservation and without affecting their viability or
functionality. TCD HPC(A) products were prepared using the Miltenyi
CliniMACS Cell Selection system according to manufacturers recommendations to reduce CD3+ T-cells approximately 4-5 logs by enriching CD34+
cells. Excess CD34+ cells were stored at refrigerated temperature (4o-6oC)
up to 10 days post processing. Stored HPC(A) products were evaluated for
CD34+ cell count and 7AAD viability at selected time points. Preliminary
analysis: n¼4 pts, >98% CD34+ cells (44-240 hrs); n¼10 pts, >90% viable
(67-192 hrs) enriched HPC(A) samples following refrigerated short-term
storage. Data show that cell viability and CD34+ cell content was maintained
for up to 10 days storage at refrigerated temperature. Our ongoing study
warrants further investigation on the short-term storage of enriched CD34+
cells at refrigerated temperatures including the impact of short-term storage
on residual immune (CD3+) cells.
388
CHARACTERIZATION OF HUMAN PLATELET LYSATE IN
CYTOKINE ANALYSIS AND PROLIFERATIVE EFFECT ON
Human platelet lysate (hPL) has emerged as a viable human-derived alternative
to fetal bovine serum (FBS) for the expansion of mesenchymal stem cells
(MSCs). Derived from human platelets, hPL contains similar growth factors
and cytokines found in FBS at comparative levels. Several methods of producing can be found in the literature, thus, hPL products differ in number of
pooled donors, lot size, consistency between lots, cytokines/growth factor
presence and/or levels, and heparin requirements. These differences significantly impact cell growth, morphology, multipotency, doubling time, and
senescence. The focus of this study was to compare a new product, COOK
HPLÔ to several commercially available hPL products in terms of physical
properties (such as turbidity and chemistry), cell proliferation, cell morphology,
and cytokine/growth factors. COOK HPLÔ is provided in two different
versions, a heparin-requiring hPL (PL-H) and heparin-free hPL (PL-NH) and
is produced at an industrial scale with high lot-to-lot consistency and purity.
PL-H was determined to have slightly higher levels of growth factors/cytokines
compared to PL-NH and was comparable to hPL from other manufacturers.
hPL from different manufactures had different levels of turbidity, growth
factors/cytokines, and cell proliferation and morphology. While the PL-H had
some differences in cytokine levels, performance in cell culture did not seem to
be affected and the ability to use without heparin provided an advantage in
efficiency if not culture results.
389
LARGE-SCALE EXPANSION OF MESENCHYMAL STEM CELLS
IN 3D CULTURES USING XENO-FREE MICROCARRIERS AND
HUMAN PLATELET LYSATE
RN Dayment1, CG Taylor1, MZ Albanna1, KN Sarchet1, TE Ichim2,
EJ Woods1
1
Cook General BioTechnology, Indianapolis, Indiana, United
States, 2Medistem, Inc, San Diego, California, United States
Fetal bovine serum (FBS) is often used as the serum-supplement in large-scale
manufacturing of animal and human cells for cell therapy applications; however, it poses several regulatory and species cross-contamination challenges
hindering its use in clinical applications. The use of serum-free media is
expensive and often requires custom development based on cell type and
source. Hence, a human-based media additive such as human platelet lysate
(hPL) can be a practical alternative. hPL has been shown to support proliferation of human and animal stem and primary cells. This study evaluates the use
of hPL to grow human mesenchymal stem cells (hMSCs) onto xeno-free
microcarriers in 3D culture. Human endometrial regenerative cells (ERCs)
were expanded in DME/F-12 low glucose supplemented with 10% COOK
HPL PL-NH. ERCs were initially isolated and expanded using 2-D culture.
When adequate numbers of cells were obtained, cells were seeded onto HillexÒ II (modified polystyrene) and grown for seven days in a 300 mL spinner
flask under standard growth environment conditions. Samples from the culture
were taken at days 1, 3, 5, and 7 and cells were evaluated using DAPI nuclear
stain for cell number and rhodamine-phalloidin cytoskeleton stain for cell
attachment and growth. On day 7, cells were harvested and surface markers and
viability were characterized by flow cytometry. DAPI demonstrated high initial
cell attachment after 1 day of seeding and nuclear counts showed progressive
cell growth over 7 days in culture with a 3-fold increase. Cytoskeleton staining
showed attachment and cell spreading on the microcarriers. After cell harvest,
flow cytometry analysis showed that ERCs maintained expression of stem cell
markers (>90% MSC markers, <5% HSC markers) with >90% viability. This
study demonstrates the ability of hPL to substitute FBS for large-scale
expansion of human MSCs in a xeno-free 3D culture and support cell
attachment and growth.
390
ISOLATION AND LARGE-SCALE EXPANSION OF BONE
MARROW-DERIVED MESENCHYMAL STEM CELLS WITH
SERUM-FREE MEDIA UNDER GMP-COMPLIANCE
SH Mei1,2, M Salkhordeh1, F Xue1, J Zhang1, I Watpool3,4, D Allan1,2,
L McIntyre3,2,4, DW Courtman1,2, DJ Stewart1,2
1
Canada, 2Faculty of Medicine, University of Ottawa, Ottawa, Ontario,
Canada, 3Clinical Epidemiology, Ottawa Hospital Research Institute, Ottawa,
Ontario, Canada, 4Critical Care, Ottawa General Hospital, Ottawa, Ontario,
Canada
Introduction: Mesenchymal stem cells (MSCs) are adult somatic stem cells
that can be isolated from bone marrow and extensively expanded in vitro.
Recent studies have shown that MSCs exhibit important “immunomodulatory” properties, which likely contribute to their ability to reduce tissue
damage and enhance repair. Our goal is to develop a safe and clinical-grade
MSC product to conduct the Phase I clinical trial, “Cellular Immunotherapy
for Septic Shock” (CISS). The CISS trial will be the first in human clinical
trial to examine the safety and tolerability of MSCs for the treatment of septic
shock.
Methods and Findings: Bone marrow cells (from Ottawa General Hospital
donors) were seeded in single-layer flasks for w10 days using a growth factor
supplemented serum-free media (Biological Industries), before passaged into a
Corning HYPERFlask (a flask with multiple gas permeable layers), which
eventually yielded w50 to 70 million MSCs per flask by the end of 6w8 days.
MSCs were microscopically observed for fibroblast-like morphology, and
confirmed for multi-lineage differentiation potentials (osteogenesis, adipogenesis, and chondrogenesis). Immune-phenotype of MSCs was confirmed by
surface marker expression of CD105 (88%), CD73 (100%), and CD90 (100%);
and lack of expression of CD45 (0.1%), CD14 (0.1%) and HLA-DR (0.1%), as
defined by ISCT guidelines. The basal ability of MSCs to inhibit human T cell
proliferation was assessed with a standard co-culture assay. Preliminary results
showed that the MSCs isolated and expanded by our protocol were able to
inhibit proliferation of T cells (co-culture at a ratio of 1:9) by 55%, by flow
cytometry analysis.
Conclusions: We have successfully developed a MSC manufacturing protocol, which uses GMP-compliant, serum-free media to produce a sufficient
quantity of MSCs that meets the ISCT criteria. Currently we are using this
protocol to develop a master cell bank of MSCs, which will be tested for their
potency before usage.
391
CHALLENGES OF SCALE IN CELL THERAPY MANUFACTURING
J Beltzer
Cell Processing, Terumo BCT, Lakewood, Colorado, United States
Moving a promising cell therapy from the initial discovery phase through to
clinical trials and therapeutic delivery poses a number of challenges. Not
only must an evolving process clear regulatory hurdles and scale to meet
dose and lot size requirements, but it must also maintain economic value. In
addition to discussing these challenges, a number of case studies will be
presented to describe how process development and economic goals were
achieved in various laboratories by utilizing automation. Particular attention
will be directed toward the process changes often needed to meet these
goals.
392
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393
PRODUCTION OF LARGE-SCALE QUANTITIES OF RARE
THERAPEUTIC CELLS: A PROGRESS REPORT
R Hulspas1, D Berglund2, C Baecher-Allan3, L Villa-Komaroff1, J Sharpe1
1
Cytonome/ST, LLC, Boston, Massachusetts, United States, 2Immunology,
Genetics and Pathology, Uppsala University, Uppsala,
Sweden, 3Neurology, Brigham and Women’s Hospital, Boston,
Massachusetts, United States
Advanced cellular therapy demands well-characterized, specific cell types
and stringent control over the entire process of manufacturing therapeutic
cell products. As flow cytometry is a powerful, established technology for
cell characterization, it is an important technology for advanced cellular
therapy. The technology’s capability to detect and analyze every cell of any
complex, characterized cell population and select a well-defined subset of
cells benefits manufacturing processes of therapeutic cell products.
Although the advantages of flow cytometry are recognized by many, it is
only rarely applied in manufacturing processes for cellular therapy because
the instrumentation is designed to support basic research only and is
incompatible with clinical workflows. As a result conventional FACS presents difficulties for the production of clinically relevant quantities of rare
therapeutic cells that include: sample throughput, sterility and safety, and
ease-of-use. We have been able to overcome the sample throughput limitation by incorporating multiple sorters into a parallel architecture that
functions as a single unit. The concept of parallel architecture has been
explored with the ultimate goal of accommodating large-scale cell purification applied specifically in cellular therapy. This approach utilizes a
parallel microfluidic sorter array, and a sterile fluidic disposable to measure
and isolate highly pure cell populations. Using a reference test, we have
shown performance characteristics to equal or exceed those of a range of
conventional FACS instruments. In one study, 1.5E5 to 1.7E6 Treg cells
were purified from 0.6 to 1.5 billion human PBMCs within 1 hour, on
average. The average % of FoxP3 expressing cells in the purified product
was 85% (range 64%-96%) with an average viability of 97%. Our data
indicates that large scale cell selection for cellular therapy can be accomplished through parallel processing of cell samples with sterile fluidic
disposables.
394
A CLOSED SYSTEM CONTAINER FOR SHIPPING NONFROZEN CELLULAR THERAPY PRODUCTS FOR DIRECT
CLINICAL USE
S Zacharias1, E Fearnot1, S Thirumala1, E Woods1,2
1
Cook General Biotechnology LLC, Indianapolis, Indiana, United
States, 2Indiana University School of Medicine, Indianapolis, Indiana, United
States
The use of closed systems for processing, storage and shipping of cell
therapy products is necessary for good manufacturing practices (GMP). The
current study evaluates the CellSealÒ closed system cryogenic vial as a
container for shipping cellular therapy products which have never been
cryopreserved. The objective of the study was to ascertain the effect on
cellular therapy product viability, integrity, sterility and functionality during
shipping overnight from a manufacturing facility to the bedside of the patient. The fresh cells were shipped overnight to the clinic in a shipper
validated to maintain a temperature of 4-8C. The autologous Mesenchymal
Multipotent Stem Cells (MSC) were derived from equine skin punch biopsies. The purpose of the cellular therapy was to treat various naturally
occurring tendon/ligament injuries of the distal extremities. A dose of 10-20
million cells were suspended in autologous peripheral plasma lysate at a
concentration of 10 million cells/mL. The recovery, viability and functionality of the cells were within the acceptable range set forth for this study.
