Abstracts

Abstracts by Category
Cell and Gene Therapy
Oral Presentation Session 1
Wednesday June 6, 11:00am – 12:15pm
Abstracts 1-2
Poster Session 2
Thursday June 7, 5:00pm – 6:00pm
Abstracts 145-151
Immunotherapy and Dendritic Cells
Oral Presentation Session 1 Wednesday June 6, 11:00am – 12:15pm Abstracts 3-5
Poster Session 1
Wednesday June 6, 5:00pm – 6:00pm
Abstracts 90-108
Translational Process Development
Oral Presentation Session 2
Wednesday, June 6th, 11:00am – 12:15pm
Abstracts 6-10
Poster Session 2
Thursday June 7, 5:00pm – 6:00pm
Abstracts 192-226
Cardiovascular Repair and Regeneration
Oral Presentation Session 3
Wednesday, June 6th, 11:00am – 12:15pm
Abstracts 11-13
Poster Session 1
Wednesday June 6th, 5:00pm – 6:00pm
Abstracts 36-45
Nervous System Repair
Oral Presentation Session 3
Wednesday, June 6th, 11:00am – 12:15pm
Abstracts 14-15
Poster Session 1
Wednesday June 6th, 5:00pm – 6:00pm
Abstracts 109-117
Basic Biology of Non-Hematapoietic Stem Cells/
Non-Hematapoietic Stem Cells Towards Clinics
Oral Presentation Session 4
Thursday, June 7th, 10:45am – 12:15pm
Abstracts 16-21
Poster Session 1
Wednesday June 6, 5:00pm – 6:00pm
Abstracts 46-89
Hematopoietic Stem Cells
Oral Presentation Session 5
Thursday, June 7th, 10:45am – 12:15pm
Abstracts 22-27
Poster Session 2
Thursday June 7, 5:00pm – 6:00pm
Abstracts 120-143
Lab Practices
Oral Presentation Session 6
Friday, June 8th, 10:45am – 11:45am
Abstracts 28-29
Poster Session 2
Thursday June 7, 5:00pm – 6:00pm
Abstracts 154-164
Legal and Regulatory Affairs
Oral Presentation Session 6
Friday, June 8th, 10:45am – 11:45am
Abstracts 30-31
Poster Session 2
Thursday June 7, 5:00pm – 6:00pm
Abstracts 165-171
Regenerative Medicine and Tissue Engineering
Oral Presentation Session 7
Friday, June 8th, 10:45am – 11:45am
Abstracts 32-35
Poster Session 2
Thursday June 7, 5:00pm – 6:00pm
Abstracts 172-191
Poster Session 1
Wednesday June 6, 5:00pm–6:00pm
Poster Session 2Thursday June 7, 5:00pm–6:00pm
Abstracts 36-117
Abstracts 120-226
AUTHOR INDEX
Primary Author Primary Author
Abstract Title
Last Name
First Name
Abdul-Alim
C. Siddiq
CHARACTERIZATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS USED AS FEEDER CELLS IN EX-VIVO CLINICAL T CELL
REPLICATIONS
Adair
Jennifer
MGMTP140K GENE-MODIFIED CD34+ CELLS ALLOW FOR INCREASED CHEMOTHERAPY ADMINISTRATION AND EXTENDED
SURVIVAL IN POOR-PROGNOSIS GLIOBLASTOMA PATIENTS
Aghdami
Nasser
Akbarzadeh
Session
196
Poster 2
1
Oral 1
CONTRIBUTON OF HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED ENDOTHELIAL CELLS IN VASCULAR REGENERATION
OF BLEOMYCIN-INDUCED SCLERODERMA MOUSE MODEL
34
Oral 7
Shiva
ENGINEERED COMPOSITE SKIN TO TREAT BURNS: A PILOT STUDY
188
Poster 2
Akel
Salem
ASSESSMENT OF LEVELS OF IGF-1, IGFBP-3 AND IGF-1/IGFBP-3 RATIOS IN UMBILICAL CORD/MATERNAL BLOOD IN RELATION
TO CELLULAR CONTENTS OF CORD BLOOD HARVESTS
137
Poster 2
Alkadi
Halah
COMPARISON BETWEEN FICOLL DENSITY GRADIENT CENTRIFUGATION AND PREPACYTE® -CB FOR NUCLEATED CELL
SEPARATION FROM UMBILICAL CORD BLOOD, AND THE EFFECT ON RECOVERY OF CD 34+ CELL SELECTION, PURITY, AND
VIABILITY.
124
Poster 2
Aqui
Nicole
COMBINATION IMMUNOTHERAPY AFTER ASCT FOR ADVANCED MYELOMA WITH MAGE-A3/POLY-ICLC IMMUNIZATION
FOLLOWED BY TRANSFER OF VACCINE-PRIMED ACTIVATED AUTOLOGOUS T CELLS
4
Oral 1
Avanzi
Mauro
OPTIMIZING MEGAKARYOCYTE POLYPLOIDIZATION INCREASES PLATELET RELEASE IN CULTURE
22
Oral 5
Bassi
Giulio
MICROPOROUS BIPHASIC CALCIUM PHOSPHATE GRANULES (MBCP+®) RETAIN IMMUNOLOGICAL PROPERTIES OF BONE
MARROW-DERIVED MESENCHYMAL STROMAL CELLS AND PROMOTE OSTEOBLASTIC DIFFERENTIATION
183
Poster 2
Berrigan
Susan
POST THAW CD34 RECOVERY AND VIABILITY OF HEMATOPOIETIC PROGENITOR CELLS IS A VALUABLE CLINICAL TOOL IN
AUTOLOGOUS BLOOD AND MARROW TRANSPLANTS.
29
Oral 6
Bifari
Francesco
STEM/PRECURSOR CELLS IN MENINGES REACT TO SPINAL CORD INJURY, MIGRATE TO THE PARENCHYMA AND CONTRIBUTE
TO GLIAL SCAR FORMATION
112
Poster 1
Birebent
Brigitte
DEALING WITH CORD BLOOD SELECTION FOR ALLOGENEIC TRANSPLANTATION: THE NUMBER OF CD34+ CELLS MATTERS
120
Poster 2
Joseph
IMPROVED IMMUNOMAGNETIC ENRICHMENT OF CD34+ CELLS FROM UMBILICAL CORD BLOOD USING THE CLINIMACS CELL
SEPARATION SYSTEM
128
Poster 2
Blaschke
Jessica
PURIFICATION AND CULTIVATION OF FUNCTIONAL HUMAN PLASMACYTOID DENDRITIC CELLS (PDC) IN A CLOSED AND
FULLY AUTOMATED SYSTEM
96
Poster 1
Bleasdale
Nicole
Blake
2
Abstract
Number
FROM REACTIVE TO PROACTIVE: THE QUALITY MANAGEMENT MATURITY MODEL
170
Poster 2
36
Poster 1
Blusztajn
Agnieszka
ENHANCED ENGRAFTMENT AND FUNCTIONAL BENEFIT OF HUMAN CARDIOSPHERE-DERIVED STEM CELLS DELIVERED IN AN
IN SITU POLYMERIZABLE HYDROGEL
Blyth
Emily
EFFECTIVE PROPHYLAXIS OF CMV DISEASE IN RECIPIENTS OF ALLOGENEIC HAEMOPOIETIC STEM CELL TRANSPLANTS USING
ADOPTIVE T CELL TRANSFER - LONG TERM FOLLOW-UP OF 50 PATIENTS
5
Oral 1
STANDARDIZED QUALITY CONTROL AND CELL EQUIVALENCY FOR XENO-FREE MULTISTEM , AN ADHERENT STEM CELL
THERAPEUTIC
35
Oral 7
IXMYELOCEL-T PROTECTS THE HEART FROM DAMAGE IN A MURINE MODEL OF HEART FAILURE
38
Poster 1
17
Oral 4
Poster 1
Bogaerts
Annelies
Booth
Erin
®
Boregowda
Siddaraju
HUMAN MESENCHYMAL STEM CELLS EXPRESS PROTEINS BELONGING TO OR RELATED TO THE IL1 FAMILY THAT CONTRIBUTE
TO THEIR ANTI-INFLAMMATORY ACTIVITY
Boregowda
Siddaraju
TWIST1 FUNCTIONS AS A PRO-SURVIVAL AND SELF-MAINTENANCE FACTOR FOR HUMAN MESENCHYMAL STEM CELLS
63
Bowersock
Joscelyn
PARTIAL T CELL DEPLETION OF MOBILIZED PERIPHERAL BLOOD AS A STRATEGY TO REDUCE INCIDENCE AND SEVERITY OF
GRAFT VERSUS HOST DISEASE
97
Poster 1
Bravery
Christopher
REGULATORY IMPLICATIONS OF ALLOGENEIC CELL BANKING STRATEGY
31
Oral 6
Brehm
Claudia
IL-2 STIMULATED BUT NOT UNSTIMULATED NK CELLS INDUCE SELECTIVE DISAPPEARANCE OF PERIPHERAL BLOOD CELLS:
CONCOMITANT RESULTS TO A PHASE I/II STUDY
91
Poster 1
Briddell
Robert
ISOLATION OF MESENCHYMAL STEM CELLS FROM UMBILICAL CORD TISSUE (TC-MSC) PRIOR TO CRYOPRESERVATION
RESULTS IN AN 8 FOLD INCREASE IN AVERAGE VIABLE TC-MSC RECOVERED WHEN COMPARED WITH THE ISOLATION AND
RECOVERY OF TC-MSC AFTER THAWING CRYOPRESERVED UMBILICAL CORD TISSUE SEGMENTS
187
Poster 2
Brindley
David
THE IMPACT OF MARKET VOLATILITY ON CELL THERAPY INDUSTRY GROWTH
167
Poster 2
99
Poster 1
Brix
Liselotte
RELIABLE ASSAY FOR MONITORING CMV-SPECIFIC T CELL IMMUNITY FOLLOWING ALLOGENEIC HEMATOPOIETIC CELL
TRANSPLANTATION
AN EX VIVO PLATFORM FOR PREDICTING RENAL SAFETY AND PHYSIOLOGIC MECHANISMS FOR NEW CHEMICAL ENTITIES
175
Poster 2
GENOMIC STABILITY OF MULTIPLE TISSUE DERIVED MSC’S DURING IN VITRO EXPANSION
207
Poster 2
Bruce
Andrew
Cakstina
Inese
Campbell
Andrew
SERUM-FREE SPHEROID SUSPENSION CULTURE OF MESENCHYMAL STEM CELLS
46
Poster 1
Campbell
Andrew
SCALABLE EXPANSION OF HUMAN MESENCHYMAL STEM CELLS USING A MICROCARRIER-BASED SYSTEM UNDER SERUMFREE AND XENO-FREE CONDITIONS
47
Poster 1
Carmen
Jessica
A MOLECULAR TEST FOR THE MEASUREMENT OF TRILINEAGE POTENTIALITY OF BONE MARROW-DERIVED HUMAN
MESENCHYMAL STROMAL CELLS
210
Poster 2
Carstens
Jason
OPTIMIZATION AND SCALE-UP OF CELL THERAPY CLINICAL MANUFACTURING PROCESSES USING CONTINUOUSLY
MONITORED AND CONTROLLED BIOREACTORS
208
Poster 2
Castegnaro
Silvia
CYTOKINE-INDUCED KILLER CELLS ARE EFFECTIVE AGAINST LOW-GRADE AND AGGRESSIVE B-CELL LYMPHOMA “IN VITRO”
104
Poster 1
Chai
Ming-Foong
HUMAN UMBILICAL CORD-DEVERIED MESENCHYMAL STROMAL CELLS RETAIN ITS PROPERTIES AFTER CRYOPRESERVATION
57
Poster 1
Chakraborty
Rikhia
LARGE-SCALE EXPANSION OF FUNCTIONAL REGULATORY T CELLS USING A GAS-PERMEABLE RAPID EXPANSION CULTURE WARE
(G-REX)
102
Poster 1
Chelluri
Lakshmi Kiran
OPTIMISATION STRATEGIES OF STEM CELL”NICHE” USING SYNTHETIC BIO-POLYMERS AND HYDROGELS FOR TISSUE
ENGINEERING
181
Poster 2
Chen
Sheng-Hsien
COMPARISON OF THERAPEUTIC EFFECTS BETWEEN HUMAN UMBILICAL CORD DERIVED MESENCHYMAL STEM CELLS AND
HUMAN UMBILICAL CORD BLOOD DERIVED HEMATOPOIETIC STEM CELLS ON EXPERIMENTAL HEATSTROKE
89
Poster 1
Cho
Seok-Goo
THERAPEUTIC POTENTIAL OF EBV-SPECIFIC CTLS IN PATIENTS WITH EXTRANODAL NK/T CELL LYMPHOMA
103
Poster 1
AUTHOR INDEX
Primary Author Primary Author
Abstract Title
Last Name
First Name
Abstract
Number
Session
Choi
Christopher
GMP IN A BOX: MANUFACTURE OF CELL THERAPY PRODUCTS USING A BARRIER ISOLATOR FOR CLINICAL INVESTIGATIONS
198
Poster 2
Chung Jr.
Francisco
UTILITY OF AUTOLOGOUS DENDRITIC CELL AS A COMPLEMENTARY TREATMENT FOR COLON CANCER: A CASE REPORT
94
Poster 1
Nathalie
CATS1 STUDY: IMMUNOMONITORING AND CLINICAL RESULTS OF ANTIGEN-SPECIFIC T REGULATORY (Treg) CELL THERAPY
FOR CROHN’S DISEASE (CD)
3
Oral 1
Culme-Seymour
Emily
ESTABLISHING THE NECESSARY DATA TO MODEL THE FUTURE RESOURCE REQUIREMENTS AND PREDICTED HEALTHCARE
TARGETS FOR CELL THERAPY AS PART OF ROUTINE CLINICAL PRACTICE
30
Oral 6
Curran
Kevin
GENETICALLY MODIFIED DONOR DERIVED EBV-SPECIFIC T CELLS FOR THE TREATMENT OF PEDIATRIC RELAPSED CD19+ ALL
POST ALLO-HSCT
148
Poster 2
Cuthbert
Richard
OPTIMISING CONCENTRATION STRATEGIES FOR PERCUTANEOUS INJECTION OF BONE MARROW DERIVED MULTIPOTENTIAL
STROMAL CELLS
86
Poster 1
Daley
Heather
NAVIGATING THE FIRST-IN-HUMAN IMPLANTABLE POLYMER-DERIVED TUMOR VACCINES INTO A CLINICAL TRIAL
8
Oral 2
Davie
Natasha
PEAK SERUM: IMPLICATIONS OF SERUM SUPPLY FOR CELL THERAPY MANUFACTURING
217
Poster 2
A NEW ROLE FOR MENINGES AS A NICHE FOR STEM/PRECURSOR CELLS WITH NEURAL DIFFERENTIATION POTENTIAL
DURING DEVELOPMENT UP TO ADULTHOOD
14
Oral 3
134
Poster 2
Poster 1
Clerget-Chossat
Decimo
Ilaria
DeJarnette
Shaun D.
ANALYSIS OF INFUSED CD34+ DOSE AND HEMATOPOIETIC RECOVERY IN PATIENTS UNDERGOING AUTOLOGOUS STEM CELL
TRANSPLANT (ASCT)
Deng
Xuewen
EX VIVO EXPANDED NATURAL KILLER CELLS CAN POSSIBLY KILL CANCER STEM CELLS
108
Deskins
Desirae
HUMAN MESENCHYMAL STEM CELLS: IDENTIFYING AASSAYS TO PREDICT POTENCY FOR THERAPEUTIC SELECTION
203
Poster 2
Desmarais
Cindy
TRACKING T CELL CLONES USING HIGH-THROUGHPUT SEQUENCING OF ANTIGEN RECEPTOR CDR3 CHAINS
90
Poster 1
Deutsch
Varda
HEMATOPOIETIC STEM CELLS AND MEGAKARYOCYTE PROGENITORS AND THEIR SUBSETS DIFFER IN NORMAL AND
MEYELOPROLIFERATIVE DISEASE STATES
23
Oral 5
DiGiusto
David
DEVELOPMENT OF QUANTITATIVE MODEL OF ENGRAFTMENT OF NOD-SCID IL2RYNULL MICE
123
Poster 2
Dornsife
Ronna
CLINICAL CORD BLOOD UNITS WASHED WITH AUTOMATED SEPAX® CELL PROCESSING SYSTEM PROVIDES HIGH QUALITY
POST-THAW RECOVERIES AND VIABILITY FROM CRYOPRESERVED UNITS
221
Poster 2
Dyson
Pamela
EVENT REPORTING AS A TOOL FOR ASSESSING STAFF ACCEPTANCE AND COMPLIANCE WITH REGULATORY REQUIREMENTS
FOR THE MANUFACTURE OF CELLULAR THERAPIES.
162
Poster 2
Eaker
Shannon
CELL CONCENTRATION AND WASHING USING HOLLOW FIBER FILTRATION AND READYCIRCUIT ASSEMBLIES FOR CELL
THERAPY APPLICATIONS
205
Poster 2
Egloff
Matthieu
SCALING-UP ADHERENT STEM CELL CULTURES: IMPORTANCE OF PHYSIOCHEMICAL ENVIRONMENT CONTROL BY MULTIPLATE
BIOREACTOR
214
Poster 2
Egloff
Matthieu
THE IMPORTANCE OF DIGITAL HOLOGRAPHIC MICROSCOPY FOR AUTOMATED, REAL-TIME MONITORING OF HUMAN ADULT
STEM CELL CONFLUENCE IN LARGE-SCALE CULTURES.
215
Poster 2
Fajardo-Orduña
Guadalupe
MESENCHYMAL STROMAL CELLS FROM UMBILICAL CORD BLOOD AND PLACENTA SUPPORT PROLIFERATION AND
EXPANSION OF HEMATOPOIETIC PROGENITOR CELLS.
50
Poster 1
ROBUST EX VIVO EXPANSION OF CD34+ CELLS FROM CORD BLOOD, BONE MARROW, AND MOBILIZED PERIPHERAL BLOOD
USING NANEX NANOFIBER SCAFFOLD
142
Poster 2
Poster 2
Fischer
Stephen
Fisk
Mary Beth
COMPARISON OF UMBILICAL CORD BLOOD POST-PROCESSING VERSUS POST-THAW VIABILITY
132
Fisk
Mary Beth
CORD BLOOD COLLECTIONS: OPERATIONAL METHODS
133
Poster 2
Fitzgerald
Erica
EVALUATION OF GRANULOCYTES IN ASSESSMENT OF HPC,A QUALITY
159
Poster 2
Poster 1
Flores
Catherine
THE USE OF HUMAN ALDHBRIGHT CELLS IN ATTENUATING INTRACRANIAL INFLAMMATION DUE TO BRAIN IRRADIATION
110
Fogarty
Janice
MESENCHYMAL STROMAL CELL THERAPY FOR REFRACTORY CROHN’S DISEASE
20
Oral 4
Foussat
Arnaud
DEVELOPMENT OF A NEW DEVICE FOR POINT-OF-CARE THAWING AND DOSING OF CELL THERAPY PRODUCTS
194
Poster 2
Gadelorge
Mélanie
PERFORMANCE STUDY OF HUMAN PLATELET LYSATE PREPARATION KIT FOR MESENCHYMAL STEM/STROMAL CELLS
EXPANSION
85
Poster 1
COMMON EXPRESSION OF STEMNESS MOLECULAR MARKERS AND EARLY CARDIAC TRANSCRIPTION FACTORS IN WHARTON’S
JELLY-DERIVED STEM CELLS AND HESCS
54
Poster 1
Gao
Lian
Gouveia de
Andrade
Ana Valeria
IN VITRO IMMUNOSUPPRESSIVE POTENTIAL OF GAMMA-IRRADIATED MESENCHYMAL STROMAL CELLS
75
Poster 1
Gouveia de
Andrade
Ana Valeria
STANDARDIZATION OF IMUNNOSUPPRESSIVE POTENCY TESTS FOR MESENCHYMAL STROMAL CELL - BASED THERAPIES
193
Poster 2
Guest
James
COMPARISON OF HUMAN SCHWANN CELL PROLIFERATION RATES IN CULTURE FROM ORGAN DONOR AND CADAVERIC
NERVES
15
Oral 3
Guest
James
PREPARATION OF HUMAN SCHWANN CELLS FOR TRANSPLANTATION. HUMAN SERUM ALBUMIN IN FINAL WASH STEPS
ENHANCES CELL VIABILITY.