Upon arrival, the veterinarian used a standard syringe and a 20g needle to
withdraw the cells from the closed CellSeal(R) system and inject them via
ultrasound guidance into the core lesion of the tendon or ligament injury. No
adverse effects were noted in the horses post injection, efficacy standards of
the cellular therapy were promising and no complications were reported by
the attending veterinarians regarding use of the CellSeal(R) closed system.
Twenty years of the International Society for Cellular Therapies: the
past, present and future of cellular therapy clinical development
STEWART ABBOTT1, GEOFF MACKAY2, MATTHEW DURDY3, SUSAN SOLOMON4 &
CLAUDIA ZYLBERBERG5
1
Celgene, Warren, New Jersey, USA, 2Organogenesis, Canton, Massachusetts, USA, 3Cell Therapy Catapult, London,
United Kingdom, 4The New York Stem Cell Foundation, New York, New York, USA and 5Akron Biotech, Boca
Raton, Florida, USA
Historical perspective
Some 20 years ago, as the International Society for
Cellular Therapies (ISCT) was being founded (1992),
it was impossible to imagine all of the scientific and
clinical advances about to occur in the subsequent
two decades.
As evidenced by the annual publication rate and
manuscript focus, the field of stem cell biology and
therapeutics has mushroomed and diversified over
the last two decades: 1992 saw 3280 “stem cell”
papers published (NCBI database). Given the pioneering work of E. Donall Thomas four decades
earlier that led to Thomas and Joseph Murray
receiving the Nobel Prize for Medicine in 1990, it is not
surprising that the majority (51%) of these papers
focused on hematopoietic cells (HSC) and HSC
transplantation; 19% of the papers were directed to
embryonic stem cell (ESC) biology, with only w1%
each on mesenchymal biology or induced pluripotency biology. By 2012 (the last full year of record),
the published stem cell manuscript tally for the year
had grown to >27,000, with 23% focusing on HSC,
19% on ESC, 6% on inducible pluripotent stem cells
(IPC) and 20% on mesenchymal cell biology. A few
of the key academic and commercial milestones for
cell and tissue therapeutics in the past two decades
are depicted in Figure 1.
The fate of two companies (Osiris Therapeutics
and Dendreon) that were founded in the same year
as the ISCT highlights some of the opportunities and
challenges facing cell therapy developers. Osiris
aimed to commercialize the early discoveries of
Arnold Caplan by developing mesenchymal stromal/
stem cell therapeutics (1). After a decade of research,
development and clinical trials, their first cell
product (Prochymal) received conditional regulatory
approval in Canada and New Zealand. Only about a
year later, Osiris’s cell therapy assets were sold to
Mesoblast for a reported $100 million, a fraction of
the cost of development. Likewise, Dendreon achieved regulatory approval, after nearly two decades
of research and development, for their autologous
dendritic cell vaccine Provenge (2). Initial expectations for the product, which garnered reimbursement
of $93,000, were high and indicated that complex
cellular therapeutics could be commercially viable.
Unfortunately, in the face of commercial and logistical challenges and perhaps overly optimistic sales
expectations, Dendrion recently downsized its
workforce and is exploring various options to reinvent a path to sustainability.
The considerable funding, time and effort required to bring cell therapy products to market,
however, has not prevented many other groups from
trying. On the contrary, in the early 1990s, approximately 10 cell therapy companies were formed
each year. A little over 2 years ago, the rate of cell
therapy startups grew to >100 per year. This growth
has been fueled in part by the burgeoning science
knowledge base (as represented by >27,000 stem
cell manuscripts published last year) and by the
successes of a few select groups in launching live cell
therapy products. Organogenesis, founded in 1985,
is notable in that it has launched multiple products,
hired more than 500 staff, and recorded annual
revenues in excess of $150 million. Although Organogesis’s products may represent one of the few
examples to date of commercially available bioengineered cell-based products in the United States,
other companies have products in development.
Correspondence: Claudia Zylberberg, PhD, Akron Biotech, 1095 Broken Sound Pkwy NW, Suite 100, Boca Raton, FL 33487, USA. E-mail: czylberberg@
akronbiotech.com
ISSN 1465-3249 Copyright Ó 2014, published by Elsevier Inc. on behalf of International Society for Cellular Therapy.
Twenty years of the ISCT
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Figure 1. Key developments in cell and tissue therapy development 1992e2013. Founding of The New York Stem Cell Foundation,
generating $100 million for stem cell research and supporting 100 scientists internationally (2005). The New York Stem Cell Foundation
opens safe-haven stem cell research laboratory (2006).
Tengion took an early lead in developing groundbreaking tissue (bladder) engineering based on
technology from Anthony Atala’s laboratory at Wake
Forest and has developed a range of innovative cell
and tissue candidates. Likewise, Humacyte is actively
developing tissue engineered vascular grafts, and
Organovo is further developing Thomas Boland’s
highly innovative three-dimensional bioprinting approaches to create organoids for drug screening and
regenerative medicine applications.
Many groups looking to repeat the success of
Organogensis are currently focusing on the emerging
fields of human embryonic stem cell (hESC) biology
(pioneered in the late 1990s by investigators including
Austin Smith, James Thomson and Rudolf Jaenisch)
and inducible pluripotent stem cell (iPSC) biology
(developed, more recently, in 2006 by Shinya Yamanaka and James Thomson). Geron Corporation, an
early adopter of Thomson’s technology and intellectual property from the University of Wisconsin
Alumni Research Foundation, led the clinical development of hESC-based therapeutics. As such, Geron
was the first to tackle and overcome some of the
substantial regulatory hurdles associated with the
clinical introduction of a novel therapeutic modality.
After extensive and protracted nonclinical studies and
discussions with the US Food and Drug Administration (FDA), Geron received investigational
new drug approval in 2010 to initiate clinical trials of
its hESC-derived oligodendrocyte product candidate
to assess safety and efficacy in patients with spinal
cord injury. A little over a year later, Geron divested
its hESC assets to Biotime to focus on oncology.
Geron’s pioneering work with the FDA provided
a service to the field of cellular therapeutics, and,
within 6 years of its founding in 2004, Advanced Cell
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S. Abbott et al.
Technologies was able to bring a hESC-based retinal
pigmented epithelial cell product candidate into dose
escalation clinical studies.
Over the past two decades, US Federal Government actions have both accelerated and decelerated the development of novel cellular therapeutics.
Arguably, President G.W. Bush’s stance on hESC
had little direct impact on nonefederally funded
development of hESC-based therapeutics, but many
companies looked for alternative sources of cells. On
the other hand, federal influence has also stimulated
development of cellular therapeutics; the Stem Cell
Therapeutics and Research Act of 2005 focused specifically on enhancing and expanding the clinical use
of stem cells derived from umbilical cord blood. The
Japanese government has been quick to realize the
substantial positive impact of clinical research and
development and regenerative medicine initiatives
and has recently developed funding and regulatory
environments that should foster the rapid development of iPSC-based therapeutics.
The therapeutic potential of HSC in settings other
than HSC transplantation for gene-modified cell
therapy is being explored. Groups including Sangamo Biosciences and bluebirdbio have developed
non-viral and virus-based gene-modification technologies to correct genetic abnormalities in a variety
of cell types. Several groups have developed highly
targeted gene-modified cell therapies through innovative interpretation of natural phenomena such as
CCR5 mutations in human immunodeficiency virus
and by harnessing the growing body of data derived
from genomic screens of rare disorders (3,4). The
synergistic technology convergence associated with
gene-modified cellular therapies should, in theory
at least, facilitate the rapid development of targeted
therapies with definable persistence and distribution
characteristics. Additionally, such characteristics may
provide a path toward clinical and commercial differentiation of gene-modified cell therapies from
small- and large-molecule therapy alternatives. Mesoblast’s recent acquisition of Osiris’s mesenchymal
cell assets and collaboration with Ziopharm/Intrexon
to develop inducible gene expression systems for
cellular therapies may be illustrative of this view (5).
has doubled over the past 5 years (6). In the UK, the
number of such studies has increased by 43%.
Data from the Cell Therapy Catapult indicates
that there are 34 ongoing cell therapy clinical trials at
the moment in the United Kingdom, with 37 at the
preclinical stage (believed to be no more than 2 years
from the clinic) (7). How these figures, and those of
other commentators, change over the ensuing years
will give a feel of the development of the cell therapy
industry in terms of quality, maturity and ability to
meet medical need.
Therapeutic activity is relatively broad-based,
with the areas being targeted including autoimmune,
cardiovascular, neurological, hepatic, ophthalmic,
bone and cartilage disorders, plus oncology, stroke
and diabetes (2,8).
On the marketed product side, since Tigenix’s
ChondroCelect for cartilage repair became the first
European advanced therapy medicinal product to be
approved in 2009, it has been followed by three
others, as illustrated in Table I. The hurdles associated with gaining reimbursement of such novel therapies across Europe have been highlighted by the
ChondroCelect case, with The Netherlands the first
to grant it in 2012, followed by Spain in 2013.
In the United Kingdom and across Europe, there
is a strong collaborative environment between academia, pharmaceutical and biotechnology companies.
This is further facilitated and encouraged by organisations such as the United Kingdom’s Cell Therapy Catapult, European Framework and Horizon 20/
20 programmes, plus research charities such as the
Wellcome Trust. Government grant schemes (eg, the
Biomedical Catalyst scheme in the United Kingdom)
also play a key role in providing soft money to meet
important industry challenges.
Deals such as the collaboration between the
United Kingdom’s University College London and
Cellectis on a preclinical allogenic T-cell product for
leukaemia in 2012 illustrate the willingness to partner. There is also merger and acquisition activity, in
the search for both marketed and clinical phase
products, as exemplified by Shire in its 2011 acquisition of Advanced Biohealing Inc for $750 million,
and its 2012 purchase of Pervasis Therapeutics (for
$200 million up front), respectively.
Current state of the UK/European market
It is fair to say that the cell therapy industry in the
United Kingdom and Europe is flourishing, although
it is not yet mature. Across the United Kingdom
and Europe, there are extensive studies of ongoing
cell therapies at the clinical and preclinical stages, in
a variety of therapeutic areas. Cell therapy data from
clinicaltrials.gov (searched under “cell therapy,”
“intervention”) suggest that the number of studies
What does the future hold for the cell therapy
industries in the United Kingdom and Europe?
We expect to see further high-quality collaboration
across the region and an increased pipeline in terms
of quantity, quality and maturity—all toward the
goal of satisfying unmet medical need. The progress
of this work should encourage investment, both
corporate and financial, in this area. In addition, as
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Table I. European advanced therapy medicinal product approvals.
Therapy
Company
ChondroCelect TiGenix
Provenge
Dendreon
Maci
Genzyme
Dermagraft
Smith &
Nephew
Source
Indication
Approval
Status
Autologous Cartilage diseases: repair of European Union,
single symptomatic
Oct 2009
cartilage defects of the
femoral condyle of the
knee (ICRS III or IV) in
adults
Rejected from reimbursement in
France; reimbursed in Belgium,
The Netherlands (previously
through a risk-sharing scheme,
but now fully), Spain and
through private payers in the
United Kingdom (at a cost of
wV20K); NICE appraisal
pending
Autologous Treatment of asymptomatic European Union,
Decision pending in the European
or minimally
April 2010 (also approved
Union; in the United States
symptomatic metastatic
in the United States)
reimbursed by CMS and private
castrateeresistant
healthcare plans (at a cost of
prostate cancer
w$93,000 per patient)
Autologous Cartilage damage
European Union,
Decision pending
June 2013
Allogeneic Diabetic foot ulcers
European Union, June
Withdrawn from European Union
2002 (also approved in
in 2005
the United States)
ICRS, International Cartilage Repair Society; NICE, UK National Institute for Health and Care Excellence; CMS, US Centers for Medicare
and Medicaid Services.
more therapies are used in the clinic and approved
and novel reimbursement mechanisms are developed, the cost-effectiveness argument will become
clearer.
One of the most therapeutically promising,
actively researched and widely debated areas of
regenerative medicine are cell-based therapies, and
with reason. Currently, cell-based therapies are the
subject of more than 1900 clinical trials around the
world. This staggering number includes almost every
imaginable disease or condition: from immune disease to metabolic diseases and cancer. The ability of
stem cells to differentiate into virtually any cell type
offers the therapeutic field the possibility of treating
diseases and conditions such as Alzheimer disease,
spinal cord injury, stroke, heart disease, diabetes and
rheumatoid arthritis, among others. Apart from being
medically significant, cell-based therapies are a
lucrative business: in 2010, according to the Alliance
for Regenerative Medicine, the top 20 cell therapy
products generated revenue in excess of $460 million,
which was expected to more than double by the end
of 2013. This rapid pace of growth is testament to the
confidence placed in cell-based therapies as viable
treatment options for many acute ailments.
As therapeutic agents, cells have capabilities that
extend beyond those of small-molecule biologics.
Cells not only sense and respond to their environment, but they use those cues to migrate to specific
sites in the body and execute complex response behaviors. Thus far, the biggest challenge to the mainstream commercialization of cell-based therapies has
been biocompatibility: controlling their actions when
placed in a therapeutic setting has been marred with
scientific and regulatory—not to mention ethical—
obstacles.
Development of successful and efficient stem
cellebased therapies is rooted in our ability to
develop efficient cellular engineering strategies to
harness the full therapeutic potential of cells while
minimizing adverse effects or reactions. The current
cell-based therapies in development include a broad
variety of cell types: from primary cells, progenitor
cells, adult and embryonic stem cell to pluripotent
stem cells. Increasing the complexity of the target
treatment in turn requires more sophisticated manufacturing processes, which results in a greater need
for engineering and funding. To achieve their full
medical promise, scientists must achieve successful
differentiation, transplantation and engraftment. For
instance, to be useful for transplant purposes, stem
cells must not only differentiate successfully into
the desired cell type, integrate into the surrounding
tissue and survive in the recipient after transplant,
but also function appropriately for the rest of the
recipient’s life.
Of more than 40 cell therapy products currently
on the market, the majority target wound-healing
(35%) and musculoskeletal (35%) applications, followed by skin (11%), cancer (10%) and ocular (7%)
applications. The development pipeline paints a
similar picture. Of the 13 late-stage, industry-sponsored clinical trials in the United States, 32% target
cancer treatment, followed by musculoskeletal (28%)
and wound-healing (28%) applications. The largest
chunk of industry-sponsored trials (n ¼ 52) is,
however, in mid-stage development. A number of
big players, such as Dermagraft, manufactured by
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S. Abbott et al.
Advanced Biohealing, and Apligraf by Organogenesis, both for wound-healing applications, record
revenues in excess of $100 million.
One of the clinical market leaders in the cell
therapy segment is Dendreon. Its flagship product,
Provenge, is the only FDA-approved autologous
immunotherapy treatment for advanced prostate
cancer. It was approved in April 2010 and has since
enjoyed healthy growth, with annual sales topping
$500 million. Although the company has seen its US
growth slow down in the past year, due to the entry of
Johnson & Johnson’s Zytiga in the prostate cancer
market in December 2012, the European Commission has just approved Porovenge for the European
Union market, thereby significantly potentially expanding the company’s market reach.
Another leader in the cell therapy arena, Mesoblast, recognized the benefit that forging strong
partnerships has on expansion early on in the game.
In 2011, the company formed a strategic alliance
with Lonza for clinical and long-term commercial
production of Mesoblast’s allogeneic adult stem cell
products. The company develops treatments for a
range of conditions—namely, systemic inflammatory
and immunology conditions, cardiovascular diseases,
orthopedic diseases of the spine and oncology—
based on mesenchymal precursor cells. The company reported positive results of a Phase 2 trial
assessing the safety and efficacy of injecting a single
dose of their mesenchymal precursor cells directly
into the heart muscle of patients with end-stage heart
failure just earlier this year.
For Advanced Biohealing, heavy investing in cell
therapies paid off. The company’s lead product,
Dermagraft, a bio-engineered skin substitute used to
treat diabetic foot ulcers, achieved sales of $146
million in 2010. It is composed of human fibroblasts,
an extracellular matrix containing fibrinogen and
fibronectin and a bioabsorbable polyglactin mesh
scaffold. This company’s success did not go unnoticed. Just days before the company was to go public,
it announced it was being acquired by Shire Plc for
$750 million.
Other strong players in the stem cell therapy field
are PluriStem, which focuses on developing stem
cell products derived from human placentas, and
Osiris Therapeutics, whose mesenchymal stromal
cell product for the treatment of Crohn disease is
currently in clinical trials.
The above evidence—compounded by a healthily
growing market—suggests that there appears to be
sufficient confidence in stem cells’ therapeutic efficacy. The road to market is, however, paved with
obstacles that extend beyond the therapies’ successes
in the clinic. One of those is safety. Unlike smallmoleculeebased therapeutics—whose safety and
efficacy is regulated by the FDA’s Center for Drug
Evaluation and Research—the FDA has not published a similar set of guidelines for cell-based
therapies.
The release of such guidelines would not only
standardize important procedures for the manufacturing of stem cellebased products but would
instill confidence in both the public, investors and the
clinicians administering such treatments that they are
a viable, well-documented pharmaceutical prospect.
This may, in turn, help turn these somewhat “exotic”
treatments into commonplace therapies.
Looking to the future
Current cutting-edge cell therapy initiatives give us a
glimpse of what the future of cellular therapies may
look like. Groups such as Novartis, Celgene and
Mesoblast have realized the technology convergence
that exists between cell and gene therapies and,
through a variety of collaborations, have started to
develop gene modified cell therapies. Mesoblast has
recently established a collaboration with Intrexon to
explore the potential of small-moleculeeinduced
gene expression systems in cell-based therapies.
Novartis and Celgene (9), respectively, have invested
in collaborations with the University of Pennsylvania
and Baylor College of Medicine and bluebird bio
to develop gene-engineered T-cell therapies for
oncology (9,10). bluebird bio is independently
developing gene-modified HSC therapies for adrenoleukodystrophy and potentially other genetic disorders. Sangamo and Cellectis have invested in the
development and acquisition of effective gene-editing technologies (ZFNs, TALENs and CRISPRs).
Some of these approaches have already demonstrated
clinical efficacy and are likely to be further refined
and optimized as academic synthetic biology initiatives such as those of Craig Venter, George Church,
Jim Collins and others develop beyond early discovery research (11,12). Collaborations between
pharma, biotech and academia are likely to play an
increasing role in the development of effective genemodified cell therapies, as exemplified by the intellectual property map in Figure 2. Novel concepts
conceived in leading academic labs will be developed
in partnership with risk-tolerant commercial
biotechnology groups. Following clinical proof of
concept by these partnerships, large pharma support
for late-stage clinical trials and commercialization
will realize the wider availability of effective genemodified cell therapies.
Although less than a decade old, the field of iPSC
biology appeared poised to eclipse the initial hype
and hope of hESC biology and advance beyond
effective application in traditional drug screening
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Figure 2. Collaboration map of published applications of top applicants, from UKSCN patent Watch Landscape 2012 (15).
into an effective therapeutic modality in its own right.
However, the development of patient-specific hESCs
through somatic cell nuclear transfer (SCNT) by
Dieter Egli and colleagues of The New York Stem
Cell Foundation Research Institute in 2011 and
diploid hESCs by Shoukrat Mitalipov and colleagues
of the Oregon Health and Science University in 2013
have reopened this conversation (13). Because of
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S. Abbott et al.
Figure 3. Current state of the US and ROW market. Source: ARM Annual Report 2013.
regulatory and financial constraints surrounding
SCNT, there are significantly more efforts in developing iPSC therapies as explored above. Whereas
iPSC research and development remains an important branch of stem cell research, the possibility of
hESC therapies has come back to the forefront of
potential therapeutic advancements (14).
In particular, the current economic and regulatory support being provided to iPSC biology by the
Japanese government appears to be set to capitalize
on the intellectual property position of academic
groups in Japan and could potentially catapult the
field. In November 2013, the Japanese Government
passed two bills into law to support regenerative
medicine. The Upper House has also passed a bill
aimed at ensuring the safety of regenerative medicine
and another to revise the pharmaceutical affairs law
to promote safe and swift treatment with the use
of iPSCs and other stem cells. The revised pharmaceutical affairs law defines medical products containing stem cells as regenerative medicine products.
It allows the government to approve such products
conditionally even when their effects are not verified,
if their safety is confirmed in clinical trials (Figure 3).
This may result in, at best, the achievement of
clinical success or, at worst, a rapid realization that
the safe and effective translation of this emerging
science is still an incredibly complex task requiring
broad collaboration within the field.
Irrespective of what approaches are taken to
develop future cell therapeutics, it is clear that
greater attention will be paid to defining and interpreting distribution, persistence and putative mechanism of action of such product candidates in
relevant animal models (FDA Guidance for Industry:
Preclinical Assessment of Investigational Cellular and
Gene Therapy Products, November 2013). Ideally, as
more sensitive and specific analytical tools and
methods are developed, the non-clinical pharmacology understanding provided by animal models
will be testable in clinical studies.
For the development of pluripotent stem cell
therapeutics, collaborative standardization initiatives
will be necessary to develop protocols for producing
fully mature adult cell types for transplantation.
Existing efforts from academic and other research
groups have produced new protocols for generating
various cell types, ushering in new possibilities for
cell transplantation. However, these protocols produce cell types that may not fully recapitulate all
cellular processes of their mature cell types. To
achieve more standardized and robust cell production, true innovation will be required. One such
initiative to achieve this is The New York Stem Cell
Foundation’s (NYSCF) Global Stem Cell Array,
a fully automated robotic system that is able to
consistently produce cell lines and adult cell types on
a large scale, allowing for more robust and standardized cell production in collaboration with academic and industry partners.