113
Poster 1
Gupta
Pawan
A PHASE I/II STUDY ASSESSING THE SAFETY AND EFFICACY OF INTRAVENOUS EX VIVO CULTURED ADULT ALLOGENEIC
MESENCHYMAL STEM CELLS IN PATIENTS WITH ST ELEVATED ACUTE MYOCARDIAL INFARCTION (STEMI)
12
Oral 3
Haque
Khawaja
TRANSPLANTING AUTOLOGOUS PERIPHERAL HEMATOPOIETIC PROGENITOR CELLS STORED AT 40C AND NOT
CRYOPRESERVED
129
Poster 2
Haylock
David
BIOREACTOR CULTURE OF CD34+ CELLS FOR PLATELET PRODUCTION; NEW SCAFFOLDS TO SUPPORT MEGAKARYOCYTE
DEVELOPMENT
179
Poster 2
Helfer
Brooke
MONITORING THERAPEUTIC INTERVENTION THROUGH INFLAMMATORY RESPONSE WITH CLINICALLY TRANSLATIONAL 19F
MRI
37
Poster 1
Helfer
Brooke
HEMATOPOIETIC STEM CELL CHARACTERIZATION WITH 19F TRACER AGENT; THE ABILITY TO EVALUATE CELLULAR
PERSISTENCE
195
Poster 2
Henderson
Christianna
UNREPORTED CORD BLOOD (CB) CD34DIM/+ CELL SUB-POPULATIONS AND THEIR POST-PROCESSING RECOVERY
122
Poster 2
3
AUTHOR INDEX
Primary Author Primary Author
Abstract Title
Last Name
First Name
Session
Hennerbichler
Simone
INFLUENCE OF HYDROXYETHYL-STARCH (HES) ON CELL RECOVERY IN CORD BLOOD PROCESSING
139
Poster 2
Henshaw
Mariluz
DEVELOPMENT OF A CLOSED FILTRATION SYSTEM FOR BATCH PROCESSING OF POOLED PLATELET LYSATE
213
Poster 2
April
PROVINCE WIDE ELECTRONIC NOTIFICATION OF TRANSFUSION REQUIREMENTS FOR ALLOGENEIC TRANSPLANT PATIENTS
ENSURES SAFE TRANSFUSION PRACTICES
154
Poster 2
Hillman
April
POST-THAW ASSESSMENT OF CD34+ CELL RECOVERY AND VIABILITY IN ALLOGENEIC CELLULAR THERAPY PRODUCTS
SUGGESTS FRESH IS BETTER THAN FROZEN
155
Poster 2
Ho
Jennifer
SYSTEMIC HUMAN ORBITAL FAT-DERIVED STEM CELL TRANSPLANTATION AMELIORATES ACUTE INFLAMMATION IN
LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY
32
Oral 7
Homer
Mary
OPPORTUNITIES FOR ADVANCED PRODUCT DEVELOPMENT AT THE BIOMEDICAL ADVANCED RESEARCH AND DEVELOPMENT
AUTHORITY
168
Poster 2
REGULATING HUMAN MESENCHYMAL STEM CELLS FOR OSTEOGENIC TISSUE REPAIR BY p63 AND p38 ISOFORMS
18
Oral 4
COMBINATION CELLULAR AND IL-2 THERAPY IMPROVES SURVIVAL OF OVARIAN CANCER BEARING MICE
105
Poster 1
STABILITY OF THAWED HEMATOPOIETIC PROGENITOR CELLS (HPC) IN DIMETHYL SULFOXIDE (DMSO) IN FILTERED AND
UNFILTERED PRODUCTS
28
Oral 6
Hillman
Howard
Guy
Ingersoll
Susan
Irani
Mehraboon
Iudicone
Paola
PATHOGEN-FREE PLATELET LYSATE FOR THE EXPANSION OF BONE MARROW DERIVED MESENCHYMAL STROMAL CELL
60
Poster 1
Zoran
FUNCTIONAL STABILITY OF HEMATOPOIETIC STEM AND PROGENITOR CELLS IN EX VIVO EXPANSION PRODUCT OF CORD
BLOOD CD34+ CELLS AT +4ºC.
226
Poster 2
USE OF THE BIOSAFE SEPAX TO WASH AND CONSOLIDATE CRYOPRESERVED HEMATOPOIETIC PROGENITOR CELL,
APHERESIS PRODUCTS FREE OF DMSO FOR INFUSION
Ivanovic
®
Jacobson
Pam
136
Poster 2
Jain
Deepak
A NEO-KIDNEY AUGMENT PRODUCT FOR KIDNEY REGENERATION IN A LARGE ANIMAL MODEL OF CHRONIC KIDNEY DISEASE
33
Oral 7
Jiang
Yajuan
EXPLORING THE USE OF HUMAN VERY SMALL EMBRYONIC-LIKE STEM CELLS ISOLATED FROM ADULT PERIPHERAL BLOOD
FOR THERAPY OF DRY AGE-RELATED MACULAR DEGENERATION
117
Poster 1
Jing
Donghui
GROWTH KINETICS OF HUMAN MESENCHYMAL STEM CELLS IN THE CELLREADY SINGLE USE BIOREACTOR
79
Poster 1
Kaur
Jasmeet
DEFINED XENO-FREE MEDIA GROWTH SUPPLEMENT FOR CULTIVATION OF PLURIPOTENT STEM CELLS
82
Poster 1
Kehoe
Daniel
INVESTIGATION OF THE EFFECTS OF SHEAR FORCES ON HUMAN MESENCHYMAL STEM CELLS
76
Poster 1
Khan
Aisha
IMPROVED HUMAN ISLET ISOLATION OUTCOMES USING A MAMMALIAN TISSUE-FREE ENZYME BLEND
173
Poster 2
LIPOXYGENASE INHIBITOR ENHANCES THERAPEUTIC EFFECT OF THE TREATMENT OF TRAIL-SECRETING MESENCHYMAL
STEM CELLS IN GLIOMA
151
Poster 2
CONCENTRATIONS OF SELECTED CYTOKINES IN PLASMA AND BONE MARROW OF PATIENTS WITH HEMATOLOGICAL
MALIGNANCIES
131
Poster 2
163
Poster 2
Kim
Seong Muk
Klabusay
Martin
Knight
Rebekah
USE OF UNRELATED DONOR COLLECTION CENTER CD34+ COUNTS DECREASES TIME TO PRODUCT INFUSION WITHOUT
COMPROMISING PATIENT OUTCOMES
Koch
Carmen
TRACKING OF REPLICATIVE SENESCENCE BY EPIGENETIC MODIFICATIONS AT SPECIFIC CGP SITES
200
Poster 2
Kolrep
Ulrike
NEW GMP-GRADE, ANIMAL-COMPONENT-FREE MEDIUM FOR ACTIVATION AND EXPANSION OF T-CELLS
107
Poster 1
Kopyov
Oleg V
HUMAN FETAL-DERIVED STEM CELLS CAN DECELERATE MOTOR DETERIORATION AND WEIGHT LOSS IN A RAT MODEL OF
CEREBELLAR ATAXIA
111
Poster 1
Kreissig
Carla
USE OF PLATELET LYSATE FROM OUTDATED PLATELET CONCENTRATES IN MESENCHYMAL STROMAL CELL CUTLURE
177
Poster 2
Kreissig
Carla
USE OF PLATELET LYSATE IN MESENCHYMAL STROMAL CELL CULTURE – CAN WE EXPECT A STANDARDISED CELL CULTURE
SUPPLEMENT?
178
Poster 2
Kreke
Michelle
PROCESS SCALE-UP AND TRANSITION FROM AUTOLOGOUS TO ALLOGENEIC MANUFACTURING OF CARDIOSPHERE-DERIVED
STEM CELLS
6
Oral 2
Kreke
Michelle
SAFETY AND EFFICACY OF INTRACORONARY INFUSION OF ALLOGENEIC CARDIOSPHERE-DERIVED STEM CELLS IN A PIG
MODEL OF MYOCARDIAL INFARCTION
11
Oral 3
Krugh
Dave
SUPPLIER AND SUPPLY QUALIFICATION – SINGLE CENTER EXPERIENCE WITH ISLET ISOLATION SUPPLIES
157
Poster 2
Kuci
Selim
GENE EXPRESSION PROFILE OF MESENCHYMAL STROMAL CELLS DERIVED FROM HUMAN BONE MARROW CD271+
MONONUCLEAR CELLS
62
Poster 1
Kuci
Zyrafete
FUNCTIONAL HETEROGENEITY OF MESENCHYMAL STROMAL CELLS DERIVED FROM HUMAN BONE MARROW CD271+
MONONUCLEAR CELLS AT THE CLONAL LEVEL.
67
Poster 1
Kumar
Vijay
QUALITATIVE AND QUANTITATIVE ANALYSIS OF BONE MARROW CONCENTRATE (BMC) PRODUCED USING RES-Q™ 60 BMC- A
POINT OF CARE MEDICAL DEVICE
143
Poster 2
Lee
Oscar
AMINE-SURFACE-MODIFIED SUPERPARAMAGNETIC IRON OXIDE NANOPARTICLES INTERFERE WITH DIFFERENTIATION OF
HUMAN MESENCHYMAL STEM CELLS
59
Poster 1
Levine
Bruce
A SINGLE INFUSION OF ZINC FINGER NUCLEASE (ZFN) CCR5 MODIFIED AUTOLOGOUS CD4 T-CELLS (SB-728-T) CORRELATES
WITH INCREASES IN CD4 COUNTS AND EFFECTS ON VIRAL LOAD (VL) IN AVIREMIC HIV-INFECTED SUBJECTS
2
Oral 1
Loudovaris
Maureen
BIOASSAYS FOR THE TESTING OF DC MANUFATURED FOR USE IN HUMAN CLINICAL TRIALS
216
Poster 2
Ludwig
Emily
IMPLICATIONS OF LOCAL WOUND ENVIRONMENT FOR CELL-BASED THERAPY
182
Poster 2
Lysak
Daniel
IMMUNOMODULATORY EFFECTS OF MESENCHYMAL STEM CELLS ON ALLOGENEIC LYMPHOCYTES
55
Poster 1
Macpherson
Janet
BUILDING A GOOD MANUFACTURING PRACTICE (GMP) FACILITY WITHIN THE PUBLIC HEALTHCARE SYSTEM
164
Poster 2
Macpherson
Janet
CURRENT STATUS OF CELLULAR THERAPY IN THE ASIA-PACIFIC REGION
165
Poster 2
Leah
CXCR4 TRANSFECTION OF MESENCHYMAL STROMAL CELLS USING CATIONIC LIPOSOME ENHANCES THEIR MIGRATION TOWARDS
SDF-1
83
Poster 1
21
Oral 4
Marquez-Curtis
4
Abstract
Number
McNiece
Ian
McPherson
Gaytha
MOBILIZATION OF MESENCHYMAL STEM CELLS FROM THE BONE MARROW TO PERIPHERAL BLOOD
HUMAN BLOOD-DERIVED RAW MATERIAL: ENABLING CONTROLLED, CONSISTENT COLLECTION
160
Poster 2
Messino
Nancy
QUALITY CULTURE AND TOOLS: SYNERGY FOR COMPLIANCE AND ACCREDITATION
169
Poster 2
AUTHOR INDEX
Primary Author Primary Author
Abstract Title
Last Name
First Name
Minullina
Izida
CHARACTERISATION AND PROPERTIES OF BONE MARROW MESENCHYMAL CELLS FROM PATIENTS WITH CHRONIC HEART
FAILURE
Abstract
Number
Session
40
Poster 1
Mohan
Sunil
CAN INDUCED PLURIPOTENT STEMCELLS CELLS REPLACE DENTAL STEM CELL BANKING
65
Poster 1
Montemurro
Tiziana
OFF-THE-SHELF CORD BLOOD, ADIPOSE AND BONE MARROW MESENCHYMAL STEM CELL BANK
189
Poster 2
Moviglia
Gustavo
IN VITRO RETINOBLAST DIFFERENTIATION FROM FAT MSC
81
Poster 1
Mura
Manuela
IRES-BASED LENTIVIRUS CO-EXPRESSING TGFb1 AND FGF2 IMPROVES CELL SURVIVAL AND ANGIOGENESIS IN BONE MARROW
DERIVED-MESENCHYMAL STEM CELLS
13
Oral 3
Mura
Manuela
CONCOMITANT OVEREXPRESSION OF IGF1 AND BMP2 IN MESENCHYMAL STEM CELLS MEDIATES CYTOPROTECTION
THROUGH BOTH AUTOCRINE AND PARACRINE ACTIVATION OF AKT, ERK1/2 AND SMAD1/5/8 PATHWAYS.
149
Poster 2
Murrell
Julie
IDENTITY AND PURITY CHARACTERIZATION OF MESENCHYMAL STEM CELLS EXPANDED IN A 3L STIRRED TANK BIOREACTOR
53
Poster 1
Musick
James
GMP CELL CULTURE MEDIA FOR EXPANSION OF MSCS PRIOR TO ALLOGENEIC OR AUTOLOGOUS TRANSPLANTATION
51
Poster 1
Newman
Robert
ISOLATION AND EXPANSION OF HUMAN BONE MARROW-DERIVED MESENCHYMAL STEM CELLS AND HUMAN ADIPOSEDERIVED STEM CELLS IN A SERUM-FREE MEDIUM
87
Poster 1
Nguyen
Kim
CULTURE OF NORMAL HUMAN DERMAL FIBROBLAST CELLS IN A FUNCTIONALLY CLOSED AUTOMATED CELL EXPANSION
SYSTEM
211
Poster 2
Niam
Madelaine
COMPARISON STUDY OF USING CULTURE BAGS AND G REX FLASKS TO GROW CIK CELLS
212
Poster 2
Ian
VALIDATION AND IMPLEMENTATION OF NON-CRYOPRESERVED TRANSPORT OF EX VIVO EXPANDED CORD BLOOD
PROGENITORS FOR CLINICAL APPLICATION
125
Poster 2
Nieda
Mie
CD56+HUMAN DENDRITIC CELLS PULSED WITH TUMOR ANTIGEN AND/OR ZOLEDRONATE EFFECTIVELY PROMOTE THE
EXPANSION OF TUMOR ANTIGEN-SPECIFIC CD56+CD8+T CELLS AND /OR CD56+GAMMA DELTA T CELLS
101
Poster 1
Niedzinski
Jerry
IMPORTANCE OF GATING OUT DEBRIS WHEN PHENOTYPING COMPLEX STEM CELL PRODUCTS BY FLOW CYTOMETRY
209
Poster 2
Nilsson
Susie
PROSPECTIVELY ISOLATED SCAVENGING BONE-MARROW SINUSOIDAL ENDOTHELIAL CELLS HOME TO, AND REVASCULARIZE
THE BONE MARROW OF TRANSPLANTED ABLATED RECIPIENTS.
Nordon
Robert
DEVELOPMENT OF MICROFLUIDIC BIOREACTORS FOR CELL EXPANSION
220
Poster 2
O’Brien
Vincent
A BROADLY-APPLICABLE MICRORNA-BASED MONITORING TOOL FOR STEM CELL QUALITY CONTROL AND DIFFERENTIATION
MONITORING
224
Poster 2
Orellana, Sr.
Maristela
Delgado
58
Poster 1
Nicoud
MEGAKARYOCYTES GENERATION FROM HUMAN EMBRYONIC STEM CELLS
16
Oral 4
Osadolor
Isaac
POLICIES REGARDING HUMAN GENETIC MODIFICATION TECHNOLOGIES IN MEXICO
171
Poster 2
Osadolor
Isaac
EFFECTS OF SILDENAFIL ON TESTICULAR TORSION/DETORSION(TD) DAMAGE: AN EXPERIMENTAL STUDY IN A RAT MODEL
172
Poster 2
Otsuru
Satoru
NOVEL METHOD TO ISOLATE MESENCHYMAL STROMAL CELLS FROM BONE MARROW
88
Poster 1
127
Poster 2
Oyer
Jeremiah
IMMUNE CELLULAR RECONSTITUTION AND DONOR CELL CHIMERISM AFTER MYELOABLATIVE AND REDUCED INTENSITY
CONDITIONING WITH IV BUSULFAN AND ALLOGENEIC STEM CELL TRANSPLANTATION (SCT)
Pacelli
Luciano
QUALITY CONTROLS OF IMMUNE REGULATORY PROPERTIES OF EX-VIVO, GMP-GRADE EXPANDED MESENCHYMAL STROMAL
CELLS FOR CLINICAL USE (EUROPEAN MULTICENTER STUDY CASCADE)
56
Poster 1
Padley
Douglas
DEVELOPMENT OF A CELLULAR THERAPY LABORATORY MANUFACTURING PLATFORM FOR ADIPOSE DERIVED MESENCHYMAL
STEM CELLS
204
Poster 2
Page
Kristin
Paramban
Rosanto
Petchdee
Soontaree
Plöderl
Karin
WHAT CLINICAL CHARACTERISTICS OF AFRICAN AMERICAN MOTHERS AND BABIES FAVORABLY INFLUENCE CORD BLOOD
POTENCY? DEFINING PARAMETERS THAT CAN GUIDE PUBLIC CORD BLOOD COLLECTION?
27
Oral 5
TOOLS AND METHODS FOR HIGH-THROUGHPUT UNBIASED CELL SURFACE MARKER SCREENING OF STEM CELLS AND THEIR
PROGENY BY FACS
222
Poster 2
INTRAVENOUS ADMINISTRATION OF MESENCHYMAL STEM CELLS EXERTS THERAPEUTIC EFFECTS ON INFARCTED HEART
MODEL OF RABBIT: FOCUSING ON POTENTIAL EFFECTS OF MYOCARDIAL REGENERATION
45
Poster 1
STANDARDIZED PROCESSING OF PLATELET LYSATE FOR USE IN CELL CULTURE
185
Poster 2
116
Poster 1
Ponemone
Venkatesh
INTRATHECAL ADMINISTRATION OF AUTOLOGOUS BONE MARROW CELLS WITH 10% HEMATOCRIT / RBCS ARE CLINICALLY
SAFE
Ponemone
Venkatesh
AUTOLOGOUS BONE MARROW DERIVED STEM CELL GRAFT FACILITATES REMODELING OF NON UNION FRACTURES
191
Poster 2
Prata
Karen
EXPANSION OF MULTIPOTENT MESENCHYMAL STROMAL CELLS FROM FETAL ADNEXA IN XENOFREE CONDITIONS
73
Poster 1
Prata
Karen
ACUTE ADVERSE REACTIONS SECONDARY TO MESENCHYMAL STROMAL CELL INFUSIONS ?