To usher in this new era of pluripotent-derived
cell therapies and personalized treatments, it will also
be critical for the patient’s voice to be heard. Equally
important to research efforts, non-profit organizations are able to address advocacy and patient representation problems seen in the private sphere.
When there are new therapies in the pipeline, patient
advocacy non-profit groups have always been at the
front lines of both regulation and implementation,
having a voice at the FDA and, more generally, a
hand in the regulatory environment. These groups
are able to accelerate access and widespread implementation of new therapeutics historically, and there
is no reason to think this pattern will change in the
future.
Deep connections into patient populations and
connections with promoting and supporting new
clinical trials are crucial in the trials’ success due to
access to patients, trust, credibility and information
dissemination, among many other reasons. Similarly,
the ability of activist non-profit organizations to
garner support and action for compassionate use of
therapeutics within the trial phases of therapeutic
approval highlight the expanding and multifaceted
role that non-profit organizations play and will play
in the future.
The past two decades of cellular therapy development have seen many clinical hopes founded and
dashed. However, given the wealth of ingenuity and
innovation in the field, coupled with a rapidly increasing knowledge base and the increasingly rational
approaches to clinical development and efficient trial
design, it seems only a matter of time until a myriad of
clinically effective and cost-efficient cellular therapies
positively affect the practice of medicine.
References
1. Lazarus HM, Haynesworth SE, Gerson SL, Rosenthal NS,
Caplan AI. Ex vivo expansion and subsequent infusion of
human bone marrow-derived stromal progenitor cells
(mesenchymal progenitor cells): implications for therapeutic
use. Bone Marrow Transplant. 1995;16:557e64.
2. Murphy G, Tjoa B, Ragde H, Kenny G, Boynton A. T-cell
therapy for prostate cancer using autologous dendritic cells
pulsed with HLA-A0201-specific peptides from prostate-specific membrane antigen. Hum Gene Ther. 1993;4:521e7.
3. Mullen CA, Snitzer K, Culver KW, Morgan RA,
Anderson WF, Blaese RM. Molecular analysis of T lympho-
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
S119
cyte-directed gene therapy for adenosine deaminase deficiency: long-term expression in vivo of genes introduced with
a retroviral vector. Hum Gene Ther. 1996;7:1123e9.
Blaese RM, Culver KW, Chang L, Anderson WF, Mullen C,
Nienhuis A, et al. Treatment of severe combined immunodeficiency disease (SCID) due to adenosine deaminase deficiency with CD34þ selected autologous peripheral blood cells
transduced with a human ADA gene. Hum Gene Ther. 1993;
4:521e7.
MesoblasteZiopharm/Intrexon press release. Available at http://
www.mesoblast.com/news-and-media/news-announcements.
Accessed November 2013.
Clinicaltrials.gov. Available at http://clinicaltrials.gov/ct2/results?
term¼cellþtherapyþintervention&Search¼Search. Accessed
November 2013.
Cell Therapy Catapult UK Clinical Trials Database. Available at
https://ctcatapult.innovateuk.org/documents/2156401/2232869/
Clinical-Trials-Database-commentary-April-2013.pdf/73fe61fe6d6b-415e-b822-0752ad2111df. Accessed November 2013.
Ellem KA, O’Rourke MG, Johnson GR, Parry G, Misko IS,
Schmidt CW, et al. A case report: immune responses and
clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy. Cancer Immunol Immunother.
1997;44:10e20.
Novartis: University of Pennsylvania press release. Available
at http://www.novartis.com/newsroom/media-releases/en/2012/
1631944.shtml. Accessed November 2013.
bluebirdbio-Celgene-Baylor College of Medicine press release.
Available at http://investor.bluebirdbio.com/phoenix.zhtml?
c¼251820&p¼irol-newsArticle&ID¼1817134&highlight¼.
Accessed November 11, 2013
Church GM. From systems biology to synthetic biology. Mol
Syst Biol. 2005;1:2005.0032.
Isaacs FJ, Dwyer DJ, Collins JJ. RNA synthetic biology. Nat
Biotechnol. 2006;24:545e54.
Noggle S, Fung HL, Gore A, Martinez H, Satriani KC,
Prosser R, et al. Human oocytes reprogram somatic cells to a
pluripotent state. Nature. 2011;478:70e5.
Tachibana M, Amato P, Sparman M, Gutierrez NM, Tippner-Hedges R, Ma H, et al. Human embryonic stem cells
derived by somatic cell nuclear transfer. Cell. 2013;153:
1228e38.
UKNSCN patent watch landscape. Available at http://www.ipo.
gov.uk/informatic-stemcells.pdf. Accessed November 2013.
Immunotherapy: opportunities, risks and future perspectives
MARTIN HILDEBRANDT1, KARL PEGGS2, LUTZ UHAREK3,
CATHERINE M. BOLLARD4 & HELEN E. HESLOP5
1
Technical University Munich, Faculty of Medicine, TUMCells Interdisciplinary Center for Cellular Therapies, Munich,
Germany, 2University College Hospital, Research Department of Hematology, London, United Kingdom, 3Charite
Universitaetsmedizin Berlin, Department of Hematology and Oncology, Berlin, Germany, 4Children’s National Health
System, Center for Cancer and Immunology Research, Washington, DC, USA, and 5Baylor College of Medicine and
Pediatrics, Houston, Texas, USA
Abstract
This review is intended to reflect upon the current status and perspectives of cell-based immunotherapy at a time when the
promise of extensive pre-clinical research has been translated into encouraging clinical responses. However, some of these
have also been complicated by significant adverse reactions. As the field moves towards definitive late stage trials, with a
growing interest from pharmaceutical companies, we realize that novel cell therapy strategies pose questions that are familiar
to traditional drug development, along with new considerations due to the potential of T cells to persist long term and to
expand after adoptive transfer. These questions address the safety of the product, the efficacy, the mode of action, and the
anticipation of risks. From different perspectives, we intend to address exciting opportunities and safety concerns in current
concepts of cellular immunotherapy.
Key Words: clinical trials, gene therapy, immunotherapy, safety, T cell receptor, treatment efficacy
Introduction
Helen E. Heslop & Martin Hildebrandt
The International Society for Cell Therapy (ISCT)
2014 Annual Meeting provides us with an opportunity
to review the current status and perspectives of cellbased immunotherapy. This comes at a time when the
promise of extensive pre-clinical research has been
translated into encouraging clinical responses with
several immunotherapy strategies (1e8). However,
some of the most impressive clinical responses have
also been complicated by significant adverse reactions
(1,2,4,9). As the field moves toward definitive latestage trials, with a growing interest from pharmaceutical companies, it is a timely moment to reflect on the
risks and benefits inherent in studies with complex
biological products.
Novel cell therapy strategies pose questions that
are familiar to traditional drug development, along
with new considerations regarding the potential of
T cells to not only persist long-term up to 10 years
(10,11) but to expand after adoptive transfer. These
questions address the safety of the product, the efficacy, the mode of action and the anticipation of risks.
Unfortunately, these questions will have even greater
weight after the occurrence of unexpected adverse
events with severe and life-threatening consequences
(9,12,13). Nevertheless, the prediction of hazards will
be informed by previous events that are analyzed in a
thoughtful and structured manner. As an example,
the adverse effects surrounding a first-in-man clinical
trial of a superagonistic T-celleactivating antibody
(14) cast a spotlight on points to be considered when
approaching cell-based immunotherapies, including
- Species specificity in vitro and in vivo
- New agents potentially having new mechanisms
of action
- The recognition of agonistic effects
- The potency when compared with a natural ligand
- A potential inactivation of physiological checkpoints
- An amplification of an induced effect
- Scientific rationale for the pre-clinical development and clinical trial
- Approriate dosage, preferably the lowest among
several calculated starting doses
- Scientific merit of a first-in-man trial versus
safety of the participants.
Correspondence: Prof Martin Hildebrandt, Technical University Munich, TUMCells Interdisciplinary Center for Cellular Therapies, Faculty of Medicine,
Ismaninger Strasse 22, Munich, Bavaria 81675, Germany. E-mail: [email protected]
(Received 20 December 2013; accepted 4 February 2014)
ISSN 1465-3249 Copyright Ó 2014, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
These recommendations (15) also have implications for cell-based immunotherapies as potential
high-risk medicinal products and encourage a broad
exchange of information to discuss and to adjust
future trials that are based on emerging data on safety
profiles of different products. The ISCT 20th Anniversary Annual meeting, and the plenary session on
Immunotherapy presented here, is such a forum for
an exchange on both the promise of cell therapy and
on unexpected events observed by the groups actively
engaging in similar research.
The authors who contributed to this review and to
the plenary session must be thanked for summarizing
current results and delineating future perspectives
in cell-based immunotherapy so thoughtfully: Karl
Peggs, London, will review the current status of
pathogen-specific adoptive T-cell therapies that have
now progressed to definitive licensing studies; Lutz
Uharek, Berlin, will summarize the history and current status of redirected T cells and natural killer
(NK) cells targeting tumors; Catherine Bollard,
Washington, will focus on anti-tumor T-cell therapies
generated by stimulation with virus or tumor antigens.
From different perspectives, they summarize three
exciting areas of immunotherapy.
Pathogen-specific adoptive T-cell therapies
Karl Peggs
The basic concepts underpinning pathogen-specific
adoptive T-cell therapies are relatively straightforward. Deficits primarily in number, but to some
degree also function, of pathogen-specific T cells
underlie the increased propensity to infection or
reactivation of a variety of viruses after allogeneic
hematopoietic stem cell transplantation. Adoptive
transfer and engraftment of cells from an appropriate
donor source followed by expansion in vivo can
theoretically hasten the restoration of immunity and
reduce the infective burden. This is technically
easiest when the original stem cell graft donor has
pre-existing immunity to the pathogen of interest. In
these cases, direct selection of pathogen-specific T
cells, or expansion of such cells in ex vivo, allows
generation of a therapeutic product. Pathogens for
which the greatest experience in clinical application
exists include cytomegalovirus (CMV), Epstein-Barr
virus (EBV) and adenovirus.
Most of the early demonstrations of proof of
concept relied on an ex vivo expansion step, making
more widespread clinical application more challenging. Nevertheless, these studies showed that it
was technically possible to expand T cells with specificity for either EBV or CMV, and more latterly
adenovirus, and that after adoptive transfer these cells
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appeared to expand, control viral infection and then
contract but persist as a memory population providing
longer-term immunity (16e18). Refinements over
subsequent years included the development of culture
conditions, allowing more rapid cell expansion
(6,19e24) and increasingly sophisticated strategies
allowing direct selection of virus-specific T cells when
donor immunity is present and precursor frequencies
are maintained at reasonably high levels. These
included selection according to secretion of cytokines
after re-stimulation with viral peptides (notably interferon [IFN]g) (25e27), upregulation of cell surface
activation markers or more direct selection on the basis
of binding of class I human leukocyte antigen (HLA)
multimers loaded with immunodominant viral peptides (28e30). Each of these approaches produces a
therapeutic product that differs either relatively subtly
or in some cases more dramatically in terms of cellular
composition (eg, CD4 versus CD8, pauci-clonal
versus poly-clonal), purity, antigen specificity and
functional characteristics. Application in subsequent
phase I-II studies has also introduced further variation
in terms of cell doses administered, timing of administration after transplantation and indication for intervention (eg, prophylactic, pre-emptive or for clinically
“resistant” infection). The result is that we have a series
of relatively small clinical studies performed with the
use of differing therapeutic products that give broadly
similar messages. In the patients included in these
studies, administration of the cellular therapeutic
results in reconstitution of (presumed) donorderived immunity related to expansion of transferred
populations; this “immunity” appears to be functionally capable of clearance of a variety of viral
pathogens, with establishment of longer-term T-cell
memory and durable immunity in the absence of
subsequent enhanced immune suppression, and the
antigen-specific T-cell populations appear to have a
low risk of inducing significant toxicities, including
graft-versus-host disease (GVHD) (31).