74
Poster 1
INFLAMMATION AND TLR LIGATION DIFFERENTIALLY AFFECT THE OSTEOGENIC POTENTIAL OF HUMAN MESENCHYMAL
STROMAL CELLS (MSC) DEPENDING ON THEIR TISSUE ORIGIN
49
Poster 1
Raicevic
Gordana
Ranjbarvaziri
Sara
MESENCHYMAL STEM CELLS TRACKING USING QUANTUM DOTS IN AN ANIMAL MODEL OF SPINAL CORD INJURY
223
Poster 2
19
Oral 4
Roelofs
Helene
MSC FUNCTIONALITY AND ROBUSTNESS OF THE MSC EXPANSION PROCESS ARE CRITICALLY DEPENDENT ON TISSUE
SOURCE AND EXPANSION CONDITIONS
Samuel
Edward
T REGULATORY CELLS ENRICHED FROM G-CSF MOBILISED APHERESATES DO NOT SURVIVE SHORT TERM CULTURE -; IMPACT
ON ADOPTIVE IMMUNOTHERAPY
93
Poster 1
Saxena
Deepa
SYNTHETIC SURFACE FOR CULTURE OF HUMAN KERATINOCYTES IN DEFINED XENO-FREE MEDIUM
180
Poster 2
Schulz
Thomas
SCALABLE SUSPENSION-BASED DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO PANCREATIC PROGENITORS
66
Poster 1
Semple
Elisabeth
DETECTION OF BACTERIAL CONTAMINATION IN CORD BLOOD: A MATTER OF SAMPLING VOLUME.
158
Poster 2
138
Poster 2
Sharma
Shanta
WONDER FILE: AUTOMATIC CALCULATION OF TOTAL BLOOD VOLUME, CIRCULATING CD34+/KG, COLLECTION EFFICIENCY AND
MORE
Shellooe
Christopher
CHARACTERIZATION OF T-CELL GROWTH IN STATIC VS. AGITATED AND FED-BATCH VS. PERFUSION CULTURE CONDITIONS
199
Poster 2
Daniel Tzu-bi
ISOLATION AND CHARACTERIZATION OF CLONOGENIC MULTIPOTENT STROMAL STEM CELLS (GSSCs) FROM GINGIVA TISSUE
FOR REGENERATIVE MEDICINE
77
Poster 1
Shih
5
z
AUTHOR INDEX
Primary Author Primary Author
Abstract Title
Last Name
First Name
Shoulars
ALDHbr content of segments from banked cord bloods predicts engraftment after cord blood
transplantation.
24
Poster
Session
Oral 5
Singh
A.P.
CURCUMIN HELPS IN TREATMENT OF NEURODEGENERATIVE DISORDERS
109
Poster 1
Slobodianski
Alex
THERAPEUTIC ANGIOGENESIS - PRE-CLINICAL AND MANUFACTURING ASPECTS
192
Poster 2
Smilee
Renee
HARVESTING LARGE VOLUMES OF TUMOR INFILTRATING LYMPHOCYTES (TIL) USING A CLOSED SYSTEM, COMMERCIALLY
AVAILABLE, DEVICE
219
Poster 2
A FULLY AUTOMATED CELL PROCESSING SYSTEM FOR VARIABLE APPLICATIONS
126
Poster 2
SYNAPSE-DIRECTED DELIVERY OF IMMUNE-STIMULANTS USING T-CELL-CONJUGATED NANOPARTICLES
100
Poster 1
OPTIMIZED MANUFACTURE OF CTLS WITH ANTI-VIRUS AND ANTI-TUMOR SPECIFICITY FOR NEUROBLASTOMA PATIENTS
POST STEM CELL TRANSPLANTATION
147
Poster 2
Spiegel
Iris
Stephan
Matthias
Sun
Jiali
Teoh
Hoon Koon
SUPPRESSION OF INTERLEUKIN-6 IN HUMAN MESENCHYMAL STEM CELLS BY ADENOVIRUS-BASED SHORT HAIRPIN RNA
TRANSDUCTION
145
Poster 2
Then
Kong-Yong
IMPROVING CORD BLOOD PROCESSING METHODS: COMPARISON BETWEEN MANUAL PROCESSING AND AUTOMATED
PROCESSING
130
Poster 2
Thirumala
Sreedhar
A NOVEL CRYOPRESERVATION SYSTEM SETTING AND METHOD FOR FREEZING HPC-CORD BLOOD
141
Poster 2
Ting
Anthony
DEVELOPMENT OF AN ANGIOGENIC POTENCY ASSAY FOR CLINICAL GRADE STEM CELL PRODUCTION
9
Oral 2
RESULTS OF RECONSTRUCTIVE SURGERY IN 6 CHILDREN WITH CLEFT PALATE WITH THE COADJUVANT USE OF AUTOLOGOUS
UMBILICAL CORD BLOOD.
Trigo
Guillermo
80
Poster 1
Valle
Ileana
WHOLE HEARTS USED TO MANUFACTURE ALLOGENEIC CARDIOSPHERE-DERIVED STEM CELLS AT A LARGE-SCALE
39
Poster 1
Vanecek
Vaclav
USE OF A BONE SCAFFOLD COMBINED WITH MESENCHYMAL STEM CELLS FOR THE TREATMENT OF VERTEBRAL BODY
DEFECTS
190
Poster 2
Vanguri
Padmavathy
QUANTITATIVE BIOASSAYS TO CHARACTERIZE MESENCHYMAL STEM CELLS DURING PROCESS DEVELOPMENT AND
MANUFACTURE
218
Poster 2
Varadaraju
Hemanthram
DEVELOPING LARGE SCALE CELL MANUFACTURING PROCESSES: THE IMPACT ON CELL QUALITY ATTRIBUTES AND COST OF
GOODS
10
Oral 2
Varettas
Kerry
VALIDATION OF BACT/ALERT FAN BOTTLES USED FOR CONTAMINATION TESTING OF HAEMATOPOIETIC PROGENITOR CELLS
161
Poster 2
Villa
Carlos
ADDITION OF PLERIXAFOR TO MOBILIZATION REGIMENS IN AUTOLOGOUS PERIPHERAL BLOOD STEM CELL TRANSPLANTS
DOES NOT AFFECT THE CORRELATION OF PRE-HARVEST HEMATOPOIETIC PRECURSOR CELL (HPC) ENUMERATION WITH
FIRST HARVEST CD34+ STEM CELL YIELD
121
Poster 2
Voorhies
Howard
CONVERSION OF STATIC FLASK EX VIVO EXPANSION METHODS TO A ROTATIONAL CULTURE METHODOLOGY RESULTS IN
SIGNIFICANT COST REDUCTION AND IMPROVED CLINICAL FEASIBILITY
206
Poster 2
Wagner
Beate
LUMINOMETRIC DETERMINATION OF PROGENITOR CELL FUNCTION IN CRYOPRESERVED PERIPHERAL BLOOD STEM CELL
HARVESTS
26
Oral 5
Wall
Donna
VACUUM FAILURE WITH IMPLOSION: A RARE BUT POTENTIALLY CATASTROPHIC RISK FOR AGING OR DAMAGED LIQUID
NITROGEN FREEZERS
156
Poster 2
Weber
Daniel
SUCCESSFUL TRANSITION FROM ISOLEX TO CLINIMACS DURING A PHASE I CLINICAL TRIAL
202
Poster 2
Weiss
Mark
IDENTIFICATION OF OPTIMAL CONDITIONS FOR GENERATING MSCS FOR PRECLINICAL TESTING: COMPARISON OF THREE
COMMERCIAL SERUM-FREE MEDIA AND LOW-SERUM GROWTH MEDIUM.
84
Poster 1
Wilson
David
THE CRYO-IMAGING SOUTION TO “WHERE DID MY CELLS GO”
7
Oral 2
Wilson
Kyle
NK DEPLETION ENHANCES MELANOMA REJECTION BY NAIVE, TUMOR-SPECIFIC CD4+ T CELLS
98
Poster 1
Wiwi
Chris
DEVELOPMENT TOWARDS THE VALIDATION OF A MULTICOLOR FLOW CYTOMETRY ASSAY FOR CELLULAR PRODUCT RELEASE
197
Poster 2
Wong
Chee-Yin
IN VITRO DIFFERENTIATION OF MESENCHYMAL STROMAL CELLS INTO MESANGIAL CELLS AFTER CO-CULTURE WITH INJURED
MESANGIAL CELLS
71
Poster 1
Wong
Chee-Yin
INTRADISCAL DELIVERY OF AUTOLOGOUS BONE MARROW-DEVIRED MESENCHYMAL STROMAL CELLS: A FEASIBLE AND SAFE
APPROACH OF CELL THERAPY IN TWO PATIENTS WITH DEGENERATIVE DISEASE
72
Poster 1
Wright
Craig
MANAGING A TGA GMP LICENCE FOR HPCS WITHIN NSW PUBLIC HEALTH
166
Poster 2
Meiting
DEVELOPING A CELL-BASED THERAPY FOR PREVENTING AND/OR REMOVING ECTOPIC CALCIFICATION IN CALCIFIC AORTIC
VALVE DISEASE (CAVD)
43
Poster 1
Wu
Dongyun
EFFICIENT INDUCED HBV SPECIFIC CTL BY HFP3 MEDIATED CORE ANTIGEN LOADING OF HUMAN DENDRITIC CELLS IN
CHRONIC HEPATITIS B PATIENTS
92
Poster 1
Ye
Lei
TROGLITAZONE UP-REGULATES PTEN EXPRESSION AND INDUCES APOPTOSIS OF PULMONARY ARTERY SMOOTH MUSCLE
CELLS UNDER HYPOXIA
41
Poster 1
Ye
Lei
ROLE OF THYMOSIN BETA4 ON SKELETAL MYOBLAST MIGRATION, PROLIFERATION, AND SURVIVAL
186
Poster 2
IMPROVED EX VIVO EXPANSION OF HEMATOPOIETIC STEM AND PROGENITOR CELLS FROM CORD BLOOD IN A NOVEL
SERUM- AND ANIMAL-COMPONENT-FREE MEDIUM
135
Poster 2
OKT3 AND CD3 PURE ANTIBODIES ARE BIOEQUIVALENT FOR THE ‘EX-VIVO’ GENERATION OF CYTOKINE-INDUCED KILLER
CELLS (CIK) FOR CLINICAL USE
201
Poster 2
ISOLATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELL (HUVEC), CD117pos MATTERS OVER ENDOTHELIAL CELLS
COUNT.
61
Poster 1
Wu
6
Kevin
Abstract
Number
Yuan
Ning
Zanon
Cristina
Zevallos
Remy
Zhang
Hong-Mei
TREGS DEPLETION WITH IL-21 ADMINISTRATION TO HCC CELL TOTAL RNA-TRANSFECTED DCS TO BOOST STRONG SPECIFIC
IMMUNE RESPONSES AGAINST HCC
95
Poster 1
Zuliani
Thomas
VALUE OF LARGE-SCALE EXPANSION OF TUMOR INFILTRATING LYMPHOCYTES IN A COMPARTMENTALISED GAS-PERMEABLE
BAG: INTERESTS FOR ADOPTIVE IMMUNOTHERAPY
106
Poster 1
Zylberberg
Claudia
NANOLITER DROPLET VITRIFICATION FOR BLOOD AND STEM CELL CRYOPRESERVATION
25
Oral 5
z
Oral ABSTRACTS
1
MGMTP140K GENE-MODIFIED CD34+ CELLS ALLOW FOR
INCREASED CHEMOTHERAPY ADMINISTRATION AND EXTENDED
SURVIVAL IN POOR-PROGNOSIS GLIOBLASTOMA PATIENTS
Jennifer E. Adair, Ph.D.1 Brian C. Beard, Ph.D.1,2 Grant D. Trobridge, Ph.D.3 Maciej M.
Mrugala, M.D., Ph.D.2 Hans-Peter Kiem, M.D.1,2 1Fred Hutchinson Cancer Research Center,
Seattle, USA, 2University of Washington, Seattle, USA, 3Washington State University, Pullman,
USA.
Drug-resistant, MGMTP140K gene-modified hematopoietic CD34+ cells
(HPCs) could circumvent myelosuppression associated with alkylating agent
chemotherapy. We are conducting a study using a nonmyeloablative dose of BCNU
(600mg/m2) to facilitate engraftment of MGMTP140K gene-modified cells followed
by post-transplant combination chemotherapy with O6-benzylguanine (O6BG)
and temozolomide (TMZ). We have achieved successful, long-term engraftment
of MGMTP140K gene-modified HPCs in 3 glioblastoma patients displaying
unmethylated MGMT promoter in tumor cells, indicative of poor response to TMZ
chemotherapy and reduced survival. Patients received, 9, 3 and 4 cycles of O6BG/
TMZ chemotherapy, respectively. We observed transient increases in circulating
gene-modified white blood cells following each cycle. Analysis of peripheral
blood CD34+ colony forming cells (CFCs) revealed increases in gene-modified
CFCs over time and with multiple cycles of chemotherapy, (Range 27% to 85%).
Moreover, we observed sustained gene marking at >18 months after transplant.
Overall survival is currently 23.7 ± 6 months (Range 18-30 months). Patient 1, who
received 9 cycles of O6BG/TMZ chemotherapy, likely the most cycles received by
any patient to date, is currently alive at >2.5 years since diagnosis with no evidence
for disease progression. Importantly, all 3 patients surpassed the median expected
survival for glioblastoma patients displaying unmethylated MGMT promoters (11.8
months). We have expanded this cohort and continue to monitor gene-modified
cell levels, clonal contribution, and response. We have identified over 12,700
unique retrovirus integration sites in these patients and no patient has displayed
abnormal hematopoiesis as a result of the gene-modified cell infusion to date. We
believe these data demonstrate MGMTP140K-modified HPCs as a safe method of
chemoprotection in patients. We further contend that administration of O6BG/TMZ
chemotherapy in the context of MGMTP140K gene-modified HPCs could potentially
extend survival in poor-prognosis glioblastoma patients.
drug therapy (ART) Treatment Interruption (TI). In the California study, 9 INR in 3
cohorts received 1x, 2x or 3x1010 cells. Mean CD4 and SB-728-T counts increased
in both IR and INR subjects from the 2 studies and persisted. Increases in CD4
over time correlated with SB-728-T engraftment (г=0.78, p<0.0001). SB-728-T
was detected in gut mucosa of 18/18 subjects biopsied (median 6%). During TI,
HIV-RNA dropped ~0.8-2.1 log from peak levels in 3 subjects. In one CCR5Δ32
heterozygous subject with a viral setpoint of 165K copies/ml, VL peaked at 6247
during Wk 6 of TI and was undetectable by Wk 12. SB-728-T expands rapidly and
homes to the gut. In one subject with the highest level of CCR5 modification, VL
was controlled (undetectable) without ART. Therefore, in addition to increases in
CD4 counts, SB-728-T may also suppress HIV replication. This justifies further
development of cell-based engineering approaches inducing robust CD4+ T-cell
resistance to HIV infection. Advantages would include significantly less expense
andtoxicity than allogeneic HSC transplantwith a CCR5-/-(Δ32) donor, and less
expense than a lifetime of anti-retroviral drug therapy.
3
CATS1 STUDY: IMMUNOMONITORING AND CLINICAL RESULTS
OF ANTIGEN-SPECIFIC T REGULATORY(Treg) CELL THERAPY FOR
CROHN’S DISEASE (CD)
Nathalie Clerget-Chossat, 1 Valérie Brun,1 Pierre Desreumaux,2 Mathieu Allez,3 Laurent
Beaugerie,4 Xavier Hébuterne,5 Yoram Bouhnik,6 Maria Nachury,7 Agnès Duchange,8 Arnaud
Foussat,1 Miguel Forte,1 Jean-Frédéric Colombel,2 1TxCell, Valbonne Sophia-Antipolis, France,
2
Claude Huriez Hospital, Lille, France, 3St-Louis Hospital, Paris, France, 4St-Antoine Hospital,
Paris, France, 5L’Archet 2 Hospital, Nice, France, 6Beaujon Hospital, Clichy, France, 7Jean
Minjoz Hospital, Besançon, France, 8Effi-Stat, Paris, France.
CATS1 study assessed the tolerability and efficacy of Ovasave, an antigen-specific
Treg therapy for patients with refractory Crohn’s Disease. Treg cells induce
immunomodulating effects through cytokines secretion and cell-cell contact.
CATS1 was an open label, 12-week, single injection, phase I/II study in 20 patients
with CD and active inflammation (4 doses of 106, 107, 108, 109 cells with respectively
8, 3, 3, 6 patients). Ovasave was produced ex vivo from patients’ PBMCs exposed
to ovalbumin followed by cell cloning, expansion and formulation for infusion.
Patients were assessed for tolerability and efficacy (CDAI responder: decrease
>=100; remission: <150). In vitro changes in peripheral blood immune cell
populations and PBMCs proliferative response to ovalbumin were also evaluated.
2
Mean age: 34.5; Baseline CDAI: 364±81 (n=20); 19/20 patients had previous failure
to immunosupressors and multiple anti-TNFs; 16/20 had previous CD surgery.
A SINGLE INFUSION OF ZINC FINGER NUCLEASE (ZFN) CCR5
MODIFIED AUTOLOGOUS CD4 T-CELLS (SB-728-T) CORRELATES
WITH INCREASES IN CD4 COUNTS AND EFFECTS ON VIRAL LOAD
(VL) IN AVIREMIC HIV-INFECTED SUBJECTS
Ovasave injections were well tolerated: 54 adverse events (2 related), 11 serious
adverse events (3 possibly related, recovered).
Bruce L. Levine, Ph.D.1 Andrea L. Brennan, M.S.1 Zhaohui Zheng, M.S.1 Julio Cotte,1 Ashley
N. Vogel, M.S.1 Dawn A. Maier, M.S.1 Anne Chew, Ph.D.1 Gabriela Plesa, Ph.D.1 David Stein,
M.D.2 Ronald Mitsuyasu, M.D.3 Jay Lalezari, M.D.4 Shelley Wang,5 Gary Lee,5 Winson Tang,5
Dale Ando, M.D.5 Pablo Tebas, M.D.6 Carl June, M.D.1 1Department of Pathology and
Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA, 2Albert Eistein
College of Medicine, Bronx, NY, USA, 3UCLA, Los Angeles, CA, USA, 4Quest Clinical Research,
San Francisco, CA, USA, 5Sangamo BioSciences, Richmond, CA, USA, 6Department of
Medicine, University of Pennsylvania, Philadelphia, PA, USA.
CCR5 is an important co-receptor for HIV-1 cellular entry. CCR5-/-(Δ32) persons
are highly resistant to HIV infection, providing rationale for genetic approaches
that target CCR5 to knock out surface expression. Designer Zinc Finger Nucleases
(ZFN’s) are chimeric DNA binding proteins/endonucleases that can edit the
genome by targeted DNA double strand breaks. CCR5 ZFN modification in CD4
T-cells (SB-728-T) may render a survival advantage to these cells in HIV infected
subjects. We report data from two clinical trials on safety, increases in CD4,
persistence and trafficking of SB-728-T, and effect on HIV viral load (VL). In the
Penn study, 6 immunologic responders (IR) and 6 immunologic non-responders
(INR) were infused with 1010 cells. At Week 4, IR underwent a 12-week anti-retroviral
Response was observed in 40% (8/20) patients at weeks 5 and 8. In the best dose
group (106 cells), response was 75% (6/8) at both time points; remission was
38% (3/8) and 25% (2/8) and the mean CDAI reduction 143.4±105 (p=0.0062)
and 131.6.3±65.4 (p=0.002) at weeks 5 and 8 respectively. Immunomonitoring
studies revealed that blood CD16+ pro-inflammatory monocytes were selectively
decreased 3 weeks after Ovasave administration in the group of responder
patients at week 5. In this group, the proliferative response of PBMC to ovalbumin
in vitro was significantly reduced 3 weeks after treatment, suggesting a direct
suppression of ovalbumin-specific immune response.
This first open label study shows that Treg cell therapy is well tolerated and
demonstrates a dose related efficacy consistent across multiple clinical and
mechanistic immunological assessment methods. Treg cell therapy may represent
an innovative value-adding opportunity for refractory CD patients.
7
Oral ABSTRACTS
4
Combination immunotherapy after ASCT for advanced
myeloma with MAGE-A3/Poly-ICLC immunization
followed by transfer of vaccine-primed activated
autologous T cells
Nicole A. Aqui, M.D.1 Aaron Rapoport, M.D.2 Edward Stadtmauer, M.D.3 Dan Vogl, M.D.3
Brendan Weiss, M.D.3 Yinyan Xu,1 Ling Cai, Ph.D.2 Hong-Bin Fang, Ph.D.2 Anne Chew,
Ph.D.3 Elizabeth Veloso, J.D., B.S.N.3 Tina Lowther, CIP3 Holly McConville, R.N., B.S.N.3
Bruce L. Levine, Ph.D.3 Carl H. June, M.D.3 1University of Pennsylvania School of Medicine,
Philadelphia, PA, USA, 2Marlene and Stewart Greenbaum Cancer Center, University of
Maryland, Baltimore, MD, USA, 3Abramson Cancer Center, University of Pennsylvania,
Philadelphia, PA, USA.
High-dose chemotherapy and autologous stem cell transplant (SCT) improves
survival for myeloma patients, but is not curative, emphasizing the need for new
therapeutic strategies. We recently reported that adoptive transfer of autologous,
hTERT-primed CD3/CD28-activated T cells (CD3/CD28TC) induces hTERTspecific immunity in ~40% of HLA-A2+ myeloma patients. Based on this response,
we designed a Phase II trial using a MAGE-A3 Trojan peptide vaccine composed
of both Class I and promiscuous Class II epitopes in advanced myeloma. All
patients receive a priming immunization with MAGE-A3 vaccine andTLR3-binding
adjuvant Hiltonol® (Poly-ICLC)and the pneumococcal conjugate vaccine (PCV)
~10 days prior to T cell collection. Patients then undergo a standard melphalan
200 mg/m2 autologous SCT, followed by CD3/CD28TC at day +2 post-SCT. At days
+14, +42, and +90, patients receive MAGE-A3 and PCV booster immunizations,
followed by lenalidomide maintenance therapy starting at approximately day +100
and two additional MAGE-A3 and PCV immunizations at days +120 and +150. To
date, 20 of 28 planned patients have enrolled. Sixteen patients have received T cell
infusions. The median number of T cells infused was 4.94 x 1010 (0.28 – 5.05). At
day +14 post-transplant, median CD3, CD4 and CD8 counts were 3483, 1468, and
1476 cells/ml respectively. In vitro assays demonstrate MAGE-A3-specific T cells
in 7/7 tested patients. Cytokine production in response to MAGE-A3 vaccine was
observed in 4/8 patients. MAGE-A3 antibodies were induced after vaccination in
8/10 patients. Higher titers appear to correlate with clinical response, though not
significantly in this small sample size (n=10, p = 0.133). Updates will be presented
at the meeting. Combination immunotherapy with MAGE-A3 Trojan vaccine
and CD3/CD28TC induces both cellular and humoral immunity and antibody
production may be a biomarker for clinical response.
5
EFFECTIVE PROPHYLAXIS OF CMV DISEASE IN RECIPIENTS
OF ALLOGENEIC HAEMOPOIETIC STEM CELL TRANSPLANTS
USING ADOPTIVE T CELL TRANSFER - LONG TERM FOLLOW-UP
OF 50 PATIENTS
Emily Blyth, 1 Leighton Clancy,1 Renee Simms,2 Jane Burgess,2 Chun Kei K. Ma,2 Kenneth P.
Micklethwaite,1 David J. Gottlieb,2 1Westmead Hospital, Westmead, Australia, 2University of
Sydney, Westmead, Australia.
Introduction: We investigated the use of donor-derived cytomegalovirus (CMV)
specific cytotoxic T cells administered early post transplant to prevent CMV
disease.
Methods: CMV CTL were generated from CMV seropositive haemopoietic stem
cell donors by three methods. Cohort 1: monocyte derived dendritic cells (mo-DC)
from peripheral blood were used to present the CMV peptide NLVPMVATV to T
cells. Cohort 2: mo-DC transfected with an adenoviral vector encoding the entire
CMV pp65 protein (Adpp65) were used to stimulate T cells from peripheral blood.
Cohort 3: peripheral blood stem cell harvest product was used with the same
method as cohort 2. A single cell infusion of 2x107 cells/m2 was administered to
transplant recipients from day 28. Patients were followed for infusional safety,
transplantation outcomes and CMV directed immune function.
8
Results: We infused 50 patients with CMV CTL between 2003 and 2011 (cohort 1
n=10; cohort 2 n=33, cohort 3 n=10). There were 36 sibling donors and 14 unrelated
donors. 45/50 were fully HLA matched and 5/50 had 1 antigen mismatch. 50% of
patients developed CMV reactivation within 100 days post transplant (17/25 preinfusion, 8/25 post infusion). There were no cases of CMV reactivation post day
100. Mean peak CMV titre was 6399 copies/ml. Three patients required treatment
with intravenous ganciclovir post T cell infusion (peak titres for these patients
were 57400, 42900 and 3160). One of fifty patients developed CMV pneumonitis
and died despite treatment with ganciclovir. 38% (19/50) developed acute GVHD
(grade I-II 15/19, III-IV 4/19). Of these, 10 were post cell infusion (grade I-II 7/10,
III-IV 3/10). Two patients (4%) died of acute GVHD post T cell infusion. Median
follow-up was 18 months post transplant (range 2 to 80). Overall survival was
70%.
Conclusion: Adoptive transfer of CMV specific CTLs is effective in preventing CMV
disease in HSCT recipients.
6
PROCESS SCALE-UP AND TRANSITION FROM AUTOLOGOUS TO
ALLOGENEIC MANUFACTURING OF CARDIOSPHERE-DERIVED
STEM CELLS
Michelle Kreke, Ph.D., Ileana Valle, Agnieszka Blusztajn, Linda Marbán, Ph.D., Rachel R.
Smith, Ph.D., Capricor Inc., Los Angeles, USA.
Intracoronary infusion of autologous cardiosphere-derived cells (CDCs) after
myocardial infarction has been shown to be safe and effective in a Phase I clinical
study (CADUCEUS, NCT00893360). Preclinical data have demonstrated that
allogeneic CDCs are equally effective and do not elicit a clinically meaningful
immune response; therefore, follow-on clinical studies will utilize allogeneic
CDCs. The manufacturing process was scaled-up and adjusted accordingly with
a focus on maintaining product and process integrity, minimizing cost, meeting
regulatory requirements for the donor (21 CFR 1271C) and master cell banks (ICH
Q5D) needed to support allogeneic production, and creating an off-the-shelf
cryopreserved product with a reasonable shelf-life. Process scale-up allowed for:
1) a 128-fold increase in the amount of tissue utilized, 2) the elimination of two
surface-coating reagents (one of which constituted 12% of the total material cost
and the other which suffered from lot-to-lot variability that affected the product),
3) a 10-fold reduction in the number of culture vessels used and a 3-fold reduction
in the vessel footprint, and 4) a 700-fold increase in number of doses generated
per donor. Total per dose costs were scaled down by 77% in comparison to
autologous production. A donor population subject to suitability criteria defined
for organ/tissue donors was selected. Appropriate QC testing was implemented,
and included viral agent testing not required for an autologous product and
microbial testing more appropriate for the allogeneic product. Products were able
to be cryopreserved in a container made of an inert material that contains no
leachables, is gas permeable, and has received market clearance from the FDA.
Products can be thawed at room temperature and remain stable for 3 hours with
no decrease in viability or recoverability. Current shelf-life is several months. The
allogeneic CDC product is appropriate for off-the-shelf clinical use.
Oral ABSTRACTS
7
THE CRYO-IMAGING SOLUTION TO “WHERE DID MY CELLS GO?”
David L. Wilson, 1 Kristin L. Sullivant,1 Patiwet Wuttisarnwattana,1 Mohammed L. Qutaish,1
Madhusudhana L. Gargesha,2 Sasidhar Katari,2 Wouter V. Hof,3 Zhenghong Lee,1 Horst v.
Recum,1 Kenneth R. Cooke,1,4 1Case Western Reserve University, Cleveland, OH, United
States, 2BioInVision, Inc., Cleveland, OH, United States, 3Athersys, Inc., Cleveland, OH,
United States, 4University Hospitals of Cleveland, Cleveland, OH, United States.
We have developed and applied a cryo-imaging system which enables assessments
of stem cell biodistribution, homing, and engraftment in preclinical models with
single cell sensitivity. Our cryo-imaging system consists of a fully automated
system for repeated physical sectioning and tiled microscope imaging of a frozen
tissue block face, providing anatomical brightfield and molecular fluorescence,
3D microscopic imaging of a large animal organ or entire mouse. Cryo-imaging
fulfills an unmet need to map cells within a 3D anatomical context over volumes
106 times larger than confocal or 2-photon, at a resolution/sensitivity much better
than in vivo. Stem cells can be fluorescently labeled with quantum dots, dyes,
and/or gene reporters. Machine learning algorithms have been created, and
rigorously validated, to detect stem cells. Recently, we have developed specialized
image analysis algorithms to obtain cell counts in specific organs (kidney, lung,
spleen, liver, bone marrow, etc.). In addition, we have developed very specialized
visualization approaches which allow one to probe the enormous (150+GB) image
volumes. For example, at the computer screen, one can examine the entire mouse
in 3D with and without stem cells visible; zoom to an organ and, for example, see
the 3D distribution of cells within the kidney; and zoom even further and examine
single cells in bone marrow within an exact anatomical context. The technology
has been applied in multiple stem cell applications including MultiStem
distribution in a GVHD model, MSC homing in heart therapy, MSC engraftment
in a cancer model, and safety studies of embryonic stem cells. In addition, dual/
triple reporters enable in vivo imaging with SPECT, PET, bioluminescence, etc.,
followed by cryo-imaging with single cell sensitivity. Finally, multispectral imaging
enables identification of multiple cell types and/or differentiation reporters. We
will describe the technology, illustrate applications, and invite vigorous discussion
of potential applications.
8
NAVIGATING THE FIRST-IN-HUMAN IMPLANTABLE POLYMERDERIVED TUMOR VACCINES INTO A CLINICAL TRIAL
Heather Daley, BS1 Linda Kelley, PhD1 Jeffrey Cram, BS1 Olive Sturtevant, MHP, MT(ASCP)
SBB,SLS, CQA(ASQ)1 Birju Patel, MS1 Jerome Ritz, MD2 Christine Canning, P.A.-C3 Glenn
Dranoff, MD3 David J. Mooney, PhD4 Sara Russell, MD5 Charles Yoon, MD, PhD5 Omar
Ali, PhD6 F. Stephen Hodi, MD7 Alexander Stafford, BS6 Des White, BSc6 Edward Doherty,
MS6 1Connell O’Reilly Cell Manipulation Core Facility, Dana-Farber Cancer Institute, Boston,
USA, 2Connell O’Reilly Cell Manipulation Core Facility, Cancer Vaccine Center, Dana-Farber
Cancer Institute, Boston, USA, 3Cancer Vaccine Center, Dana-Farber Cancer Institute, Boston,
USA, 4School of Engineering and Applied Sciences, Harvard University, Cambridge, USA,
5
Division of Surgical Ongology, Brigham and Women’s Hospital and Dana-Farber Cancer
Institute, Boston, USA, 6Wyss Institute for Biologically Inspired Engineering, Boston, USA,
7
Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, USA.
Cell-based vaccines have been used to activate the immune system to generate
anti-tumor effects. New findings suggest that creating an infection mimicking
microenvironment by presenting exogenous cytokines (GM-CSF) and danger
signals (CpG-ODN) in concert with cancer antigens (tumor lysate) may provide
a methodto precisely control the number and timing of dendritic-cell (DC)
activation and trafficking, in vivo. Polymers (poly [lactide-co-glycolide]) have been
designed as a physical, antigen-presenting structure to which DC are recruited,
and reside while they are activated. The final implantable vaccine is a small
polymer disc similar in size and appearance to an aspirin and was developed at
the Wyss Institute at Harvard. During technology transfer to our manufacturing
facility we were challenged by unique process, methods and raw materials
qualification and validation. In-house assays were developed to qualify the raw
materials including GM-CSF-microspheres, sieved sucrose and oligonucleotides.
New processes were validated including tumor lysate preparation, lysate/
microsphere preparation, component compounding, compression into a tablet
and CO2 foaming. In addition to standard testing for sterility and stability, the final
product was tested for GM-CSF, oligonucleotide content, total protein content,
as well as surface porosity using electron microscopy. Our experience confirms
that technology transfer of a complicated and novel therapy using convergent
technologies can be successfully performed in an academic medical center to
support IND submission for a Phase I clinical trial.
9
Development of an Angiogenic Potency Assay for
Clinical Grade Stem Cell Production
Anthony E Ting, PhD, Nicholas Lehman, BA, Rochelle Cutrone, MS, Amy Raber, MS, Robert
Perry, MS, Wouter Van’t Hof, PhD, Robert Deans, PhD, Juliana Woda, PhD, Athersys, Inc.,
Cleveland, USA.
Delivery of stem cells after ischemic injury has been shown to provide therapeutic
benefit through trophic support to injured tissue by regulating immune and
inflammatory cells, limiting apoptosis, stimulating neo-angiogenesis, and
recruiting host tissue for repair. MultiStem®, an adherent multipotent progenitor
cell population derived from bone marrow, has been shown to be beneficial
in animal models when delivered following ischemic injury such as AMI and
PVD. Previous results suggest that MultiStem induces neo-vascularization by
promoting angiogenesis. Production of clinical grade stem cell products requires
the development of lot release criteria based on potency assays that directly reflect
on the fundamental mechanistic pathway underlying therapeutic response to
verify manufacturing process and create pass/fail criteria to verify potency. Using
an in vitro endothelial tube formation assay, a potency assay has been developed
that reflects MultiStem’s proangiogenic activity. Serum free conditioned media
collected from MultiStem was found to induce endothelial tube formation in
vitro, reflecting MultiStem’s in vivo angiogenic activity. Three cytokines, CXCL5,
Il-8 and VEGF, have been identified in the conditioned media as being required
for this angiogenic activity. Depletion of any of these factors from the media
prevents tube formation. By adding back increasing amounts of these cytokines
into the depleted serum free conditioned media, the lower limits of each of these
cytokines required to induce angiogenesis was defined. Therefore, the detection
of these factors in the spent media from manufacturing production runs was
defined as a potency assay for MultiStem’s angiogenic activity. By correlating the
levels of these cytokines required in the serum free conditioned media to induce
tube formation in vitro with levels of these factors found in the spent media from
MultiStem manufacturing production runs, lot release criteria was established for
the angiogenic potency of MultiStem based on the detection of VEGF, CXCL5 and
IL-8 in spent media.
9
Oral ABSTRACTS
10
DEVELOPING LARGE SCALE CELL MANUFACTURING PROCESSES:
THE IMPACT ON CELL QUALITY ATTRIBUTES AND COST OF
GOODS
Hemanthram Varadaraju, Research Engineer, Jacob Pattasseril, Engineering Manager, Jessica
Carmen, Scientist, Elizabeth Misleh, Research Associate II, Jon Rowley, Innovation Director,
Cell processing Technologies, Lonza Inc, Walkersville, USA.
There is an industry demand for larger lot sizes of anchorage-dependent cells
such as human Mesenchymal Stem Cells (hMSCs).Adherent therapeutic cells are
currently manufactured in 10-layer vessels producing lot sizes of approximately
8-12 billion cells per lot.The goal of this study was to evaluate the impact of scaling
the lot size of MSCS to approximately 4 times the current surface area on the
critical quality attributes (yield, phenotype, function) and the overall cost of goods
(COGs). Multiple donors of hMSCs were cultured (Lonza MSC-GM) in 10-layer
and 40-layer vessels and evaluated for total yield and viability. Yield at harvest
was comparable at 25 ± 3 thousand cells/cm2 and 28 ± 5 thousand cells/cm2
respectively (p > 0.05).Flow marker expression (CD105, CD166 and CD45) and
secreted cytokine analysis (IL-6, IL-8 and VEGF) showed little variation between
manufacturing platforms suggesting that there would be a low comparability risk
in identity or biofunctionality during process scale up. We used 40-layer vessels
manipulated with Automated Cell Factory Manipulator robotics (ACFM, Nunc)
to develop and model a completely disposable and closed system process that is
scalable to 1.5-2 million cm2 of surface area per lot, or 35-45 billion cells per lot.
Superpro Designer® process modeling software was used to model large scale
10-layer and (4X scale) 40-layer manufacturing processes.A 4X scale up process
simulation demonstrated that with constant yields and media usage the total
number of labor hours required per billion cells produced would be decreased
by approximately three times, impacting overallCOGs per billion cells produced
by 25%.Importantly, the modeling shows that media will remain as a major cost
driver of overall CoGs.We propose that the integration of bioprocess engineering
and proper cell characterization and comparability testing is critical during scaleup and streamlining of manufacturing processes.
11
Safety and Efficacy of intracoronary infusion of
Allogeneic Cardiosphere-derived Stem Cells in a Pig
Model of Myocardial Infarction
Michelle Kreke, Ph.D.1 Konstantinos Malliaras, M.D.2 Hideaki Kanazawa, M.D.2 Christene
A. Huang, Ph.D.3,4 Ileana Valle,1 Agnieszka Blusztajn,1 Kristine Yee, D.V.M.2 David H. Sachs,
M.D.3,4 Ioannis Terrovitis, M.D.1 Linda Marbán, Ph.D.1 Rachel R. Smith, Ph.D.1 1Capricor Inc.,
Los Angeles, USA, 2Cedars-Sinai Medical Center, Los Angeles, USA, 3Massachusetts General
Hospital, Boston, USA, 4Harvard Medical School, Boston, USA.
Autologous cardiosphere-derived cells (CDCs), delivered intracoronary in a Phase
I clinical study to patients after myocardial infarction (MI), proved safe and
effective. The present translational study examined the use of allogeneic CDCs. In
order to establish a robust allogeneic model, all pigs were swine leukocyte antigen
typed. A male donor and female recipients with full 2-haplotype mismatch were
used. A master cell bank capable of generating 856 doses was created. Two weeks
after MI creation, CDCs (n=8) or vehicle (n=6) were infused using a standard
balloon catheter. Some animals were sacrificed 2 weeks post-infusion to assess the
immune response, and some 2 months post-infusion to assess cardiac function.
Cardiac enzymes and systemic inflammation peaked 1 day post-infusion for both
CDC and vehicle groups; however, there were no differences between groups
(Fig. 1A&B). Signs of a cellular and humoral immune response were assessed
by grading histological sections on a clinical rejection scale and quantifying
circulating donor-specific antibodies, respectively, and found to be undetectable
(Fig. 1C&D). Additionally, there were no systemic histological findings related to
CDCs. As expected, CDCs did not permanently engraft, with <0.1% (detection of
10
Y chromosome) persisting 2 weeks post-infusion and none detectable 2 months
post-infusion. Trends for functional benefits in CDC-treated animals were seen
2 months post-infusion and the magnitude of effect was similar to that seen in
a prior study using autologous CDCs. Ejection fraction tended to remain stable
in CDC-treated animals relative to pre-infusion and decline in vehicle-treated
animals regardless of whether the cell source was autologous or allogeneic (Fig.
1E). Infarct size tended to be somewhat reduced in CDC animals compared to
vehicle animals after either autologous or allogeneic treatment (Fig. 1F). Overall,
results demonstrate that allogeneic CDCs are equivalent to autologous in terms
of efficacy, and elicit no detectable immunological response or safety concern.