In most cases, results have been compared with
outcomes in historical control cohorts, either formally
or in the context of the discussion of the results.
Although this is not unreasonable for phase I-II
studies, it does highlight some of the difficulties in
interpretation of the data. Notably, clinically significant active GVHD is an exclusion criterion in all
studies. It is well established that CMV infection rates
are higher in patients with GVHD, and infection episodes are likely to be more prolonged and clinically
more problematic in these cases. Thus, there is a selection bias occurring for exclusion of those who are
likely to have the greatest problems. Furthermore, it is
only possible to administer a cellular therapeutic when
one can be generated. Low frequencies of virus-specific T cells in the donor graft are reported to correlate
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M. Hildebrandt et al.
with poorer post-transplant immune reconstitution but
will also probably correlate an increased risk of failure
to generate a product. Because very few of the study
reports detail how frequently there was a failure to
generate a therapeutic product, we can surmize that
there is at least some selection bias occurring. These
considerations highlight the pressing need for randomized confirmatory studies.
These considerations form the basis for two randomized confirmatory studies currently being performed in the United Kingdom assessing the utility of
CMV-specific T cells: one in the sibling donor setting
(Immunoprophylactic Adoptive Cellular Therapy
Study or IMPACT) and one in the unrelated donor
setting (Alternate Donor Study of Pre-Emptive
Cellular Therapy Study or ASPECT). Both include
only CMV-seropositive recipients receiving Tcelledepleted grafts because this patient group has (i)
a very high incidence of CMV infection (ie, high
clinical need and less chance of an unused preselected product in a prophylactic or pre-emptive
study) and (ii) a low baseline incidence of GVHD (ie,
reducing again the chance of an unused pre-selected
product and providing a baseline for assessing potential toxicity of the cellular therapy). Patients are
excluded if they develop grade >1 acute GVHD
before the indicated time point for cellular therapy.
The control groups receive standard viral polymerase
chain reactionebased surveillance, with standardized
criteria for intervention and stoppage of antiviral
drugs. The active intervention arms receive CMVspecific T cells selected either by HLA streptamers
(IMPACT/ASPECT) or by gamma-catch technology
(IMPACT) according to HLA type.
Although such studies may prove the value of
virus-specific T cells in a model transplant setting,
many questions will remain unanswered. For example, what is the role of such therapies in the Treplete setting, in which problematic CMV infection
more generally occurs in association with GVHD?
How well will such cells perform in patients with
slightly greater evidence of GVHD, both in terms of
toxicity and in terms of function in the setting of
enhanced immune suppression, for example, corticosteroid use? How will the products be best integrated into transplant practice—as prophylaxis, as
pre-emptive therapies or only in those with resistant
or prolonged infections? What is the best approach
when the donor is CMV-naive, for example, cord
blood or seronegative donor? Nevertheless, it is
hoped that the current studies will provide a more
solid basis for further evaluation of these questions.
With respect to the issue of treating patients
without available seropositive donors, there are a
number of potential solutions being evaluated. The use
of “third-party” virus-specific cells offers the intriguing
possibility of being able to generate a bank of cell lines
that would be rapidly available for use directed by a
“best available HLA match” algorithm (wherein the
transferred cells must recognize the pathogen in the
context of a shared HLA allele). Potential issues here
pertain to possible alloreactivity in either a graft-versushost direction, resulting in third-party GVHD if the
cells engraft robustly, or in the opposite host-versusgraft direction, resulting in rejection of the adoptively
transferred populations before they can exert the
desired effect. Nevertheless, proof of concept exists in
the setting of EBV-related post-transplant lymphoproliferative disorders (32) and has been demonstrated
more recently for a CMV, EBV and adenovirus after
hemopoietic stem cell transplantation (HSCT) (33).
No significant toxicities have been reported. The lack
of detectable persistence of transferred cells raises
interesting questions regarding the mechanism of
therapeutic effect that should be addressed in future
studies. One possible mechanism is that the thirdparty cells engender more rapid reconstitution of
second-party immunity derived from the T cells of the
original stem cell donor and that the third-party cells
are acting either directly as a cellular vaccine or
indirectly through a brief burst of lysis of virally
infected host cells. Clearer resolution of the value of
this approach should be provided in randomized
controlled studies. An alternative strategy relies on
induction of primary immune responses ex vivo, with
subsequent expansion and adoptive transfer (34).
Finally, although currently limited in terms of widespread application by technological constraints and
costs, proof-of-concept studies evaluating genetically
modified T cells transduced to express virus-specific
T-cell receptors are currently being undertaken.
Whereas such mono-pathogenespecific therapies
can address the more frequently occurring viral infections, it is well recognized that severely immunosuppressed patients are at risk from multiple pathogens.
Therapeutic products with multiple specificities may
therefore be advantageous in certain clinical situations
(35). An even broader repertoire of immune reconstitution against both known and unknown pathogens
may be achievable by transfer of memory T-cell populations depleted of the naive compartment that contains most of the alloreactive potential of the graft (36).
The results of ongoing clinical studies evaluating such
products are keenly anticipated.
The role of redirected T cells and NK cells in
tumor medicine
Lutz Uharek
In 1909, Paul Ehrlich proposed that the immune
defense system can identify and eliminate nascent
tumor cells. Exactly 100 years later, this principle
was successfully applied to treat patients with refractory leukemia by use of their chimeric antigen
receptor (CAR)-modified T cells (1,2,9). Previous
attempts to exploit lymphocytes for tumor therapy
were either associated with significant side effects,
as graft-versus-host disease (GVHD) in the case
of allogeneic T cells, or turned out to be very
cumbersome as the use of tumor-infiltrating lymphocytes. Now we have the technology to expand
and process T cells and NK cells in a way that allows
specific targetting of a huge variety of antigens and
cell surface molecules. Is this the beginning of a new
era of tumor medicine in which cytotoxic drugs will
be replaced by individually designed cellular products? What are the realistic chances and what are the
risks of this approach? Which are the central questions that still must be adressed in preclinical studies?
Is CAR or T-cell receptor technology superior?
Currently, two approaches for redirecting T-cell
specificity are used: (i) T-cell receptors (TCRs), in
which variable a- and b-chains are cloned from
T cells with specificity against a tumor antigen (37),
and (ii) CARs, in which tumor antigens were recognized through antibody single-chain variable
fragments (scFvs) linked to intracellular signaling
domains (38). The TCR approach was the first
demonstration of redirected T-cell specificity (39). It
relies on the natural way of T-cell function and has
the major advantage that a large number of mutated
intracellular proteins can be targeted. Its major difficulties are a low cell surface expression of TCRs
and so called “mispairing,” which occurs by formation of TCRs formed by of one endogenous and one
transduced TCR chain.
In contrast to TCR technology, the CAR technology requires no antigen processing and is HLAindependent. The major restriction preventing its
broader application is the limited number of suitable
antibody-targeted tumor surface antigens. Other
problems of so-called “third-generation” CARs
include cytokine release induced by low-avidity “offtarget” binding and immunogenicity (the ScFv
portion is generally mouse-derived), which may
result in immune responses and early clearance of
CAR-engineered T cells.
What are immunological acceptance criteria for
T-cell and NK-cellebased biopharmaceuticals?
The optimal target antigen has the following properties: it is immunogenic, it is completely tumor-specific
with no significant expression on normal tissues, it is
highly expressed on all tumor cells (including tumor
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stem cells), it is essential for survival or proliferation
of the tumor cell, it has multiple epitopes and it
is expressed on the tumor surface. For clinical applications, it is not necessary that all of the abovementioned criteria are fulfilled. The importance of
each criterion will depend on the balance between
aspects of safety, reliability and effectiveness in a given
clinical situation.
In addition, the functional quality is determined
by its ability to lyse tumor cells expressing a particular marker, the affinity with which the introduced
receptor binds its antigen, the level of receptor
expression on the cell surface, the in vivo expansion
and persistence of the T cells, the lack of off-target
toxicities. Full functionality is only ensured if both
the afferent function (tumor recognition) as well as
the efferent function (tumor kill) are operative. Assays to measure T-cell activation on exposure to
patient-derived tumor tissue may help to determine
the individual effectiveness in vitro (40).
The assessment of off-target toxicity is critical
for a product without self-limiting properties. For
CARs, it is relatively easy to determine whether the
antibody shows cross-reactivity and binds to the
surface of tissues not expressing the targeted antigen.
Similar tests for cross-reactivity of TCR-based biopharmaceuticals are more difficult to establish
because all relevant HLA types must be considered.
Which is the optimal technology for gene
transduction?
Retroviral or lentiviral vectors can be used to transfer
TCR or CAR coding genes into T cells. Both yield
a high level of stable transgene expression and
permanent gene expression. The most important
advantage of retroviral vectors is long-term experience in clinical trials (11). Whereas retroviral transduction can be performed only on efficiently dividing
cells, lentiviral vectors are also capable of integrating
into non-dividing cells. An additional albeit theoretical advantage is their lower risk of damaging insertions. It is therefore very likely that lentiviral
vector systems will be used more often. Recently,
transposon systems such as Sleeping Beauty (41)
have been developed as simple and inexpensive
methods for a stable non-viral genetic modification.
In contrast to viral vectors, they do not have an
intrinsic capacity to cross the cellular membranes
and must be delivered either by different non-viral
strategies or by vector systems. Transposons have the
advantage that they do not require cell pre-activation,
have a low immunogenicity and have a relatively
large cargo capacity. Their major disadvantage is
limited clinical experience and little knowledge about
their oncogenic potential in vivo.
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How to improve the effectiveness of redirected
T cells and NK cells
Five different approaches to improve the effectiveness of redirected T cells and NK cells are currently
discussed: (i) in vitro genetic engineering to modify
T-cell and NK-cell features (transduction of interleukin [IL]-2 gene, induction of anti-apoptotic proteins, introduction of specific chemokine receptors),
(ii) in vitro cytokine activation (IL-2, IL-7, IL-15 and
IL-21), (iii) pre-selection of T-cell and NK-cell
subsets (EBV- or CMV-specific T cells, central
memory T cells, mature/activated NK cells), (iv)
modifying the host environment by preconditioning
before cell transfer or (v) by supportive treatment
after T-cell transfer.
Thus far, only some studies used immunomagnetic selection techniques to restrict the T-cell or
NK-cell pool. However, there is no doubt that the in
vivo survival and effectiveness of adoptively transfused
cells depends on subtype and differentiation status. In
the past, most of the clinical trials have used effector
memory T cells (TEMs) as a result of cell culture
technologies leading to a rapid differentiation into
late-stage effector cells (today mostly by utilization
of a CD3/CD28 activation procedure). Although in
vivo TEMs show more cytotoxicity when compared
with central memory T cells, TEMs might not ensure
long-term tumor surveillance. Gattinoni and colleagues (42) identified stem cell memory T-cells
(TSCM) as a very attractive population for adoptive
cell transfer because of their self-renewal capacity and
their ability to generate TEM, central memory T cells
and effector T cells in vivo.
Environmental variables, including the presence
of regulatory T cells (Tregs) or myeloid-derived
suppressor cells (MDSCs), can also be important for
the success of adoptive T-cell transfer. Host preparative lymphodepletion, introduced by Dudley and
colleagues (43), has been proposed to ensure optimal
environmental conditions such as reduction of Tregs
and MDSCs, decrease in endogenous lymphocyte
competition for cytokines or access to antigenpresenting cells. Systemic administration of cytokines
such as IL-2, IL-7, IL-12 and IL-15 or IFN-g after
adoptive T-cell transfer has also been used to enhance
T-cell and NK-cell effector function. However, such
treatments are often associated with relevant side
effects, and their potential advantages have not been
formally demonstrated.
How to improve the safety of genetically
engineered cells
The risk of side effects caused by on- and off-target
toxicity has risen with increasing effectiveness of
genetically modified T cells and NK cells. Therefore,
different approaches to allow on-demand cell destruction by application of a substance that can switch
on a suicide gene have been developed. Herpes
simplex virus thymidine kinase (HSV-TK) can be
regarded as a reference strategy (44); it is currently
under investigation in a phase III clinical trial. Other
strategies include inducible caspase 9 (iCasp9) (45)
and application of CD20 (46).
Which tumors should be targeted, and what is
the optimal administration schedule of the
cell product?
The targeted tumor should be immunogenetic and
sensitive to cell-induced apoptosis (either on the basis
of in vitro data or experience from allogeneic DLI).
There is still an urgent need to define and standardize
reliable biomarkers and feasible tests to determine the
sensitivity of tumor entities toward T-cell and NKcellemediated cytotoxicity. To allow the induction of
an immune response, slowly growing tumors are
preferred. Tumor entities, tumor stages, and clinical
situations in which no alternative treatments are
availabe should be chosen for early-stage trials.
Thus far, there are no conclusive data concerning
the optimal number of TCR- or CAR-transduced
cells. Regarding conventional anti-cancer drugs,
dosing and application schedules will certainly
depend on specific characteristics of the cell product
and host factors, such as tumor recognition (immunogenicity) and sensitivity of the tumor toward
cellemediated (perforin) lysis, as well as dynamics of
tumor proliferation and tumor sensitivity toward
chemotherapy or other immunotherapies.
What kind of additional and supportive
treatment can increase effectiveness and reduce
risks?
Cytotoxic therapies (conditioning) before adoptive
cell transfer can enhance effectiveness. The aim is (i)
to “make space” for the newly administered cells, (ii)
to reduce the number of host lymphocytes to prevent
alloreactivity and rejection and (iii) to induce tumor
cell apoptosis to reduce the number of tumor cells
and increase their immunogenicity. Decisions on the
type and intensity of the conditioning must consider
the relative impact of each of these three goals in a
particular clinical setting as well as the individual
situation of the patient. This makes it very difficult to
give general recommendations.
Side effects of T-cell transfer should be addressed
by means of a careful risk assessment and establishment of risk-oriented prophylactic and therapeutic
procedures. Because a considerable variation in the
magnitude of the induced immunological effect must
be expected, massive tumor destruction with subsequent potentially lethal tumor lysis syndrome can
occur. Therefore, prior tumor reduction, careful
monitoring of markers for tumor destruction (LDH)
and renal function as well as prophylactic application
of allopurinol and adequate hydration is essential to
prevent fatal outcomes.
The second clinical problem that must be anticipated is an anaphylactic reaction, probably directed
against xenogeneic proteins of the transferred T-cell
product (47). As the result of sensitization against
these antigens, the risk of an anaphylactic shock increases after the second or following administrations
and application of the product should take place in
an environment in which patient monitoring and
equipment for adequate emergency interventions are
ensured.
The third critical side effect of T-cellebased
therapeutics is the induction of a cytokine release
syndrome (4), which often cannot be discriminated
easily from an anaphylactic reaction because major
aspects of clinical manifestation (hypotension) are
similar. The release of cytokines, in particular of
IL-6, IL-10 and IFN-g, is not only caused by direct
release by activated cytotoxic T cells but could also
represent the consequences of a macrophage activation syndrome, which might be reversible with the
use of IL-6R inhibitors. Prophylactic administration
of steroids could also reduce the intensity of anaphylactic reactions and cytokine release syndrome
but could negatively influence the effectiveness of
the transferred cells and are therefore usually not
administered. Standards for immediate diagnosis
and treatment for all three major side effects should
be incorporated into the study protocol.
How should safety aspects be considered in
clinical trials?
Adverse effects are categorized as on-target effects (also
referred to as target-related, exaggerated pharmacology or mechanism-based) or off-target effects (as a
result of unspecific modulation of other targets). The
discrimination between on- and off-target toxicity is
important because high or unaccaptable off-target
toxicity should be a subject of further improvements
in cell engineering and production, whereas excessive
or life-threatening on-target toxicity raises general
questions concerning the choice of the antigen. However, in some cases, the level of on-target toxicity can
be reduced by changing dose and application schedule.
Long-terminal repeats of the viral vector system
can increase the expression not only of the transduced genes but also of neighboring genes. Inserted
near an oncogene, retroviral vectors may thus drive
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oncogenesis. However, all oncogenic events have
occurred during gene transfer to stem cells, and it
appears that mature lymphocytes harbor only a very
low risk for insertional mutagenesis and are resistant
to retroviral transformation (48). Long-term safety
data are availble over a time span of more than 10
years (11).
Results of clinical trials with TCR-modified T
cells include cardiological (12,49) and neurological
(50) toxicities. Whereas the cardiac toxicity was off-target and probably caused by the CDR2 mutations
that enhance major histocompatibility complex binding of the TCR, resulting in recognition an epitope in
titin (12), the neurological toxicity was “on-target” in
that the identical epitope was expressed in MAGE-A3
and MAGE-A12 (50). The previously unrecognized
expression of MAGE-A12 in human brain underlines
the importance of elaborated antigen-expression profiles, that is, on the basis of deep sequencing technology. No autoimmune symptoms that could be
related to TCR “mispairing” have been reported thus
far. Nevertheless, careful monitoring of GVHD-like
syptoms is appropriate. In most trials with CARtransduced T cells (mostly directed against B-cell
antigens), no severe on-target side effects have been
reported (51e54). However, especially T cells transduced with first-generation CARs often failed to
persist, limiting the number of patients bearing CARmodified T cells with long-term follow-up.
Concerning on-target toxicities, the example of
CD19 demonstrates that even life-threatening side
effects (cytokine storm, tumor lysis and long-lasting
B-cell depletion) can be accepted in a particular
clinical setting (2,4,9). Regarding every conventional
drug, the magnitude and probability of side effects
must be determined as accurately as possible and
must be outweighed against the expected clinical
benefit for each individual patient.
Conclusions
Innovative technologies to transfer tumor antigen
specificity to lymphocytes are currently opening the
door to a new field of anti-cancer therapies. Although
the value of redirected T cells and NK cells still
must be demonstrated in controlled studies, the
substantial tumor regression seen in first clinical
trials impressively demonstrates the potential of these
next-generation cell products. In the future, it will be
important to determine product, target and clinical
requirements for the successful implementation of
this new approach into treatment pathways. Only the
combination of high-quality products, professional
risk management with a careful evaluation and
consideration of all relevant safety aspects and welldesigned clinical trials will successfully ensure the
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transfer of redirected T-cell and NK-cell therapies
from the bench to bedside.
T-cell therapies for hematologic malignancies:
use of a nonegene transfer approach
Catherine M. Bollard
Although hemopoietic stem cell transplantation has
increased survival significantly for patients who have
moderate risk disease and achieve a complete remission before HSCT, those with persistent disease
continue to have a dismal prognosis despite HSCT
of less than 10% survival at 2 years. Furthermore,
patients who relapse after HSCT also have a poor
prognosis, with less than 20% surviving 2 years
despite a second HSCT. Even for patients who enter
HSCT for high-risk malignancies in complete remission, the prognosis remains guarded, with less than
35% surviving to 5 years. Thus, despite the allogeneic
graft-versus-leukemia (GVL) or graft-versus-tumor
(GVT) effect, relapse represents the major cause of
treatment failure in these patients, and novel therapies
are critical for patients with high-risk leukemia or
lymphoma with relapsed or refractory disease.
Approaches to harness and increase the GVL effect
include use of donor lymphocyte infusions, suicide
geneetransduced T cells and T cells selectively depleted of GVHD reactivity. Although these approaches
increase the GVL effect, they carry a risk of causing
GVHD. Although tumor vaccines or dendritic cells
loaded with vaccine can avoid GVHD, results have
been largely unsuccessful after HSCT, in part because
of impaired T-lymphocyte and B-lymphocyte immune
reconstitution (55). More recently, T cells genemodified to express a CAR directed to a specific antigen have been developed. Although the data from
several groups are promising (1,2,4,9,52,56), CAR
treatment has limitations: (i) tumors can downregulate
or mutate the surface expressed CAR receptor and
evade CAR cell attack; (ii) in hematologic malignancies, the paucity of suitable surface antigen
expression has restricted CAR therapy largely to targeting CD19 on B-cell tumors; (iii) CAR infusions
have significant inflammatory toxicities, which limit
their safe application. Alternative and safer T-cell
therapy strategies that overcome these obstacles and
target a broad range of tumor antigens are thus still
needed to overcome these obstacles.
Tumor antigens
The identification of antigens on the tumor cells
that might be targets is a prerequisite for developing
immunotherapeutic approaches. Many tumor antigens can also be weakly expressed in healthy cells
from the same lineage, although they may be
upregulated or dysregulated in the malignant cell.
Successful eradication of tumor may thus be
complicated by loss of healthy circulating cells
expressing the antigen. Antigens used for anti-tumor
immunity must be chosen carefully from examples
that are (i) unique to tumors, (ii) highly expressed in
tumor cells, minimizing the normal tissue damage, or
(iii) expressed on healthy cells that may be deleted
for some time without major complication (eg, B
cells).
Targeting viral antigens
Many lymphomas are associated with viruses, presenting unique, often highly immunogenic epitopes
as T-cell targets. Latent EBV infection is found in
non-Hodgkin lymphoma, including Burkitt’s lymphoma, NK-T lymphomas and lymphoproliferative
disease (LPD) and a subset of Hodgkin disease (57).