12
A PHASE I/II STUDY ASSESSING THE SAFETY AND EFFICACY
OF INTRAVENOUS EX VIVO CULTURED ADULT ALLOGENEIC
MESENCHYMAL STEM CELLS IN PATIENTS WITH ST ELEVATED
ACUTE MYOCARDIAL INFARCTION (STEMI)
Pawan Kr Gupta, Dr Anoop C H, MD, Dr Anjan Das, MS, MCh, Dr Raviraja N S, PhD, Umesh
Baikunje, M Pharm, Dr Anish Sen Majumdar, PhD, Stempeutics Research, Bangalore, India.
Background: In recent years, stem cell treatment of MI has elicited great
enthusiasm upon scientists and physicians alike, thus making the finding of a
suitable stem cell type a compulsory subject for modern medicine. Methods:
This study was a double blinded, randomized, placebo controlled, single dose
(2 million MSCs / kg body wt), study in patients with STEMI (n=20) with a two
year follow-up. The trial was approved by Indian FDA & IRBs of participating
centres and registered at clinicaltrials.gov (NCT00883727). Patient included were
of STEMI status two days post percutaneous coronary intervention, LVEF of <
50% and > 30% and patients of acute anterior MI / acute inferoposterior MI.
Primary end point was safety as assessed by type and number of AEs. Secondary
end points were improvement in LVEF, ESV & EDV by ECHO, assessment of
regional myocardial perfusion by SPECT & assessment of percentage change in
infarct size by MRI. Results: 41 treatment emergent AEs were seen in the study.
Eighteen (43.9%) were reported by 7 patients in the cell arm and 23 (56.09%) AEs
were experienced by 7 patients receiving placebo. All the AEs in the cell arm were
related to the underlying disorder. Three SAEs were reported in the placebo arm.
Oral ABSTRACTS
14
A NEW ROLE FOR MENINGES AS A NICHE FOR STEM/
PRECURSOR CELLS WITH NEURAL DIFFERENTIATION POTENTIAL
DURING DEVELOPMENT UP TO ADULTHOOD.
Ilaria Decimo, PhD1 Marijana Kusalo, MSc1 Giorgio Malpeli, PhD2 Eliana Amati,3 Aldo Scarpa,
PhD3 Valeria Berton, MSc1 Emanuela Bersan, PhD1 Marzia Di Chio, MSc1 Guido Fumagalli,
MD1 Mauro Krampera, MD, PhD4 Francesco Bifari, MD, PhD4 1Department of Public
Health and Community Medicine, Section of Pharmacology, University of Verona, Verona,
Italy, 2Department of Pathology, Section of Pathological Anatomy, University of Verona,
Verona, Italia, 3Department of Pathology, Section of Pathological Anatomy, University of
Verona, Verona, Italy, 4Department of Medicine, Stem Cell Research Laboratory, Section of
Hematology, University of Verona, Verona, Italy.
Renal, hepatic and haematological parameters were not different in between the
two arms. There was a mean increase of 4.75% in LVEF in cell arm as compared
to 1.89% in placebo arm (p=0.2678). No statistically significant difference was
observed between EDV & ESV, total perfusion score by SPECT (p=0.3095) and
volume of infarct by MRI (p=0.0562) between the two groups. Conclusion:
Intravenous use of allogeneic MSCs are safe in AMI but larger patient population
with different routes of administration need to be carried out to prove the efficacy
of these cells.
13
IRES-BASED LENTIVIRUS CO-EXPRESSING TGFb1 AND FGF2
IMPROVES CELL SURVIVAL AND ANGIOGENESIS IN BONE
MARROW DERIVED-MESENCHYMAL STEM CELLS
Manuela Mura, PhD1 Giuseppe Malpasso, Bs1 Federica Pisano, Bs1 Federica Longo,2 Patrizia
Danieli, PhD1 Elisabetta Cervio, PhD1 Massimiliano Gnecchi, MD, PhD2,3 1Fondazione IRCCS
Policlinico San Matteo , Pavia, Italy, 2University of Pavia, Pavia, Italy, 3Fondazione IRCCS
Policlinico San Matteo, Pavia, Italy.
Background. Bone marrow mesenchymal stem cells (BM-MSC) repair infarcted
hearts mainly through paracrine mechanisms. However, donor age negatively
influences the production of paracrine factors from BM-MSC. In particular, we have
shown that in BM-MSC from elderly patients the expression of TGFb1 (T) and FGF2
(F) is decreased. We hypothesized that the overexpression of these two factors
involved in cytoprotection and angiogenesis may improve MSC repair capacity.
Methods. Rat BM-MSC were transduced with control virus (GFP-MSC) or TF
virus (TF-MSC). To study cytoprotective paracrine properties H9c2 cells were
exposed to 24h of hypoxia in the presence of control medium (CTRL-M) or media
conditioned by GFP- (GFP-CM) or TF-MSC (TF-CM). Cell viability was measured by
MTS assay. Apoptosis was evaluated through caspase-3 activation (luminometric
assay and western blot). Angiogenesis was assessed by quantifying HUVEC tube
formation on Matrigel. Transcriptional levels of known survival genes in H9c2
cells and of soluble factors other than transgenes in MSC were measured by RTPCR. Activation of FGF2 and TGFb1 pathways (Akt, ERK1/2, and SMAD2) was
evaluated by western blot.
Results. Compared with CTRL-M, TF-CM increased H9c2 viability (+46,3%
p<0.001), while GFP-CM had no effect. Caspase-3 activation was reduced by TFCM of 60,3% (p<0.001) vs CTRL-M and of 44,7 % (p<0.05) vs GFP-MSC. H9c2
treated with TF-CM showed a strong activation of Akt, ERK1/2 and SMAD2 antiapoptotic pathways, enhanced expression of Bcl-2 and Stat3 pro-survival genes,
and inhibition of FasL and TNFa pro-apoptotic genes. HUVEC tube formation
were enhanced by TF-CM of 70% (p<0.01) vs CTRL-M and of 59% (p<0.05) vs
GFP-CM. Finally, we documented that TF-MSC upregulated transcriptional levels
of other soluble factors like IGF1, PDGF-β, BMP2, VEGF and IL11.
In this work, we asked whether theimmature nestin-positive precursors with
neural differentiationpotential, were developmentally conservedin meningesfrom
embryo to adult.
Changes in distribution and in the expression of cellular and extracellular matrix
antigens were analyzed in meningesfrom embryo (E14, E20), perinatal (P0, P15)
and adult ratsbylaser capture microscopy, electron and confocal microscopy.We
found cells expressing the stem cell marker nestin inmeningesas early as E14.
The number, density and proliferation rate of these cells significantly decreased
with the animal age and represent the 13.3±4.4% of the adult rat brain meningeal
cells.Finally, we described the in vitro neural differentiation potential of meningeal
stem/precursors during development up to adulthood.
We show that the meninges are a putative new stem cell niche capable of housing
and maintaining up to adulthood a population of stem/progenitor cells with
neural differentiation potential.Further investigation will elucidate any functional
role of the meningeal stem cell niche in brain development and in adult.
15
COMPARISON OF HUMAN SCHWANN CELL PROLIFERATION
RATES IN CULTURE FROM ORGAN DONOR AND CADAVERIC
NERVES
Gagani Athauda, MD1,2 Adriana Brooks-Perez, BSc1,2 Aisha Khan, MBA PhD1,3 Dalton Dietrich,
PhD1,2 Pat Wood, PhD1,2 James Guest, MD PhD1,2 1University of Miami Miller School of
Medicine, Miami, USA, 2The Miami Project to Cure Paralysis, Miami, USA, 3Diabetes
Research Institute, Miami, USA.
Introduction: Schwann cell (SC) transplantation is a promising therapy for
nerve and spinal cord repair. Preclinical studies of human nerve-derived cells
are important to optimize cell culture methods and to validate the protocol for
manufacturing a SC product acceptable for clinical use. We compared the growth
curves of human SC isolated from organ donor nerves with growth curves of
human SC isolated from cadaveric nerves. Nerve sources for such studies include
organ donors and autopsy donors. In addition, we have compared the purity and
viability of cells isolated from these two sources.
Methods: Sural nerves were obtained from organ donors after appropriate
consent within 3h of death. Cadaveric nerves were obtained 8-28hrs after
death. SC-rich nerve fascicles were dissected, cultured in growth medium and
enzymatically dissociated after 8 days. The P0 cells were plated onto mouse
laminin coated flasks at a density of 1 x 106 viable cells in the same medium.
At 60-80% confluency cells subjected to differential adhesion purification to
improve SC/fibroblast ratio and re-plated at 0.5 x 106 viable cells/T-75cm2 flasks.
Cells were passaged to P4 and total cell counts and purity were assessed at each
passage by staining the cells for Syto24 and Sytoxgreen (live/dead cells). Purity
was determined by immunostaining for S-100. Results:
Conclusions. Simultaneous overexpression of TF transgenes improves
cytoprotective and pro-angiogenic paracrine properties and enhances expression
of soluble factors in BM-MSC.
11
Oral ABSTRACTS
Figure 1.Cell proliferation:
Table 2. Schwann cell purity (% of total cells):
and 6.9±0.41% n=22, 8.1±1.4% n=7 and 3.8±0.4% n=12, respectively) after 15h
in a non-ablated setting. Transplant studies using FITC+CD31+B220+/- BMSEC
from RFP donor mice revealed that this population actively contributed to BM
revascularisation in ablated recipients within 4 weeks, with the appearance of
blood vessels lined with RFP+ endothelial cells, which maintained their endocytic
function. The number of donor cells and vessels was significantly enhanced
when the transplanted cells were of endosteal origin compared to their central
BM counterparts. Furthermore, the addition of BMSEC to a HSC transplant
significantly shortened the reconstitution time to normal BM cellularity. BMSEC
were hierarchically organized, with only the FITC+CD31+B220- cells being able to
recapitulate all subpopulations. Conversely, the FITC+CD31+B220+ subpopulation
could only give rise to itself post transplant. Overall, the data demonstrates that
prospectively isolated BMSEC are transplantable, give rise to blood vessels whist
maintaining their endocytic capacity and in combination with HSC shorten the
time to normal BM cellularity.
17
HUMAN MESENCHYMAL STEM CELLS EXPRESS PROTEINS
BELONGING TO OR RELATED TO THE IL1 FAMILY THAT
CONTRIBUTE TO THEIR ANTI-INFLAMMATORY ACTIVITY
Table 3.Viability
Discussion: Significant differences between the growth of hSC from organ-donor
and cadaveric nerves was not observed. Conclusion: For preclinical studies, either
organ donor or cadaveric nerve source is useful, validating prior data that SC
remain viable within cooled cadaveric tissues.
16
PROSPECTIVELY ISOLATED SCAVENGING BONE-MARROW
SINUSOIDAL ENDOTHELIAL CELLS HOME TO, AND
REVASCULARIZE THE BONE MARROW OF TRANSPLANTED
ABLATED RECIPIENTS.
Peter McCourt, PhD2 Ana Oteiza, PhD1,2 Melonie Storan, PhD1 Brenda Williams,1 Andrea
Rietsma,1 Chad Heazlewood,1 Dani Cardozo,1 Susie K. Nilsson, PhD1 1CSIRO, Melbourne,
Australia, 2University of Tromsø, Tromsø, Norway.
Hemopoietic stem cells (HSC) reside in bone marrow (BM) stem cell niches
and produce all circulating blood cells. Interfacing blood and the niche are
sinusoidal endothelial cell (SEC) lined vessels, with immense endocytic capacity
for soluble waste. Recently, we developed a method to exploit this endocytic
capacity as a functional marker for the isolation and characterization of BMSEC,
demonstrating that prospectively isolated BMSEC can be used together with
standard BM transplantation, as they home to the BM and actively contribute to
revascularization when transplanted.
12
Mice were injected intravenously with aFITC-labeled reagent and BMSEC
prospectively isolated from the central and endosteal BM using FACS after
60min. FITC+ cells comprised 13% of BM and were sub-fractionated using B220
and CD31: 1% CD31+B220-, 60% CD31+B220+ and 34% CD31-B220-. Homing
studies demonstrated significantly more endosteal CD31+B220+ and CD31-B220cells homed compared to their central marrow counterparts (10.1±0.57%, n=18
Siddaraju V Boregowda, Donald G Phinney, PhD, The Scripps Research Institute , Jupiter ,
USA.
Previously we reported that mouse mesenchymal stem cells (MSCs) ameliorate
lung inflammation following acute injury via expression of interleukin 1 family
member 3 (IL1F3/IL1RN). Herein we report that human MSCs also protect mice
against acute lung injury by blocking inflammation but express ~100-fold lower
levels of IL1F3 as compared to mouse MSCs. Although human MSCs expressed
IL1R1 and responded to IL1F2 exposure by activation of NK-kB signaling, this had
only a modest effect on IL1F3 secretion. Alternatively, human MSCs were found
to express IL1F7 and IL18BP and conditioned media from human MSCs blocked
IL18-stimulated release of INF-Γ from mouse splenocytes. In addition, exposure
of hMSCs to IL1F2 resulted in up regulation of IL1F1, IL1F2 and to a lesser extent
IL1F3, IL1F4, IL1F5 and IL18BP but strongly depressed IL1F7 expression. In contrast,
FGF2 up regulated expression of IL1F1, IL1F2, and IL1F3 to a greater extent than
IL1F2, and suppressed expression of IL1F4, IL1F5, and IL1F7. Consistent with
these findings, pre-exposure of human MSCs to FGF2 significantly reduced their
capacity to block lung inflammation in response to acute injury in vivo. This
effect was linked to FGF2-induced down regulation of TWIST1, which resulted in
increased expression of IL1F2 by MSCs. Collectively, these results demonstrate
that human MSCs express a broad repertoire of anti-inflammatory proteins
belonging to or related to the IL1 family and that TWIST1 regulates inflammatory
cytokine signaling in MSCs via an FGF2-dependent mechanism.
18
REGULATING HUMAN MESENCHYMAL STEM CELLS FOR
OSTEOGENIC TISSUE REPAIR BY p63 AND p38 ISOFORMS
Guy A. Howard, PhD1,2 Kevin M. Curtis, PhD1 Kristina K. Aenlle, PhD1 Ketian Chen, PhD1
Bernard A. Roos, MD1,2 1GRECC and Research Service, Miami VA Medical Center, Miami,
USA, 2University of Miami Miller School of Medicine, Miami, USA.
Hepatocyte growth factor (HGF) and 1,25-dihydroxyvitamin D (1,25OHD)
regulate human mesenchymal stem cell (hMSC) maturation. HGF is secreted
by hMSCs promoting their migration and proliferation. p63, a member of
the p53 family, mediates the cooperative actions of HGF and 1,25OHD, upregulating the vitamin D receptor (VDR) and driving hMSC differentiation.
Moreover, 24,25-dihydroxyvitamin D (24,25OHD) promotes hMSC proliferation,
alkaline phosphatase activity and mineralization, and decreases 1α-hydroxylase
expression, potentially limiting the ability to convert 25OHD to 1,25OHD.
1,25OHD activatesboth VDR and p63 gene expression, while HGF induces p63 to
Oral ABSTRACTS
bind the VDR promoter increasing VDR gene expression. p63 has multiple variants
due to alternative promoters and RNA splicing, leading to formation of TA- and
∆Np63 isoforms and α,β,Γ splice variants. During progression of hMSC toward
the osteogenic phenotype, there is a switch from TAp63α,β to TAp63Γ. 1,25OHD
up-regulates the ∆Np63 isoforms, while 24,25OHD increases TA/∆Np63Γ mRNA
and TAp63α protein expression. These results link 1,25OHD up-regulation of
∆Np63, required for differentiation, and 24,25OHD up-regulation of p63Γ, known
to regulate VDR expression.
Activation of HGF signaling promotes osteogenic markers and mineralization –
likely due, in part, to activation of p38 in the MAPK signaling pathway. p38 consists
of four isoforms (α,β,Γ,δ), and p38α and p38β are important for skeletogenesis.
Inhibition of p38α and p38β in hMSCs leads to reduced alkaline phosphatase
activity/expression, and decreased mineralization. Our results also demonstrate
that HGF promotes the phosphorylation of total p38 and differential regulation of
specific p38 isoforms. HGF treatment increases p38α, -β and -δ, while decreasing
p38Γ mRNA. HGF treatment also increases the protein level of p38β.
Thus the actions of vitamin D on osteogenic maturation are likely due to a
regulatory relationship between p63 gene products and unique 24,25OHD /
1,25OHD effects, while through the activation of specific p38 isoforms, HGF
primes hMSCs for osteogenic maturation.
19
MSC FUNCTIONALITY AND ROBUSTNESS OF THE MSC
EXPANSION PROCESS ARE CRITICALLY DEPENDENT ON TISSUE
SOURCE AND EXPANSION CONDITIONS
Ruurd Torensma,2 Henk-Jan Prins,3 Bas Jansen,2 Ellen Schrama,1 Eugene Verwiel,4 Anton
Martens,3,5 Helene Roelofs, 1 1Leiden University Medical Center, Dept. Immunohematology
and Blood transfusion, Leiden, Netherlands, 2Radboud University Nijmegen Medical Centre,
Dept. Tumorimmunology, Nijmegen, Netherlands, 3University Medical Center Utrecht, Dept.
Immunology , Utrecht, Netherlands, 4Radboud University Nijmegen Medical Centre, Dept.
Human Genetics, Nijmegen, Netherlands, 5University Medical Center Utrecht, Dept. Cell
Biology , Utrecht, Netherlands.
For an increasing number of clinical applications MSCs are collected from various
tissues and culture-expanded using various procedures. The choices for tissue
source and expansion condition are primarily based on logistics, tissue availability
and local laboratory routine, rather than on suitability for a specific clinical
application. However, inter-laboratory differences in MSC functionalities are
often attributed to these variables as well as to inter-donor variation. For clinical
application, product consistency is very important and a robust production
process is essential.
In order to assign phenotypic and functional differences between MSC populations
to tissue source, expansion procedure, inter-donor variation or laboratory-linked
conditions, we performed a comparative analysis on MSC populations from
five bone marrow (BM)- and five adipose tissue (AT)-donors. The MSCs were
expanded in both fetal calf serum (FCS)- and human platelet lysate (hPL)-based
medium and distributed and further expanded at three different locations. The
analyses included CFU-frequency, expansion rate, surface marker expression, in
vitro and in vivo differentiation capacity, T-cell proliferation inhibitory capacity and
microarray gene expression profiling.
Hierarchical clustering of the gene expression profiles shows that tissue source is
the main contributor to MSC product variation, followed by expansion condition.
We confirm that expansion to clinically relevant cell numbers can be achieved in a
shorter time period using hPL medium. For AT, this is due to a substantially higher
clonogenicity in hPL medium and a slightly higher expansion rate, indicating a
higher product heterogeneity. Culture condition cross-over experiments and the
gene expression profiling also indicate that AT-derived MSC populations expanded
in hPL-based medium are more heterogeneous and that MSC expansion from BM
results in more reproducible products. T-cell proliferation inhibitory capacity was
not dependent on the expansion medium. As opposed to the BM-derived MSCs,
the AT-derived MSCs were incapable of in vivo bone formation.
20
MESENCHYMAL STROMAL CELL THERAPY FOR REFRACTORY
CROHN’S DISEASE
Janice Fogarty, 1 Geoff Forbes, Dr2 Adrian Cummins, Professor3 Rupert Leong, Associate
Professor4,5 Kathryn Shaw,1 Janina Pawlik, Ms2 Marian Sturm, Dr1,6 Richard Herrmann,
Professor1,6 1Cell & Tissue Therapies WA, Royal Perth Hospital, Perth, Australia,
2
Department of Gastroenterology, Royal Perth Hospital, Perth, Australia, 3Queen Elizabeth
Hospital, Adelaide, Australia, 4Concord & Bankstown Hospital, Sydney, Australia, 5University
of New South Wales, Sydney, Australia, 6University of Western Australia, Perth, Australia.
Recent advances in treatment of Crohn’s disease includes the use of
immunosuppressive monoclonal antibody therapy (biologics). However, despite
this advance for steroid and immunomodulator -refractory Crohns disease, there
remain a large group of patients who need surgery as a result of adverse effects,
failure of induction, or loss of response to biologic therapy. Approximately 23% of
patients will need major abdominal surgery. The immunomodulatory properties
of mesenchymal stromal cells (MSC) and our experience with MSC in the
treatment Graft versus Host Disease (GVHD), particularly skin and gut, has been
the impetus for this study.
This study is a phase II open label multicentre Australian study to establish efficacy
and safety of MSC infusions in the treatment of infliximab- and adalimumabrefractory moderate to severe colonic or small intestine Crohn’s disease with a
disease activity index (CDAI) >250.
Bone marrow derived MSC are manufactured from allogeneic donors under GMP
in CTTWA. Patients receive four infusions of 2 x 106/kg MSC at weekly intervals and
are observed and reassessed clinically at each study visit to the study end point
at day 42. The primary endpoint is a clinical response (CDAI decrease of >100)
at day 42; secondary endpoint include remission (CDAI <150) and endoscopic
improvements. Peripheral blood (PB) samples and colonic biopsies are collected
pre MSC therapy and at end point for the evaluation of immunopathological
changes. The study will accrue 30 patients.
To date, 9 patients have been recruited with 6 having completed therapy. Clinical
response has occurred in three patients (CDAI reduction of 102,147 and 189),
clinical remission in two (CDAI 130 and 122) and endoscopic improvement in
one. No significant adverse events have occurred. Immunopathological analyses
of colonic biopsies and peripheral blood are underway for these initial patients.
This early data suggest that MSC may have therapeutic efficacy and safety in
Crohn’s disease.
21
MOBILIZATION OF MESENCHYMAL STEM CELLS FROM THE
BONE MARROW TO PERIPHERAL BLOOD
Ian K McNiece, PhD, Santhosh K Sivajothi, Yingchun Wang, MD, University of Miami, Miami,
USA.
Cytokine-induced cell mobilization offers a readily available source of stem cells
that can be easily collected by apheresis for reinfusion or could provide circulating
stem cells for tissue repair providing a non-invasive delivery procedure. Reports
have shown that treatment with G-CSF leads to egress of hematopoietic stem and
progenitor cells (HSC and HPCs) from the bone marrow (BM) to the peripheral
circulation. As the BM contains a second stem cell population, mesenchymal
stem cells (MSCs), we evaluated G-CSF mobilized peripheral blood progenitor cell
products (PBPC) from normal human donors and demonstrated that these cells
do not generate MSCs. Therefore we have evaluated growth factors alone and in
combination for their potential to mobilize MSCs into the peripheral circulation.
Treatment of mice with rhG-CSF (250 μg/kg) alone did not result in mobilization
of MSCs while treatment with Substance P (10 μg/kg) or AMD3100 (100 μg)
resulted in significant levels of MSCs in the peripheral blood. Combinations
13
Oral ABSTRACTS
of G-CSF plus SP or G-CSF plus AMD3100 resulted in synergistic increases in
mobilization of MSCs. MSCs were isolated from the blood of animals treated
with these combinations and MSC lines generated. The morphology, phenotype
and differentiation potential of the cells were consistent with MSCs. We further
tested the combination of G-CSF plus SP and G-CSF plus AMD3100 in a non
human primate model and demonstrated similar mobilization of MSCs. In
summary our data demonstrate that combinations of growth factors synergize to
mobilize MSCs into the peripheral circulation and may provide alternate delivery
of therapeutic cells for regenerative medicine approaches. Clinical trials are being
conducted to evaluate the potential of mobilized MSCs.
22
OPTIMIZING MEGAKARYOCYTE POLYPLOIDIZATION INCREASES
PLATELET RELEASE IN CULTURE
Mauro P Avanzi, MD, Beau Mitchell, MD, New York Blood Center, New York, United States.
Introduction: Laboratory production of platelets for transfusion is a goal of
stem cell research. High-ploidy megakaryocytes are capable of releasing more
platelets; however, cultured human megakaryocytes are typically low-ploidy.
Polyploidization results from a combination of cellular mechanisms that inhibit
cytokinesis. In this study we have variably combined the inhibition of distinct
cytokinesis mechanisms with the goal of driving polyploidization, extending the
demarcation membrane system (DMS), and increasing platelet formation.
Methods: Umbilical cord blood-derived megakaryocytes were cultured with single
and combinations of cytokinesis inhibitors: Rho-Rock inhibitor (RRI), Y27632; Srcinhibitor (SI), SU6656; Nicotinamide (NIC); Aurora-B inhibitor (ABI), ZM447439;
and Myosin Light Chain Kinase Inhibitor (MLCKI). DMS was analyzed with Di-8
ANEPPS. Morphology was studied with Electron Microscopy (EM). Mature
megakaryocytes were stimulated to release proplatelets and platelets, which were
counted by microscopy and Advia-120 cytometry, respectively.
Results: All treatments increased megakaryocyte ploidy, except MLCKI. RRI
reached the highest ploidy (p=0.0007), followed by NIC (p=0.003), SI (p=0.026)
and ABI (p=0.018). Combinations all significantly increased polyploidization;
however the only combination that equaled RRI alone was the combination of all
of the other inhibitors (p<0.0001). EM showed normal megakaryocyte structure.
DMS quantification showed that high-ploidy megakaryocytes had more extensive
DMS (p<0.02) and also released more proplatelets and platelets than control and
low-ploidy cells (p=0.01).
Conclusion: RRI was most effective in driving megakaryocyte polyploidization; the
summation of all of the other cytokinesis inhibitors increased polyploidization
only to the same extent as RRI. This is likely due to the Rho/Rock pathway
overlapping all of the pathways studied. The cultured megakaryocytes were
morphologically normal. High-ploidy megakaryocytes with an extended DMS
extended more proplatelets and released more platelets. Our results show that
induction of high ploidy megakaryocytes involves a combination of distinct
cytokinesis pathways and underlines an important strategy to increase platelet
production for transfusion.
14
23
HEMATOPOIETIC STEM CELLS AND MEGAKARYOCYTE
PROGENITORS AND THEIR SUBSETS DIFFER IN NORMAL AND
MEYELOPROLIFERATIVE DISEASE STATES
Varda R Deutsch, PhD, Sigi Kay, PhD, Michal Cipok, PhD, Sofia Maizel, MSc, Elizabeth
Naparstek, Tel Aviv Sourasky Medical Center, Tel Aviv, Israel.
Prolonged thrombocytopenia following transplant is due to the lack sufficient
megakaryocyte progenitors (Mk-p).We characterized MK-p and their subpopulations
in normal and myeloproliferative diseases by high definition flow cytometry (HDFC)
and quantified these progenitor populations. The recently described CD41high/ SSC low /
CD45 dim/neg
Mk-p (1) were compared inBM, CB, and PBSC and in BM from CML and
ETandITP. Mk-p were sorted and CFU-Mk capacity tested. We demonstrate for the
first time that the proportion of early Mk-p including CD41high/ SSC low /CD45 dim/neg vary
under different physiological conditions.CD34+ and CD34+/CD41+ cells were increased
in PBSC and decreased in CB, correlating with the shorter platelet nadir in patients
transplanted with PBSC and the known protracted thrombocytopenia following CB
transplant. The same MK-p population in CB contained no detectable CD34+ cells
pointing again to reduced numbers of transplantable early Mk-p in CB.We further
resolved increased Mk-p subpopulations that maintained CD34 in ITPand CML.
While inET, which is characterized by MK maturation and increase platelet production,
fewer CD34+ cells were detected, implying accelerated maturation and loss of CD34.
The proportion of CD41high/ SSC low /CD45 dim/neg Mk-p was increased 10 fold over normal
BM.CD41high/ SSC low /CD45 dim/neg MK-p were sorted and CFU-MK assays performed. The
sorted cells displayed uniform stem cell like morphology (figure 1) with a 5- 10X
enrichment in CFU-MK over the unsorted fraction. Sorted Mk-p with JAK mutations
were increased 11 fold and independent of TPO. The expansionofdifferent Mk-p is
currently underway. Studying Mk-p under normal and myeloproliferative states has
provided new information about these rare key contributors to thrombopoiesis.
Further investigation of purified Mk-p may allow new insights into the regulation of
normal and aberrant thrombopoiesis, and the ability to efficiently expand thesecells
for transplantation.
24
ALDHbr content of segments from banked cord
bloods predicts engraftment after cord blood
transplantation.
Kevin Shoulars, Ph.D., Tracy Gentry, Kristin Page, M.D., Andrew Balber, Ph.D., Joanne
Kurtzberg, M.D., Duke University Medical Center, Durham, NC, USA.
Cryopreserved, unrelated donor cord blood units (CBU) provide an option for
transplantation for patients lacking otherwise suitable donors. A major complication
of umbilical cord blood transplantation is graft failure experienced by up to 20% of
patients.A large portion of this is likely due to decreased potency of CBUs. Thus, an
assay to assess potency on a thawed CBU prior to release from the cord blood bank
is needed. Previous studies demonstrated that the number of post-thaw colony
forming units (CFU) measured on the thawed CBU is a strong predictor of overall
survival, neutrophil and platelet engraftment. However, the usefulness of CFU as a
predictive potency assay is limited by time and assay variability. Preliminary studies
indicate that CFUs measured on thawed CBU segments correlate with the number
of cells (ALDHbright, ALDHbr) expressing high levels of the enzyme aldehyde
dehydrogenase, a known marker of hematopoietic stem and progenitor cells.
Retrospective studies of transplanted CBUs indicated that the ALDHbr cell dose
correlated with neutrophil engraftment.We have developed an ALDHbr assay that
measures the content of ALDHbr [Aldecount®], CD34+, CD45+, glycophorin A+ and
viability (7-AAD+) along with CFUs. This assay can be performed on segments from
CBUs when confirmatory HLA typing (CT) is performed so information on potency is
available prospectively. From 3/10-present, we have assayed all segments requested
for CT (n=1473) from the Carolinas Cord Blood Bank. Outcome data from158
Oral ABSTRACTS
patients transplanted with these cords is available. ALDHbr content of segments
measured at the time of CT correlates well with days to absolute neutrophil count
(ANC) >500/μL (t ratio =-2.55 p = 0.0122). Conversely, neither CFU nor viable CD34+
content of segments correlated significantly with ANC500/μL.ALDHbr content of
thawed CBU segments correlates well with neutrophil engraftment and, therefore,
shows promise as a potency assay for cord blood transplantation.
25
Nanoliter Droplet Vitrification for Blood and Stem
Cell Cryopreservation
Rami El Assal,2 Umut Atakan Gurkan,2 Vasily Giannakeas,2 Fatih Inci,2 Burcu Erkmen,2
Wendy Fuld,3 Aida Nureddin,2 April Holland,2 Neal Lindeman,3 Claudia Zylberberg, 1 Utkan
Demirci,4 13Akron Biotechnology, LLC, Boca Raton, USA, 2Bio-Acoustic-MEMS in Medicine
(BAMM) Laboratory, Center for Biomedical Engineering, Department of Medicine, Brigham
and Women’s Hospital, Harvard Medical School, Boston, USA, 3Department of Pathology,
Brigham and Women’s Hospital, Harvard Medical School, Boston, USA, 4Harvard-MIT
Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, USA.
Stem cell (SC) therapy has the potential to treat many disease conditions and
may revolutionize the face of medicine. Human SCs have emerged as one
of the most promising therapeutic approaches in regenerative medicine. For
example, hematopoietic stem cells can be used to treat leukemia and neoplastic
lymphoproliferative disorders. However, the field is facing challenges in finding
the right modality and agents for long term preservation of stem cells for future
therapeutic use. Blood banking and blood-derived stem cells will become common
practice in the near future. The challenges are in the choice of cryoprotectants and in
the post-thaw viability based on cryopreservation methods. Current cryopreservation
protocols for stem cells use a toxic dimethyl sulfoxide (DMSO) as a cryoprotective
agent (CPA) which is associated with clinically significant side-effects for humans
including uncontrolled differentiation of SCs. These methods also suffer from high
throughput limitations. Therefore, we developed a high throughput vitrification
method utilizing cell encapsulating droplets, which could overcome the limitations
of previous methods by lowering the required cryoprotectant agent concentrations
and achieving ultra-rapid cooling rates in small droplet volumes. Furthermore, we
assessed this cryopreservation system by utilizing whole blood. We also evaluated
naturally occurring non-toxic agents, i.e., Ectoin, to vitrify nanoliter droplets.
In this study, we used a nanoliter droplet generation system consisting of a coflow stream of RBC-CPA solution and nitrogen gas flowing through an ejector
developed in our laboratory. Our system generates droplets with 0.25nL volumes,
which reduces potential damage by using relatively low CPA levels, allowing rapid
cooling and warming rates.
In summary, we developed a nanoliter droplet cryopreservation method and
evaluated a naturally occurring non-toxic agent for scalable vitrification of
blood and SCs. This approach has the potential to improve the efficiency of SCs
cryopreservation and enable new technologies in the field of stem cells therapy.
26
LUMINOMETRIC DETERMINATION OF PROGENITOR CELL
FUNCTION IN CRYOPRESERVED PERIPHERAL BLOOD STEM CELL
HARVESTS
Beate Wagner, 1 Jutta Goßner,1 Bianka Lowigus,1 Peter Schramm,1 Reinhard Henschler,1
Helmut Ostermann,2 1Dept. Transfusion Medicine, Cell Therapy Products &
Hemostaseology, University Hospitals of Munich, Munich, Germany, 2Dept. of Internal
Medicine III, University Hospitals of Munich, Munich, Germany.
The quality of hematopoietic stem cell transplants is routinely determined by nonfunctional and functional assays. Colony-forming unit (CFU) assays in cytokinesupplemented semisolid media have been used as standard for measuring
hematopoietic progenitor cell (HPC) proliferation potential. Microscopic scoring
is prone to investigator-dependent bias thus hampering standardization.
Moreover, results are not available until two weeks. Luminometry allows for the
quantification of cellular ATP. It has recently been suggested as an alternative tool
to investigate the proliferation potential of HPC.
Patients and Methods: Hematopoietic reconstitution (WBC >1/nl, PLT >20/
nl) in 24 patients with multiple myeloma, NHL and sarcoma receiving high
dose chemotherapy and autologous HPC transplantation was determined
retrospectively. 5,000 light density cells/well from 24 cryopreserved HPC
transplants were inoculated in liquid media supplemented by various
hematopoietic cytokines and incubated for 7 days at 37°C and 5% CO2. ATP was
determined after cell lysis by readout in a microplate luminometer. The results
of this HALO(Hematopoietic/Hemotoxicity Assays Via Luminescence Output)
method were compared to CFU-GM as assessed with semisolid media after 14
days, and CD34+ quantification by flow cytometry.
Results: Luminometric HALO readouts were consistently dependent on the
number of input cells. With 4 different media, stimulating growth of HALO-CFUGM, -CFU-MK, -BFU-E and -CFU-GEMM, respectively, values correlated well
within the same patient (R between 0.74 and 0.97 in all permutations). Moreover,
HALO-CFU-GEMM correlated with conventional CFU-GM (R= 0.79) and CD34+
counts (R= 0.62). The median reconstitution of WBC and platelets lasted 10.5
and 12.0 days, respectively. As expected, only the results of the functionally
determined HPC assays (CFU-GM, HALO-CFU-GEMM) were inversely correlated
to these intervals (R between -0.25 and -0.37).
Conclusion: The luminometric method proved as a reliable innovative tool for
measuring HPC function. Hence, the results correlated better with standard CFUGM than with CD34+ HPC. Its earlier and standardized readout may benefit HPC
transplantation programmes.
27
WHAT CLINICAL CHARACTERISTICS OF AFRICAN AMERICAN
MOTHERS AND BABIES FAVORABLY INFLUENCE CORD BLOOD
POTENCY? DEFINING PARAMETERS THAT CAN GUIDE PUBLIC
CORD BLOOD COLLECTION
Kristin M. Page, MD1 Brigid Betz-Stablein,2 Adam Mendizabal,2 Stephen Wease,2 Kevin
Shoulars, PhD1 Jessica Sun, MD1 Tracy Gentry,1 Andrew E. Balber, PhD1 Joanne Kurtzberg,
MD1 1Duke University Medical Center, Durham, NC, U.S.A., 2The Emmes Corporation,
Rockville, MD, U.S.A..
Cord blood (CB) has improved access to hematopoietic stem cell transplantation
especially for racial/ethnic minorities. Increased total nucleated cell (TNC), CD34+
and colony forming unit (CFU) content are well-recognized characteristics of
superior CB units (CBUs). In the US, the C.W. Bill Young Cell Transplantation
Program’s National CB Inventory established criteria defining quality CBUs.
Applying current criteria, the ratios of collected to bankable CBUs for Caucasian
and African-American (AA) donors are 2:1 and 5:1, respectively. We hypothesized
that certain maternal/donor characteristics may predict high cell content.
Identifying these characteristics would be beneficial by improving the yield of
quality CBUs from AA donors.
Methods: We analyzed CBUs processed by the Carolinas CB Bank between
9/07-7/09 using standard eligibility criteria. Technical characteristics routinely
measured post-processing (TNC, CD34+ and CFU) were correlated with clinical
characteristics [maternal age, delivery type, gender, birth weight (BW), gestational
age, race/ethnicity and collected volume]. Univariate and multivariate models
were created dichotomizing for cellular content. In the overall cohort, AA race
predicted lower cell content. Therefore, AA subgroup (n=1067) analyses were
performed.
Results: Comparing to the Caucasian cohort, AA donors were younger (p<0.0001),
smaller (p=0.0042) and more likely to be born to younger mothers (p<0.0001)
by Caesarian delivery (p=0.0017). Processed CBUs had lower median TNC, CFU
(both p<0.0001) and CD34+ (p=0.04) content (Table 1). In univariate analysis
of AA donors, Caesarian delivery [OR 1.41 (95%CI 1.08-1.83) p=0.0093], volume
15
Oral ABSTRACTS
collected [>80ml, OR 4.59 (95%CI 3.46-6.10); p<0.0001] and BW [>3500g, OR
1.66 (1.28-2.15) p<0.0001] predicted high TNC (>1x109). In multivariate analysis,
BW [OR 1.43 (95%CI 1.09-1.89); p=0.01] and collected volume [OR 4.41 (95%CI
3.30-5.84); p<0.0001] remained significant predictors.
Conclusions: Collecting from AA infants weighing >3500g and maximizing
collection volume increases the likelihood of meeting banking criteria. These
findings suggest increased AA donor collections is needed to achieve a robust,
high quality inventory.
29
28
Post Thaw CD34 Recovery and Viability of
Hematopoietic Progenitor Cells is a Valuable Clinical
Tool in Autologous Blood and Marrow Transplants.
STABILITY OF THAWED HEMATOPOIETIC PROGENITOR CELLS
(HPC) IN DIMETHYL SULFOXIDE (DMSO) IN FILTERED AND
UNFILTERED PRODUCTS
Susan M Berrigan, MLT, Debbie L Kury, MLT, Hana Stastna, Laboratory Specialist, April
ME Hillman, BSC/MLT, Allison K Kornylo, BSC/MLT, Joanne Luider, BSc MLT, Nicole L
Prokopishyn, PhD, Calgary Laboratory Services, Calgary, Canada.
Mehraboon S. Irani, MD1 Robert Endres, PhD1 Robin Medis, CHS(ABHI)1 Melissa Marlowe,
MT(ASCP, CHS(ABHI)1 Stephen Hunt, BS1 Roberta Bruhn, MS, PhD2 Frank A. Nizzi, DO3
1
Blood Systems Laboratories, Tempe, AZ, USA, 2Blood Systems Research Institute, San
Francisco, CA, USA, 3Blood Systems, Inc , Scottsdale, AZ, USA.