The mechanism of EBV-induced lymphomagenesis
is well established in the EBV lymphoproliferative
diseases in immunosuppressed individuals but is
poorly defined in other EBV-associated lymphomas
in which EBV may represent a passenger virus.
Nevertheless, the presence of EBV in tumor cells
presents a target for immunotherapy. Donor-derived
EBV-specific T cells have been used for some time to
prevent and treat EBV-associated lymphoma after
HSCT (7). Targeting this highly immunogenic tumor
achieves remarkable response rates without toxicity or
GVHD even when the infused T cells are specific for
multiple viruses (18). Adapting these immunotherapy
approaches to type II latency tumors, however, is
challenging because a more restricted array of subdominant EBV antigens is expressed and the frequency of clones recognizing latent membrane
protein (LMP)1 or LMP2 antigens expressed on
these tumors is low in polyclonal EBV cytotoxic T
lymphocyte (CTL) lines generated with the use of
LCL (58). EBV-associated Hodgkin disease and nonHodgkin lymphoma that develop in the immunecompetent host show type II latency in which viral
gene expression is limited to LMP1 and LMP2,
EBNA 1 and EBERs. Expression of a minimal subset
of genes, which are weak targets for CTL activity,
therefore allows the malignant cells to evade the immune system. Nevertheless, the subdominant EBV
antigens EBNA1, LMP1 and LMP2 may serve as
targets for immunotherapy approaches. In a phase I
dose-escalation study, we evaluated the use of autologous EBV-specific CTL for patients with EBVpositive Hodgkin disease and showed persistence of
these cells for up to 1 year, with a response rate of
20% (59). However, the response rate was less
impressive than that seen in EBV-LPD after HSCT
and only seen in patients with relatively low tumor
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burden. This may have been due to a lack of specificity of the EBV-specific CTL for the immunosubdominant LMP1 and LMP2 antigens present on
the Hodgkin tumor. In addition, the tumor produces
inhibitory factors such as tumor growth factor
(TGF)-b, which affect CTL and antigen-presenting
cell activity (60).
In a clinical trial, we generated LMP1- and
LMP2-specific CTL lines in patients with EBVþve
Hodgkin disease or EBV-positive B-cell or T/NK cell
non-Hodgkin lymphomas (3,61). Patients received
doses of 4 107 CTL/m2 to 1.2 108/m2. No
immediate toxicity was observed, and 28 of 29 patients without radiological evidence of disease who
received CTL as adjuvant therapy after SCT or
chemotherapy remain in remission and 13 of 21
patients with active relapsed disease had a tumor
response, which was complete in 11 patients (3).
Therefore, immunotherapy with autologous LMPCTL was well-tolerated in patients with relapsed
EBVþve Hodgkin disease/non-Hodgkin lymphoma, and infused LMP-CTL cells accumulated
at tumor sites and induced clinical responses. In
a follow-up study, we are now attempting to overcome TGF-beinduced suppression and utilizing
this approach after HSCT with the use of donorderived LMP-CTL.
Disclosure of interests: Helen E. Heslop reported
research and licensing agreements with Celgene and
CellMedica. The other authors have no commercial,
proprietary, or financial interest in the products or
companies described in this article.
Targeting leukemia-associated antigens
References
In addition to allo-antigens, there are many leukemia- and lymphoma-associated antigens. They can
be classified as (i) antigens common to many malignancies, for example, cancer-testis antigens
MAGE, BAGE, GAGE and NY-ESO-1; (ii) antigens overexpressed by malignant cells, for example,
Her2/neu, AFP, Telomerase, WT1, RHAMM and
PRAME (62,63); (iii) antigens specific for hematopoietic lineages, for example, primary granule
proteins proteinase 3 and cathepsin G in myeloid
malignancies.
Lymphoma-associated antigenespecific T cells
We have devised several strategies that consistently
expand leukemia- and lymphoma-directed T cells from
healthy donors for targeting AML through WT1, PR3,
NE and MAGE-A3; ALL through WT1, MAGE-A3,
PRAME and survivin; and Hodgkin lymphoma/
non-Hodgkin lymphoma through PRAME, NY-ESO,
survivin and MAGE A4. The resultant lines were
predominantly CD3þ T cells (mean, 98%) with an
effector memory phenotype. When these multispecific
T-cell lines were co-cultured with primary leukemia
blasts or lymphoma cells matched at one or more class I
or class II HLA antigens, we found specific tumor
recognition and elimination, even with single HLA
class I or class II alleleematched target cells. We also
expanded such TAA-specific T cells from more than
50 patients with ALL or lymphoma (Hodgkin lymphoma or non-Hodgkin lymphoma) and evaluated
their cytolytic activity against autologous blasts. Ex
vivoeexpanded CTL lines had specificity for a median
of two antigens (64e66). We also demonstrated that
patient-derived multieTAA-CTL could efficiently kill
autologous tumors and that this effect was increased in
the presence of demethylating agents (67,68). This
approach of infusing TAA-specific T cells before and/
or after vaccines might therefore be able to overcome
the critical obstacle of vaccine therapy when there are
limited immune responses as a result of increased
immunosuppression after chemotherapy or SCT.
Furthermore, infusion of briefly expanded polyclonal
donor TAA-specific CTL donor T cells recognizing a
broad number of TAAs to reduce immune escape
may overcome the treatment failures associated with
single-antigenedirected CD19-CAR cells.
1. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A,
et al. T cells with chimeric antigen receptors have potent
antitumor effects and can establish memory in patients with
advanced leukemia. Sci Transl Med. 2011;3:95ra73.
2. Brentjens RJ, Davila ML, Riviere I, Park J, Wang X,
Cowell LG, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute
lymphoblastic leukemia. Sci Transl Med. 2013;5:177ra38.
3. Bollard CM, Gottschalk S, Torrano V, Diouf O, Ku S, Hazrat
Y, et al. Sustained complete responses in lymphoma patients
receiving autologous cytotoxic T lymphocytes targeting
Epstein-Barr virus latent membrane protein [published online
ahead of print December 16, 2013]. J Clin Oncol.
4. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH,
Spaner DE, Maric I, et al. B-cell depletion and remissions of
malignancy along with cytokine-associated toxicity in a clinical
trial of anti-CD19 chimeric-antigen-receptor-transduced
T cells. Blood. 2012;119:2709e20.
5. Rosenberg SA, Yang JC, Sherry RM, Kammula US,
Hughes MS, Phan GQ, et al. Durable complete responses in
heavily pretreated patients with metastatic melanoma using
T cell transfer immunotherapy. Clin Cancer Res. 2011;17:
4550e7.
6. Peggs KS, Thomson K, Samuel E, Dyer G, Armoogum J,
Chakraverty R, et al. Directly selected cytomegalovirus-reactive donor T cells confer rapid and safe systemic reconstitution
of virus-specific immunity following stem cell transplantation.
Clin Infect Dis. 2011;52:49e57.
7. Bollard CM, Rooney CM, Heslop HE. T-cell therapy in the
treatment of post-transplant lymphoproliferative disease. Nat
Rev Clin Oncol. 2012;9:510e9.
S128
8. Chapuis AG, Ragnarsson GB, Nguyen HN, Chaney CN,
Pufnock JS, Schmitt TM, et al. Transferred WT1-reactive
CD8þ T cells can mediate antileukemic activity and persist in
post-transplant patients. Sci Transl Med. 2013;5:174ra27.
9. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL,
Rheingold SR, et al. Chimeric antigen receptor-modified T
cells for acute lymphoid leukemia. N Engl J Med. 2013;368:
1509e18.
10. Heslop HE, Slobod KS, Pule MA, Hale GA, Rousseau A,
Smith CA, et al. Long-term outcome of EBV-specific T-cell
infusions to prevent or treat EBV-related lymphoproliferative
disease in transplant recipients. Blood. 2010;115:925e35.
11. Scholler J, Brady TL, Binder-Scholl G, Hwang WT, Plesa G,
Hege KM, et al. Decade-long safety and function of retroviral-modified chimeric antigen receptor T cells. Sci Transl
Med. 2012;4:132e53.
12. Linette GP, Stadtmauer EA, Maus MV, Rapoport AP,
Levine BL, Emery L, et al. Cardiovascular toxicity and titin
cross-reactivity of affinity enhanced T cells in myeloma and
melanoma. Blood. 2013;122:863e71.
13. Morgan RA, Yang JC, Kitano M, Dudley ME,
Laurencot CM, Rosenberg SA. Case report of a serious
adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2.
Mol Ther. 2010;18:843e51.
14. Suntharalingam G, Perry MR, Ward S, Brett SJ, CastelloCortes A, Brunner MD, et al. Cytokine storm in a phase 1 trial
of the anti-CD28 monoclonal antibody TGN1412. N Engl J
Med. 2006;355:1018e28.
15. Expert Scientific Group on phase one clinical trials. 11-302006. ISBN-10 0 11 703722 2.
16. Heslop HE, Ng CY, Li C, Smith CA, Loftin SK, Krance RA,
et al. Long-term restoration of immunity against Epstein-Barr
virus infection by adoptive transfer of gene-modified virusspecific T lymphocytes. Nat Med. 1996;2:551e5.
17. Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME,
Greenberg PD. Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science. 1992;257:238e41.
18. Leen AM, Myers GD, Sili U, Huls MH, Weiss H, Leung KS,
et al. Monoculture-derived T lymphocytes specific for multiple viruses expand and produce clinically relevant effects in
immunocompromised individuals. Nat Med. 2006;12:
1160e6.
19. Peggs KS, Verfuerth S, Pizzey A, Khan N, Guiver M,
Moss PA, et al. Adoptive cellular therapy for early cytomegalovirus infection after allogeneic stem-cell transplantation
with virus-specific T-cell lines. Lancet. 2003;362:1375e7.
20. Peggs KS, Verfuerth S, Pizzey A, Chow SL, Thomson K,
Mackinnon S. Cytomegalovirus-specific T cell immunotherapy promotes restoration of durable functional antiviral
immunity following allogeneic stem cell transplantation. Clin
Infect Dis. 2009;49:1851e60.
21. Einsele H, Roosnek E, Rufer N, Sinzger C, Riegler S,
Loffler J, et al. Infusion of cytomegalovirus (CMV)-specific T
cells for the treatment of CMV infection not responding to
antiviral chemotherapy. Blood. 2002;99:3916e22.
22. Trivedi D, Williams RY, O’Reilly RJ, Koehne G. Generation
of CMV-specific T lymphocytes using protein-spanning pools
of pp65-derived overlapping pentadecapeptides for adoptive
immunotherapy. Blood. 2005;105:2793e801.
23. Blyth E, Clancy L, Simms R, Ma CK, Burgess J, Deo S, et al.
Donor-derived CMV-specific T cells reduce the requirement
for CMV-directed pharmacotherapy after allogeneic stem cell
transplantation. Blood. 2013;121:3745e58.
24. Gerdemann U, Katari UL, Papadopoulou A, Keirnan JM,
Craddock JA, Liu H, et al. Safety and clinical efficacy of
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
rapidly-generated trivirus-directed T cells as treatment for
adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant. Mol Ther. 2013;21:2113e21.