Background: DMSO is considered toxic to HPCs after thawing, so infusion of
cryopreserved cells is typically accomplished by bedside-thawing and immediate
infusion. We compared the viability and growth potential of HPC products and
cryovial samples stored in 5% DMSO for up to three hours after thawing and we
compared cell loss between unfiltered and filtered bag samples.
Methods: The study was carried out on samples from 10 cryovials and 11 bags,
thawed in a 37 C water-bath. Colony forming units (CFU), CD45+ cell viability by
7-amino-actinomycin-D (7AAD), and CD34+ cell concentration were assessed to
see if they significantly changed when the cell samples were left at 1 to 10 C for 3
hours. The samples were diluted in an equal volume of a mixture of 25% albumin
and Plasmalyte A (one part 25% albumin and four parts Plasmalyte A). A 170 to
210 micron filter was used for the bags (standard blood filter).
Results: The results showed no significant difference between the bag and cryovial
samples with respect to CD45+ viabilities, CD34+ cell concentration and CFU at
0 and 3 hours respectively, with the sample stored at 1 to 10 C, (Table 1 and 2).
Also there was no significant difference in these parameters between the filtered
and unfiltered samples with the exception of CFU at 0 hours which was increased
after filtration (Table 3).
Discussion/Conclusion: This study shows that HPCs are stable when thawed
and appropriately diluted for up to three hours at 1 to 10 C. This would allow
laboratories to perform viability and CFU assays within three hours of thawing,
provided the samples are diluted and kept refrigerated. The study also suggests
that filtration does not decrease the number of cells or CFU in the product which
may allow routine use of standard blood filters for infusion.
High quality cryopreserved cellular therapy products (CTPs) are essential to
successful engraftment of autologous transplant patients. Rapid and consistent
assessment of cell recovery and viability following cryopreservation provides
insight into the quality and nature of the CTPs and can be utilized as a prospective
tool for engraftment outcome prior to conditioning of patients. Previously, we have
reported use of a simplified dilution method and modified enumeration protocol
for assessment of CD34+ cell recovery and viability in representative quality
control (QC) samples of Autologous HPC, Apheresis CTPs. Our laboratory now
utilizes post-thaw analysis of cryopreserved CTPs as a routine quality assessment
measure. The week following cryopreservation, QC samples of CTPs are thawed
in the laboratory at 370C and immediately diluted 1:4 in a solution of 10% LMD40
and 5% Human Serum Albumin (HSA). Diluted QC samples are analyzed by flow
cytometry for CD34+ cell counts and viability using a modified ISHAGE protocol for
gating and analysis (Calgary protocol). Average post-thaw recovery of CD34+ cells
(as compared to pre-freeze values) was 116 %±19% with an average CD34+ cell
viability of 98%±9% and TWBC recovery was 64%±23% in the samples analyzed.
The efficient methods described result in consistent and reliable determination
of CD34+ cell counts and viability in products post-thaw. Our cryopreservation
method typically generates products with high post-thaw recovery of viable CD34+
cells. Assessment of QC samples prior to conditioning and transplant of patients
can rapidly alert us to products that may pose a probable risk of delayed or poor
engraftment in patients.. Any products that have CD34+ cell recovery and/or
viability below average levels are flagged and transplant physicians are informed.
Contingency plans are in place for these recipients should engraftment fail. The
implementation of this post-thaw assessment of cryopreserved products is aimed
to ensure the utmost in patient safety.
30
Establishing the necessary data to model the future
resource requirements and predicted healthcare
targets for cell therapy as part of routine clinical
practice
Emily J. Culme-Seymour, 1 Natasha L. Davie,2,3,4 David A. Brindley,2,4,5 Chris Mason,1,4 1London
Regenerative Medicine Network, London, UK, 2Harvard Stem Cell Institute, Cambridge, MA,
USA, 3Harvard Medical School, Boston, MA, USA, 4University College London, London, UK,
5
Harvard Business School, Boston, MA, USA.
Stem cell science has advanced to where patient benefits are starting to emerge.
However, if cell therapies are to realize their full potential and become routine
clinical practice for large numbers of patients, additional science coupled to
commercial translation will be essential. The cell therapy industry (CTI) is
presently a small but potentially rapidly growing new global healthcare sector.
Success is totally dependent on resolving a number of factors unique to cells
as therapies including: mechanisms of action, manufacturing, regulation and
clinical trials. To understand how to solve these challenges, it is essential to
robustly forecast the size and resource demands of the sector over the next two
decades. Due to the highly regulated nature of medicines, one reliable method
16
Oral ABSTRACTS
is to analyze the therapies that are currently undergoing clinical trials – the
future pipeline. A search was performed on the website clinicaltrials.gov using
the embedded search-engine and key terms relating to ‘cell therapy’. 17,362 files
were extracted (27/06/2010) and individually screened for relevance using the
British Standard Institute (BSI) definition of ‘cell therapy’. The resulting 2,765
trials were then categorized and core information collated including: trial phase,
cell source (autologous/allogeneic), current activity of the trial and responsible
national regulatory agency. Key results: [1] Near equal number of autologous and
allogeneic trials, [2] Majority of trials are late-stage, [3] Significantly larger number
of transient cell therapies as opposed to permanent cell replacement. This
poster highlights all the key findings and discusses the implications for discovery
scientists, clinicians, businesses and governments.
31
REGULATORY IMPLICATIONS OF ALLOGENEIC CELL
BANKING STRATEGY
Christopher A Bravery, PhD, Consulting on Advanced Biologicals Ltd, London, UK.
The business model for allogeneic cell therapies can appear attractive since
it closely follows that of traditional biotech products. In most cases a single
donation can be used to manufacture multiple batches of many doses with the
benefit of greater batch-to-batch consistency due to the use of a cell banking (CB)
system. However, unlike traditional biotech, these CBs are rarely large enough
to last for the anticipated product life-cycle, meaning that more than one donor
will be required. Qualification of each CB can easily exceed 6 months’ work and
$0.5 million, even before factoring in the time and cost of agency approval, and
with the potential risk that products derived from multiple CBs aren’t comparable.
How this issue is tackled during early development will have ramifications both
for market authorisation and the subsequent regulatory burden of maintaining
the product license; and consequently profitability on-market.
One objective of product development should be to provide the tools to allow
comparability following process changes, including the introduction of new CBs,
without the need to resort to clinical confirmation. The sheer complexity of cell
therapy products means this objective can be a significant challenge and some
clinical qualification of subsequent CBs is likely to be requested by regulators, at
least during development. To avoid this multiple CBs from multiple donors could
be evaluated in clinical trials, yet until relevant quality parameters, are established,
avoidance of extensive clinical trials with multiple CBs brings the risk that the
CBs are not equivalent, and the resulting clinical data inconclusive. Several broad
strategies are available, each of these are discussed along with their benefits and
limitations. While a one-size-fits-all solution is not possible, by understanding the
ramifications of the various options the best solution for both development and
on-market can be selected.
32
SYSTEMIC HUMAN ORBITAL FAT-DERIVED STEM CELL
TRANSPLANTATION AMELIORATES ACUTE INFLAMMATION IN
LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY
Ming-Hsien Chien, Jennifer H Ho, Wan Fang Hospital, Taipei Medical University, Taipei,
Taiwan.
Acute lung injury (ALI) results in acute respiratory distress syndrome (ARDS).
There is no standard therapy for ARDS but supportive care. Stem cells offer a new
therapeutic potential for tissue regeneration due to the self-renewal, multipotency,
and paracrine effect. The objective of this study is to investigate the effects
and the mechanisms of systemic human orbital fat-derived stem/stromal cell
(OFSCs) transplantation on lipopolysaccharide (LPS)-induced ALI. In this study,
twenty-five μg LPS in 50 μl sterile saline or 50 μl of sterile saline was delivered into
male BALB/c mice via intra-tracheal injection. Twenty minutes later, the animals
were further randomized into subgroups that received either a tail vein injection
of 3×105 OFSCs in 50 μl of phosphate buffer solution (PBS) or 50 μl of PBS. We
demonstrated that systemic OFSC transplantation did not trigger an immune
response in BALB/c mice. OFSCs significantly reduced LPS-induced pulmonary
inflammation, which was evidenced by a decrease in total protein concentration
and neutrophil counts in alveolar fluid via bronchoalveolar lavage (BAL), which
reduced endothelial and alveolar epithelial permeability, as well as neutrophil
(Ly6G expressing cells) and macrophage (CD68 expressing cells) infiltration.
The LPS-induced expression of CD14, inducible nitric oxide synthase (iNOS), and
transforming growth factor-β (TGF-β) in lung tissue was significantly inhibited by
OFSCs. OFSCs not only reduced the circulation numbers of macrophages and
neutrophils (CD11b expressing cells), but also decreased systemic macrophagereleased pro-inflammatory chemokine levels such as macrophage inflammatory
protein-1-gama (MIP-1g), B-lymphocyte chemoattractant (BLC), interleukin-12
(IL-12), and subsequent circulation helper T cell (CD4 expressing cells) numbers.
Moreover, few human OFSCs were detectable in the recipient lung after acute
inflammation subsided. Taken together, systemic OFSC transplantation was
effective in modulating inflammation during acute lung injury. The therapeutic
effect was mainly attributed to the inhibition of the macrophage-mediated
inflammatory response.
33
A NEO-KIDNEY AUGMENT PRODUCT FOR KIDNEY
REGENERATION IN A LARGE ANIMAL MODEL OF CHRONIC
KIDNEY DISEASE
Deepak Jain, Richard Payne, Joydeep Basu, Craig Halberstadt, Toyin Knight, Neil Robbins,
Darell McCoy, Elias Rivera, Christopher Gengeimer, Andrew Bruce, Rusty Kelley, Timothy A.
Bertram, John Ludlow, Kelly Guthrie, Kelly Guthrie, Tengion Inc., Winston Salem, USA.
Chronic kidney disease (CKD) is continued loss of renal function over time.
Current renal therapies include dialysis and kidney transplant. An urgent need
exists for new treatments to restore renal function thereby delaying dialysis/
transplant. Regenerative medicine approaches hold the promise of fulfilling this
unmet medical need. Efforts by other laboratories to regenerate diseased kidneys
have focused on the application of mesenchymal stem cells to reconstitute both
kidney structure and function. In contrast, we have identified populations of tubular
epithelial cell-enriched primary renal cells (Selected Renal Cells, SRC) that positively
affect the disease phenotype in rodent CKD models, potentially by interfering with
onset of tubulo-interstitial fibrosis and mobilizing host renal stem cell populations.
This study reports on the development of a Neo-Kidney Augment (NKA) product
containing a population of SRC and a natural biomaterial. SRC are obtained from
a kidney biopsy and density gradient separation of cells and have been shown to
provide a significant regenerative stimulus in the rodent models of CKD, delaying
disease progression and reduced disease-related mortality. Gelatin-based hydrogel
biomaterials provide stability and targeted delivery for SRC and may facilitate cell
engraftment, infiltration and creation of a microenvironment for cell retention and
expansion. A canine nephrectomy model was used for evaluation in the large animal
model of CKD. Treatment with SRC resulted in a statistically significant increase in
uromodulin and a decrease of vitamin D binding protein in the urine, indicative of
restoration of tubular cell function. Histological evaluation in the dogs (47-week
post implantation) revealed that NKA product prototypes were well tolerated. Taken
together, these observations provide evidence that selected renal cells and gelatin
based hydrogel biomaterials may be effective for kidney tissue regeneration in
chronic kidney disease. In addition, this model may provide a useful approach to
evaluate the efficacy of potential therapeutics for CKD.
17
Oral ABSTRACTS
34
35
CONTRIBUTON OF HUMAN INDUCED PLURIPOTENT STEM CELL
DERIVED ENDOTHELIAL CELLS IN VASCULAR REGENERATION OF
BLEOMYCIN-INDUCED SCLERODERMA MOUSE MODEL
STANDARDIZED QUALITY CONTROL AND CELL EQUIVALENCY
FOR XENO-FREE MULTISTEM®, AN ADHERENT STEM CELL
THERAPEUTIC
Manizheh Ajdari, MSc3 Mohammad Reza Baghban Eslami Nejad, PhD3 Hossein Baharvand,
PhD3,4 Nasser Aghdami, MD., PhD1,2 1Department of Regenerative Medicine, Cell Science
Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran,
2
Royan Cell Therapy Center, Tehran, Iran, 3Department of Stem Cells and Developmental
Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology,
ACECR, Tehran, Iran, 4Department of Developmental Biology, University of Science and
Culture, ACECR, Tehran, Iran.
Annelies Bogaerts, Bart Vaes, David Craeye, Jef Pinxteren, ReGenesys, Heverlee, Belgium.
Systemic sclerosis (scleroderma) is an autoimmune disorder with vascular
dysfunction. Lack of normal vasculogenesis and angiogenesis despite of general
increase in many potent angiogenic factors emphases the role of endothelial
progenitor cells (EPCs) in pathogenic vascular in SSc. So, in this study, we
examined the human iPS derived endothelial cells contribution in vasculogenesis
of sclerotic skin in mice model.
Methods: Undifferentiated human iPS cells were cultured for 6 days on type-IV
collagen-coated dishes and Flk-1(+) cells sorted by fluorescence activated cell
sorting (FACS). hiPSC-ECs were obtained by sub-culturing these on collagen
type-I in EGM-2 medium. Phenotype and function of CD31-positive sorted cells
were studied by reverse transcription-polymerase chain reaction, flow cytometry,
immunocytochemistry, DiI-labeled acetylated low-density lipoprotein uptake, and
Matrigel tube formation assay.
Scleroderma was induced in BALB/c mice by daily subcutaneous injection of
Bleomycin for 4 weeks. Then, hiPSC-EC were injected subcutaneously in three
equidistant sites of the sclerotic skin. Presence of fluorescent DiI marked cells and
skin fibrosis were evaluated by histologic and biochemical methods.
Results: Sorted CD31-positive hiPSC-ECs exhibited typical endothelial cobblestone
morphology and stained positively for CD144, CD31, and CD146 as EC markers.
Forming capillary-like structures in matrigel and incorporating acetylated-LDL
confirmed their endothelial function in vitro. The transplantation of hiPSC-ECs to
the bleomycin-induced scleroderma mouse model contributed to the vessels and
reduced collagen deposition, the number of total and degranulated of mast cells
and improves injury in a mouse model of SC four weeks post-transplantation.
Conclusion: Our data show that transplantation of ECs derived from hiPSCs can
provide some benefit in the setting for scleroderma regeneration.
18
An indispensable part of the development and manufacturing of an advanced
cell therapy product is quality control. The legislation concerning the quality
requirements of these types of products is still under development and new, more
stringent guidelines are expected to be released in the near future. ReGenesys
therefore aims to be first in class by designing a high level QC pipeline. A set of
standards such as screening for growth, marker expression, immunosuppression,
multipotent differentiation and chromosome stability (SNP arrays) is conducted
on each newly produced batch of cells. This pipeline is sufficient to guarantee
the identity and quality of MultiStem®. However, since we are advanced with
the development of xeno-free MultiStem® and are seeking novel production
technologies, it is essential to prove that such modifications in the manufacturing
process do not lead to altered phenotypes with reduced therapeutic capacity. We
are currently extending our QC pipeline with different genome-wide screening
methods to comprehensively characterize the molecular phenotype of our
product. Next-generation sequencing is employed to analyze DNA methylation
of MultiStem® in order to prove epigenetic stability during cell expansion. It is
very important to define DNA methylation-specific markers that discriminate
MultiStem® from competing products based on e.g. mesenchymal stem cells.
Screening for gene expression and miRNA patterns, and subsequent pathway
analyses are done to investigate cultured cells and fine-tune their manufacturing.
Here, we present our QC pipeline that implements these genomics tools to ensure
that no intrinsic changes occur and that the identity of MultiStem® is secured.
POSTER ABSTRACTS
36
Enhanced Engraftment and FUNCTIONAL Benefit of
Human Cardiosphere-derived STEM Cells Delivered in
an In Situ Polymerizable Hydrogel
Agnieszka Blusztajn, 1 Ke Cheng, Ph.D.2 Deliang Shen, M.D.2 Tao-Sheng Li, Ph.D.2 Baiming
Sun,2 Giselle Galang,2 Thomas I. Zarembinski, Ph.D.3 Glenn D. Prestwich, Ph.D.4 Eduardo
Marbán, M.D. Ph.D.2 Rachel R. Smith, Ph.D.1 Linda Marbán, Ph.D.1
1
Capricor Inc., Los Angeles, USA, 2Cedars-Sinai Medical Center, Los Angeles, USA, 3BioTime,
Inc., San Francisco, USA, 4University of Utah, Salt Lake City, USA.
Cardiosphere-derived cells (CDCs) induce functional benefits in animal models of
myocardial infarction (MI), despite the fact that the majority of cells delivered into
the heart die within 24 hours, a universal problem in cardiac cell therapy. Alternative
methods for delivery are needed in order to enhance retention, which will in turn
enhance engraftment and functional benefits. The present study tested a hydrogelbased delivery approach. An in situ polymerizable hydrogel consisting of hyaluronan
and porcine gelatin (HyStem-C™) was formulated as a liquid to gel within 20 minutes.
In vitro, CDC viability in HyStem-C was 87±9% after 72 hours in culture and remained
high after one week (84±19%). Trans-membrane migration assays established that
the CDCs’ ability to migrate was not compromised by the HyStem-C. In vivo, MI was
created in mice and CDCs were delivered in either saline or Hystem-C. Control mice
received saline or Hystem-C sans cells. Mice underwent echocardiography at baseline
and 3 weeks post-MI. Ejection fractions (EFs) were comparable across all groups at
baseline indicating similar ischemic injury. After 3 weeks, EF deteriorated in the saline
only group (Fig. 1A, 9.4±4.4%) and was preserved in the HyStem-C only and saline
+ CDCs groups. The HyStem-C + CDC group showed a substantial increase in EF
(+16.6±4.3%, p<0.05). CDC retention 24 hours post-MI or engraftment 3 weeks postMI was quantified using real-time PCR. CDCs delivered in saline showed minimal
retention (Fig. 1B, 5.8±2.2%). HyStem-C delivery increased retention substantially
(35.0±25.2%, p<0.05). This magnitude of enhanced engraftment persisted for 3 weeks
post-MI (Fig. 1C, 1.2±0.4% vs. 5.9±0.5%, p<0.05). Morphometry revealed greater LV
remodeling in the HyStem-C + CDC group which had the smallest infarct size and the
thickest infarct walls (Fig. 1D&E). The CDC + Hystem-C formulation demonstrates
superior engraftment and functional benefits in contrast to a traditional delivery
method in vivo.
37
MONITORING THERAPEUTIC INTERVENTION THROUGH
INFLAMMATORY RESPONSE WITH CLINICALLY TRANSLATIONAL
19F MRI
Brooke M. Helfer, 1 Anthony Balducci,1 Eric T. Ahrens,2 David Schwartzman,3 Amy Wesa,1
1
Celsense, Inc, Pittsburgh PA, USA, 2Carnegie Mellon University, Pittsburgh PA, USA,
3
University of Pittsburgh, Pittsburgh PA, USA.
In the case of acute myocardial infarction (AMI), the degree of inflammation and
potential damage to the heart are areas of great interest for both preventative
and regenerative medical intervention. The ability to non- invasively monitor
inflammation related to AMI and to evaluate treatment is critically needed. V-Sense,
a perfluorocarbon tracer agent that labels circulating inflammatory cells, which
are then recruited out of circulation to sites of inflammation, enables detection by
19F MRI. Due to negligible 19F background in the host, detection of 19F and thus
inflammation is highly-specific. To demonstrate utility of this reagent, a collageninduced arthritis model was selected, where the inflammatory process and response
to therapeutic intervention are well understood. Disease progression in the rat hind
limbs was monitored by caliper measurements and 19F MRI on days 14, 21 and 28,
including the height of clinically symptomatic disease. Naïve rats served as controls.