Feuchtinger T, Matthes-Martin S, Richard C, Lion T,
Fuhrer M, Hamprecht K, et al. Safe adoptive transfer of virusspecific T-cell immunity for the treatment of systemic
adenovirus infection after allogeneic stem cell transplantation.
Br J Haematol. 2006;134:64e76.
Feuchtinger T, Opherk K, Bethge WA, Topp MS,
Schuster FR, Weissinger EM, et al. Adoptive transfer of pp65specific T cells for the treatment of chemorefractory cytomegalovirus disease or reactivation after haploidentical and
matched unrelated stem cell transplantation. Blood. 2010;
116:4360e7.
Icheva V, Kayser S, Wolff D, Tuve S, Kyzirakos C, Bethge W,
et al. Adoptive transfer of Epstein-Barr virus (EBV) nuclear
antigen 1-specific T cells as treatment for EBV reactivation
and lymphoproliferative disorders after allogeneic stem-cell
transplantation. J Clin Oncol. 2013;31:39e48.
Cobbold M, Khan N, Pourgheysari B, Tauro S, McDonald D,
Osman H, et al. Adoptive transfer of cytomegalovirus-specific
CTL to stem cell transplant patients after selection by HLApeptide tetramers. J Exp Med. 2005;202:379e86.
Schmitt A, Tonn T, Busch DH, Grigoleit GU, Einsele H,
Odendahl M, et al. Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8þ
T cells leads to virus clearance in patients after allogeneic
peripheral blood stem cell transplantation. Transfusion. 2011;
51:591e9.
Uhlin M, Gertow J, Uzunel M, Okas M, Berglund S, Watz E,
et al. Rapid salvage treatment with virus-specific T cells for
therapy-resistant disease. Clin Infect Dis. 2012;55:1064e73.
Melenhorst JJ, Leen AM, Bollard CM, Quigley MF,
Price DA, Rooney CM, et al. Allogeneic virus-specific T cells
with HLA alloreactivity do not produce GVHD in human
subjects. Blood. 2010;116:4700e2.
Haque T, Wilkie GM, Jones MM, Higgins CD, Urquhart G,
Wingate P, et al. Allogeneic cytotoxic T-cell therapy for EBVpositive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial. Blood. 2007;110:
1123e31.
Leen AM, Bollard CM, Mendizabal AM, Shpall EJ,
Szabolcs P, Antin JH, et al. Multicenter study of banked thirdparty virus-specific T cells to treat severe viral infections after
hematopoietic stem cell transplantation. Blood. 2013;121:
5113e23.
Hanley PJ, Cruz CR, Savoldo B, Leen AM, Stanojevic M,
Khalil M, et al. Functionally active virus-specific T cells that
target CMV, adenovirus, and EBV can be expanded from
naive T-cell populations in cord blood and will target a range
of viral epitopes. Blood. 2009;114:1958e67.
Gerdemann U, Keirnan JM, Katari UL, Yanagisawa R,
Christin AS, Huye LE, et al. Rapidly generated multivirusspecific cytotoxic T lymphocytes for the prophylaxis and
treatment of viral infections. Mol Ther. 2012;20:1622e32.
Teschner D, Distler E, Wehler D, Frey M, Marandiuc D,
Langeveld K, et al. Depletion of naive T cells using clinical
grade magnetic CD45RA beads: a new approach for GVHD
prophylaxis. Bone Marrow Transplant. 2014;49:138e44.
Nicholson E, Ghorashian S, Stauss H. Improving TCR gene
therapy for treatment of haematological malignancies. Adv
Hematol. 2012;2012:404081.
Davila ML, Brentjens R, Wang X, Riviere I, Sadelain M. How
do CARs work? Early insights from recent clinical studies
targeting CD19. Oncoimmunology. 2012;1:1577e83.
Clay TM, Custer MC, Sachs J, Hwu P, Rosenberg SA,
Nishimura MI. Efficient transfer of a tumor antigen-reactive
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
TCR to human peripheral blood lymphocytes confers antitumor reactivity. J Immunol. 1999;163:507e13.
Schroten C, Kraaij R, Veldhoven JL, Berrevoets CA, den
Bakker MA, Ma Q, et al. T cell activation upon exposure to
patient-derived tumor tissue: a functional assay to select patients for adoptive T cell therapy. J Immunol Methods. 2010;
359:11e20.
Singh H, Manuri PR, Olivares S, Dara N, Dawson MJ,
Huls H, et al. Redirecting specificity of T-cell populations for
CD19 using the Sleeping Beauty system. Cancer Res. 2008;
68:2961e71.
Gattinoni L, Lugli E, Ji Y, Pos Z, Paulos CM, Quigley MF,
et al. A human memory T cell subset with stem cell-like
properties. Nat Med. 2011;17:1290e7.
Dudley ME, Yang JC, Sherry R, Hughes MS, Royal R,
Kammula U, et al. Adoptive cell therapy for patients with
metastatic melanoma: evaluation of intensive myeloablative
chemoradiation preparative regimens. J Clin Oncol. 2008;26:
5233e9.
Ciceri F, Bonini C, Stanghellini MT, Bondanza A,
Traversari C, Salomoni M, et al. Infusion of suicide-geneengineered donor lymphocytes after family haploidentical
haemopoietic stem-cell transplantation for leukaemia (the
TK007 trial): a non-randomised phase I-II study. Lancet
Oncol. 2009;10:489e500.
Di Stasi A, Tey SK, Dotti G, Fujita Y, Kennedy-Nasser A,
Martinez C, et al. Inducible apoptosis as a safety switch for
adoptive cell therapy. N Engl J Med. 2011;365:1673e83.
Introna M, Barbui AM, Bambacioni F, Casati C, Gaipa G,
Borleri G, et al. Genetic modification of human T cells with
CD20: a strategy to purify and lyse transduced cells with antiCD20 antibodies. Hum Gene Ther. 2000;11:611e20.
Maus MV, Beatty AR, Albelda SM, Levine B, Liu X, Zhao Y,
et al. T cells expressing chimeric antigen receptors can cause
anaphylaxis in humans. Cancer Immunol Res. 2013;1:26e31.
Newrzela S, Cornils K, Li Z, Baum C, Brugman MH,
Hartmann M, et al. Resistance of mature T cells to oncogene
transformation. Blood. 2008;112:2278e86.
Cameron BJ, Gerry AB, Dukes J, Harper JV, Kannan V,
Bianchi FC, et al. Identification of a Titin-derived HLA-A1presented peptide as a cross-reactive target for engineered
MAGE A3-directed T cells. Sci Transl Med. 2013;5:197ra103.
Morgan RA, Chinnasamy N, Abate-Daga D, Gros A,
Robbins PF, Zheng Z, et al. Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.
J Immunother. 2013;36:133e51.
Brentjens RJ, Riviere I, Park JH, Davila ML, Wang X,
Stefanski J, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with
relapsed or chemotherapy refractory B-cell leukemias. Blood.
2011;118:4817e28.
Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ,
Carrum G, et al. CD28 costimulation improves expansion
and persistence of chimeric antigen receptor-modified T cells
in lymphoma patients. J Clin Invest. 2011;121:1822e6.
Kershaw MH, Westwood JA, Parker LL, Wang G, Eshhar Z,
Mavroukakis SA, et al. A phase I study on adoptive immunotherapy using gene-modified T cells for ovarian cancer.
Clin Cancer Res. 2006;12(20 Pt 1):6106e15.
S129
54. Louis CU, Savoldo B, Dotti G, Pule M, Yvon E, Myers GD,
et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma.
Blood. 2011;118:6050e6.
55. Rezvani K, Grube M, Brenchley JM, Sconocchia G,
Fujiwara H, Price DA, et al. Functional leukemia-associated
antigen-specific memory CD8þ T cells exist in healthy individuals and in patients with chronic myelogenous leukemia
before and after stem cell transplantation. Blood. 2003;102:
2892e900.
56. Cruz CR, Micklethwaite KP, Savoldo B, Ramos CA, Lam S,
Ku S, et al. Infusion of donor-derived CD19-redirected virusspecific T cells for B-cell malignancies relapsed after allogeneic stem cell transplant: a phase 1 study. Blood. 2013;122:
2965e73.
57. Cohen JI. Benign and malignant Epstein-Barr virus-associated
B-cell lymphoproliferative diseases. Semin Hematol. 2003;40:
116e23.
58. Young LS, Murray PG. Epstein-Barr virus and oncogenesis:
from latent genes to tumours. Oncogene. 2003;22:5108e21.
59. Bollard CM, Aguilar L, Straathof KC, Gahn B, Huls MH,
Rousseau A, et al. Cytotoxic T lymphocyte therapy for
Epstein-Barr virusþ Hodgkin’s disease. J Exp Med. 2004;200:
1623e33.
60. Bollard CM, Rossig C, Calonge MJ, Huls MH, Wagner HJ,
Massague J, et al. Adapting a transforming growth factor betarelated tumor protection strategy to enhance antitumor immunity. Blood. 2002;99:3179e87.
61. Bollard CM, Gottschalk S, Leen AM, Weiss H, Straathof KC,
Carrum G, et al. Complete responses of relapsed lymphoma
following genetic modification of tumor-antigen presenting
cells and T-lymphocyte transfer. Blood. 2007;110:2838e45.
62. Dao T, Scheinberg DA. Peptide vaccines for myeloid leukaemias. Best Pract Res Clin Haematol. 2008;21:391e404.
63. Quintarelli C, Dotti G, Hasan ST, De AB, Hoyos V,
Errichiello S, et al. High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic
and leukemic-precursor cells. Blood. 2011;117:3353e62.
64. Weber G, Caruana I, Rouce RH, Barrett AJ, Gerdemann U,
Leen AM, et al. Generation of tumor antigen-specific T cell
lines from pediatric patients with acute lymphoblastic leukemia: implications for immunotherapy. Clin Cancer Res. 2013;
19:5079e91.
65. Weber G, Gerdemann U, Caruana I, Savoldo B, Hensel NF,
Rabin KR, et al. Generation of multi-leukemia antigen-specific T cells to enhance the graft-versus-leukemia effect after
allogeneic stem cell transplant. Leukemia. 2013;27:1538e47.
66. Gerdemann U, Katari U, Christin AS, Cruz CR, Tripic T,
Rousseau A, et al. Cytotoxic T lymphocytes simultaneously
targeting multiple tumor-associated antigens to treat EBV
negative lymphoma. Mol Ther. 2011;19:2258e68.
67. Shafer JA, Cruz CR, Leen AM, Ku S, Lu A, Rousseau A,
et al. Antigen-specific cytotoxic T lymphocytes can target
chemoresistant side-population tumor cells in Hodgkin lymphoma. Leuk Lymphoma. 2010;51:870e80.
68. Cruz CR, Gerdemann U, Leen AM, Shafer JA, Ku S, Tzou B,
et al. Improving T-cell therapy for relapsed EBV-negative
Hodgkin lymphoma by targeting upregulated MAGE-A4.
Clin Cancer Res. 2011;17:7058e66.

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