The capacity of V-Sense to assess the effectiveness of therapy was studied in a cohort
of rats administered oral prednisolone on days 14 to 28. Comparison of caliper
measurements to the 19F accumulation, from 19F MRI, validated the quantitative
nature of this approach. Translating the process into a porcine infarct model
demonstrated the ability to pinpoint cardiac infarct related inflammation. These
studies support the use of V-Sense to monitor and quantify the inflammatory process
in disease, guide treatment, and monitor therapeutic interventions.
38
Ixmyelocel-T Protects the Heart from Damage in a
Murine Model of Heart Failure
Erin A Booth, Josh Osborne, Nikki Murphy, Judith Schmitt, Frank Zeigler, Ronnda L Bartel,
Aastrom Biosciences, Ann Arbor, USA.
Ixmyelocel-T is a patient specific, bone marrow derived expanded multicellular
therapy. The multicellular composition of the product results in multi-functional
properties including: tissue remodeling, immuno-modulation & the promotion of
angiogenesis, which is targeted to address the multiple underlying causes of many
severe cardiovascular diseases. The ability of ixmyelocel-T to promote tissue salvage
indicates that it may be protective to the ischemic heart. While there is currently
no accepted animal model for dilated cardiomyopathy (DCM), a murine model of
chronic left anterior descending (LAD) coronary artery occlusion was used to evaluate
ixmyelocel-T as a potential treatment for DCM.
Since ixmyelocel-T is a human bone marrow-derived product, an immunodeficient
murine model of heart failure was used. Mice underwent permanent occlusion of
the LAD and were allowed to recover. After two weeks, the chest was opened and
vehicle or ixmyelocel-T was injected in the border of the infarct (0.25x106 cells/heart).
Hearts were analyzed four weeks following injection. Ixmyelocel-T treatment resulted
in a significant decrease in infarct length (mm) compared to vehicle control (Donor
1: 2.72+/- 0.77 vs. 6.14+/- 0.43, P<0.001; Donor 2: 3.80+/- 0. 37 vs. 7.34+/- 0.47,
P<0.001). Vehicle had no effect on infarct size compared to the non-treated surgical
ischemic controls. Mice treated with ixmyelocel-T demonstrated reduced mortality
compared to vehicle treated mice with a 8-36% mortality in the treatment groups and
37-50% mortality in the vehicle groups (P=NS).
This study demonstrates the protective effect of ixmyelocel-T in the murine model of
heart failure resulting in a decrease in infarct size and increase in survival. Preliminary
data suggest that ixmyelocel-T does not increase capillary density and reduces
myocyte apoptosis suggesting ixmyelocel-T exerts a protective effect without inducing
angiogenesis. It is possible that this reduction in apoptotic cell death may contribute
to the decreased infarct size observed with ixmyelocel-T treatment.
39
WHOLE HEARTS USED TO MANUFACTURE ALLOGENEIC
CARDIOSPHERE-DERIVED STEM CELLS AT A LARGE-SCALE
Ileana Valle, Agnieszka Blusztajn, Michelle Kreke, Ph.D., Linda Marban, Ph.D., Rachel R.
Smith, Ph.D., Capricor Inc., Los Angeles, USA.
Allogeneic cardiosphere-derived cells (CDCs) are safe and effective in animal
models of myocardial infarction (MI). The present study demonstrates the utility
of an allogeneic source using a method designed for autologous production, the
feasibility of scaling-up the method to manufacture products for clinical use, and
19
POSTER ABSTRACTS
the potency of the products. Whole hearts (n=3) were collected from eligible,
consenting donors via a research interchange or organ procurement organization.
CDCs were generated by the 3-stage culture process (Fig. 1A) from each donor
at either a small- or large-scale (Fig. 1B) and showed the expected phenotype.
Manufacturing process scale-up incorporated new equipment and culture
vessels. Tissue chopping, performed with scissors and a scalpel in the small-scale
method, was performed with a tissue slicer and tissue chopper to enable rapid
creation of viable, uniform tissue explants. Two types of culture vessels, each
available with two types of surface treatment, were incorporated into the explantderived cell outgrowth, cardiosphere formation, and CDC expansion stages to
reduce vessel footprint, reduce the total number of vessels, and eliminate two
surface-coating reagents. Tissue storage proved feasible for 3 days at refrigerated
temperatures or indefinitely when cryopreserved with tolerable impacts on CDC
yield. Tissue acquisition from any region of the heart proved possible with a
slight yield and potency advantage seen with tissue from the atria. Potency was
tested using a mouse model of MI. Allogeneic CDCs had potency equivalent to
that seen previously with autologous products. Absolute improvement in ejection
fraction from baseline to 3 weeks post-MI was >5% and significantly different from
control (Fig. 1C, p<0.05). At the current scale, 32 grams of tissue can be readily
processed to yield 700 doses of 25 million CDCs per dose from each donor, even
after accounting for necessary QC testing losses, and the remaining 50-300 grams
cryopreserved for future use.
40
Characterisation and properties of bone marrow
mesenchymal cells from patients with chronic
heart failure
Izida Minullina, PhD, Renata Dmitrieva, PhD, Sergey Anisimov, PhD, Andrey Zaritskey, Prof,
Almazov Federal Heart, Blood and Endocrinology Centre, St Petersburg, Russia.
Mesenchymal stem cells (MSC) are gaining popularity as experimental therapy for
a number of conditions, including cardiovascular disease. There is evidence that
MSC mediated protective effect is realized via the release of paracrine factors. We
hypothesized that MSC from patients with heart failure (HF) may display differences
in expression of key regulatory molecules compared to healthy donors (D). All
subjects (11 D and 38 HF) were enrolled under SICA-HF study (7FP). Experiments
were conducted with MSC at passages 3-4 that underwent 15-16 in vitro population
doublings. We evaluated the expression and secretion of several cytokines
modulating cardiac remodeling, neovascularization, and inflammatory response.
VEGF-A and TGFβ expression and secretion did not differ in MSC from HF patients
compared to D. IL-6 and IL-8 were up-regulated in HF patients compared to D at
both protein (2629±277 pg/ml vs 1672±343 pg/ml, p=0.11 for IL-6; 460±127 pg/
ml vs 56.2±10.5 pg/ml for IL-8, p=0.12) and RNA level (fold change 9.94±1.25 vs
5.29±1.17, p=0.059 for IL-6; 13.07±2.23 vs 8.14±2.57 for IL-8, p=0.25), though the
difference did not reach statistical significance. There was a correlation between the
levels of MSC secretion (r=0.57, p=0.0004) and expression (r=0.56, p=0.0001) of
IL-6 and IL-8. A similar pattern was observed in MSC production of immunoreactive
Angiopoietin2 and HGF (r=0.459, p=0.0038). Secretion of Angiopoietin2 and HGF
20
was higher in HF patients compared to D, though the difference was not always
significant (251.9±34.4 pg/ml vs 123.0±43.7 pg/ml, p=0.09 and 3100±363.8 pg/
ml vs 1313±433.3 pg/ml, p=0.02). There was a tendency to increased expression
of Angiopoietin2 in MSC from HF patients (fold change 120.2±28.56 vs 22.2±6.56,
p=0.1). These data show that paracrine-mediated effects of MSC can be altered
in HF patients that should be taken into account in MSC-transplantation-based
therapeutic approaches.
41
Troglitazone up-regulates PTEN expression and
induces apoptosis of pulmonary artery smooth
muscle cells under hypoxia
WeiFeng Pi, MD, PhD2 Xue-Jun Guo, MD2 LiPing Su,3 Lei Ye, MD, PhD1 Wei-Guo Xu, MD2
National University of Singapore & University of Minnesota, Shanghai, China, 2XinHua
Hospital Affiliated JiaoTong University, Shanghai, China, 3National University of Singapore,
Singapore, Singapore.
1
Background and Aims: Increased proliferation and decreased apoptosis of pulmonary
artery smooth muscle cells (PASMCs) are main causes for hypoxemic pulmonary
hypertension. The study investigates the role of troglitazone (PPARΓ agonist) in
regulation of PTEN expression and apoptosis of PASMCs under hypoxia.
Methods: Normal human PASMCs were cultured in growth medium (GM) and
treated with troglitazone from 0.5-80 uM and cultured under hypoxia (5% CO2+
94% N2+1% O2) for 72 hours. Gene expressions of PTEN, AKT-1 and AKT-2 were
determined by quantitative RT-PCR (QRT-PCR). Protein expression levels of
PTEN, AKT and phosph-AKT (pAKT) were determined by western blot. Apoptosis
of PASMCs were determined by measuring activities of caspases-3, -8 and -9.
Results: Proliferation rate of PASMCs showed dose dependence of troglitazone,
the lowest proliferatiion rate was achieved at 60 uM under hypoxia (59.3±8.3%).
Gene expression level of PTEN was significantly increased, while AKT-1 and AKT2 did not change. Consistently, the PTEN protein expression also showed dose
dependence of troglitazone. Though AKT was unchanged in all cell samples,
reduced pAKT was found in troglitazone treated PASMCs. Troligazone increased
caspase-3, -8 and -9 activities of of PASMCs. Pre-treat PASMCs with bpV(HOpic)
and GW9662 (PPARΓ inhibitor) inhibited PTEN protein expression and recovered
PASMCs proliferation rate.
Conclusion: Troglitazone can increase PTEN expression under hypoxia in a dose
dependent pattern. Troglitazone can increase apoptosis of PASMCs under hypoxia.
The increased PTEN expression is mediated through PPARΓ signalling pathway.
42
Will not be presented
43
DEVELOPING A CELL-BASED THERAPY FOR PREVENTING AND/
OR REMOVING ECTOPIC CALCIFICATION IN CALCIFIC AORTIC
VALVE DISEASE (CAVD)
Meiting Wu, Cameron Rementer, Hsueh-Ying L. Yang, Anthony Blau, Cecilia M. Giachelli,
University of Washington, Seattle, USA.
Calcific aortic valve disease (CAVD) is the most common indication for surgical
valve replacement in the US. Ectopic calcification is particularly destructive to the
mechanical functions of the vasculature, and is the major cause of valve failure
in patients with CAVD. The major treatment for calcification in CAVD is valve
replacement therapy, which is associated with substantial morbidity and mortality,
as well as a significant re-implantation rate. There are currently no drugs or cell
therapeutics that target CAVD. A treatment aimed at preventing and/or removing
calcification in CAVD would have enormous health benefits for patients who suffer
from this problem. In this study, we report the development of a bioengineered
POSTER ABSTRACTS
cell therapy approach to control monocyte-derived macrophage differentiation
to osteoclasts, the main mineral resorbing cells in the body. Oligomerization
of receptor activator of nuclear factor κB (RANK) is known to be essential for
osteoclast differentiation from monocyte/macrophage precursors. We engineered
a murine monocytic cell line, RAW264.7, to express a fusion protein comprising
the intracellular RANK signaling domain and FK506-derived dimerization
domains that bind to the chemical inducer of dimerization (CID), AP20187. Virally
infected cells containing these constructs were treated with the CID and dosedependent induction of tartrate-resistant acid phosphatase (TRAP) activity as
well as multinucleated osteoclast formation were observed. Furthermore, NF-κB
signaling was upregulated in a CID-dependent fashion, demonstrating effective
RANK intracellular signaling. Functionally CID-induced osteoclasts had robust
mineral resorptive activity in both two-dimensional and three-dimensional in
vitro resorption assays. Most importantly, the CID-induced osteoclasts died when
the CID was withdrawn, providing an efficient on/off switch to control extent
of mineral resorption. In summary, these studies are the first use to apply CID
technology to control osteoclast differentiation, survival, and mineral resorption,
and provide the basis for future development of an engineered autologous cell
therapy to treat calcification in CAVD.
44
Will not be presented
45
INTRAVENOUS ADMINISTRATION OF MESENCHYMAL STEM
CELLS EXERTS THERAPEUTIC EFFECTS ON INFARCTED HEART
MODEL OF RABBIT: FOCUSING ON POTENTIAL EFFECTS OF
MYOCARDIAL REGENERATION
Soontaree Petchdee, Dr.1 Nirachada Limsuwan, Dr.2 Taweesak Songserm, Assoc. Prof.1
Petcharin Srivatanakul, Dr.3 1Faculty of Veterinary Medicine, Kasetsart University, Kamphaeng
Saen, Thailand, 2Kasetsart University Veterinary Teaching Animal Hospital, Kamphaeng Saen,
Thailand, 3BioEden Asia Tooth Cell Bank, Bangkok, Thailand.
Heart disease is the world’s leading cause of death, threatening more human lives
than any other disease. Advanced symptoms usually include myocardial infarction
(MI) due to atherosclerosis of coronary arteries. Even after successful coronary
revascularization, cell death continues and the loss of cardiomyocytes ultimately
leads to progressive ventricular dilation and heart failure. To repair or regenerate
lost myocardium and coronary vasculature, stem cell transplantation is a promising
therapeutic approach for the treatment of coronary heart diseases. In this study, the
therapeutic effects of multipotent Stem Cells from Human Exfoliated Deciduous
teeth (SHED) were examined. These dental-tissue-derived stem cells have
mesenchymal-stem-cell (MSC) qualities, including the capacity for self-renewal
and multilineage differentiation potential. Thirteen adult male New Zealand White
rabbits underwent a left thoracotomy approach for producing chronic infarcted
heart. The marginal branch of the left circumflex coronary artery was ligated over
8 weeks to produce an ischemic area of 30-40% of the left ventricle (LV). MSCs
were freshly prepared and 0.5 ml of 106 cells were injected to each of six rabbits
via the marginal ear vein. Heart rate variability (HRV) was measured to reflect
cardiac autonomic modulation. The infarcted size measurements were performed
at the end of each experiment using ImageTool software version 3.0. Here we show
significant improvement in cardiac autonomic tone and reduction in infarcted
size in the MSC-treated group. MSC transplantation confirmed a recovery of heart
function. The results suggest that MSC could provide an alternative selection of
the precursor cells for cardiac repair. Keyword: Mesenchymal stem cell, myocardial
infarction, cardiac repair.
46
SERUM-FREE SPHEROID SUSPENSION CULTURE OF
MESENCHYMAL STEM CELLS
Andrew M. Campbell, 1 Stella Alimperti,2 Yuan Wen,1 Pedro Lei,2 Stelios Andreadis,2 1Life
Technologies Corporation, Grand Island, USA, 2Department of Chemical and Biological
Engineering, University at Buffalo, The State University of New York, Amherst, USA.
Mesenchymal stem cells (MSC) offer great therapeutic value, especially for
immune-mediated diseases. As more and more clinical trials are initiated using
MSC, expansion of MSC in large quantities has been increasingly in demand in
order to satisfy the projected clinical requirement of ~109 cells per treatment.
Current methods involve growth of MSC on 2-dimensional (2D) tissue culture
treated surfaces either in flasks or on micro-carriers using bioreactors. However,
these 2D culture approaches present difficulties to efficiently expand MSC in large
scale, as the expansion ratio is largely restricted by the area of the surface for cell
attachment. Here we present a novel serum-free medium that allows human bone
marrow derived MSC to proliferate in a non-adherent manner as spheroids (cell
aggregates) in suspension. This regulatory-friendly serum-free system does not
require a surface for MSC attachment and therefore allows the efficient expansion of
MSC at high density. In a custom serum-free medium, cell growth achieved ~7 fold
cell expansion in 7 days and maintained higher than 95% cell viability throughout
the culture. Importantly, flow cytometry analysis revealed that the MSC expanded
in suspension expressed the expected MSC surface markers, namely CD 44,
CD 73, CD 90 and CD 105. When the cells expanded in suspension were plated
back to adherent culture, the cells displayed morphology similar to adherently
maintained MSC. When induced to differentiate both morphological analysis and
colorimetric staining of the differentiated cells showed successful differentiation
toward osteoblast, chondrocyte and adipocyte lineages. Taken together, these
results demonstrate that the novel serum-free medium supports MSC growth in
suspension while maintaining the desired differentiation potential. This work may
be applied to efficiently produce large quantities of high-quality undifferentiated
MSC for cell therapies.
47
SCALABLE EXPANSION OF HUMAN MESENCHYMAL STEM CELLS
USING A MICROCARRIER-BASED SYSTEM UNDER SERUM-FREE
AND XENO-FREE CONDITIONS
Andrew M. Campbell, 1 Claudia Lobato da Silva,2 Francisco dos Santos,2 Pedro Z. Andrade,2
Shayne Boucher,3 Mohan Vemuri,3 Joaquim Cabral,2 1Life Technologies Corporation, Grand
Island, USA, 2Department of Bioengineering and IBB - Institute for Biotechnology and
Bioengineering, Instituto Superior Técnico, Lisboa, Portugal, 3Life Technologies Corporation,
Frederick, USA.
The potential demand for clinical and commercial scale numbers of human
mesenchymal stem cells (MSC) for cellular therapies requires a large-scale, fully
monitored and controlled culture system for MSC production. Previous results
from our laboratories have demonstrated the feasibility of MSC expansion on
microcarriers using a low (2%) serum-containing medium in a spinner flask
system (Eibes, G., et al. J Biotechnol. 2010). Although very promising, the use of
fetal bovine serum poses a regulatory risk for cell therapy applications.
Here we report the expansion of human MSC in a microcarrier-based system using
commercially available serum-free and xeno-free reagents (StemPro® MSC SFM
XenoFree, Life Technologies). Spinner flask studies demonstrated the ability of a
xeno-free system to support expansion of MSC from bone marrow (BM MSC) and
adipose tissue (ADSC) while maintaining the expected phenotype and differentiation
potential. After 14 days of culture, BM MSC reached a maximum cell density of 2.0x105
cells/ml (fold increase of 18) while ADSC expanded to 1.4x105cells/ml (fold increase of
14). The scale-up of this system was successfully achieved for BM MSC in a 1 L fullycontrolled stirred bioreactor. The cells maintained tri-lineage differentiation potential
and retained the MSC immunophenotypic profile. Process optimization strategies
21
POSTER ABSTRACTS
and the incorporation of a fed-batch approach have been incorporated to increase the
efficiency of the system.
This work demonstrates the ability of a commercially available, GMP, serumfree and xeno-free medium to support microcarrier-based large-scale expansion
of human MSC. This system can produce large numbers of high quality MSC,
representing a feasible and more efficient alternative to the traditional cell
expansion protocol for clinical-scale manufacture of MSC.
48
Will not be presented
49
INFLAMMATION AND TLR LIGATION DIFFERENTIALLY AFFECT
THE OSTEOGENIC POTENTIAL OF HUMAN MESENCHYMAL
STROMAL CELLS (MSC) DEPENDING ON THEIR TISSUE ORIGIN
Gordana Raicevic, 1 Mehdi Najar,1 Karlien Pieters,1 Cecile De Bruyn,1 Nathalie Meuleman,1
Dominique Bron,1 Michel Toungouz,2 Laurence Lagneaux,1 1Institut J. Bordet, Université
Libre de Bruxelles, Bruxelles, Belgique, 2Hôpital Erasme, Université Libre de Bruxelles,
Bruxelles, Belgique.
Mesenchymal stromal cells (MSC) can be isolated not only from bone marrow
(BM) but also from other tissues including adipose tissue (AT) and umbilical cord
Wharton’s Jelly (WJ). Thanks to their ability to differentiate into various cell types,
MSC are considered as attractive candidates for cell based regenerative therapy.
In degenerative clinical settings, inflammation or infection are often involved. In
the present work, we hypothesized that an inflammatory environment and/or TLR
ligation could affect the MSC differentiation potential.
MSC were isolated from BM, AT and WJ. Inflammation was mimicked by a
cytokine cocktail and TLR activation was induced through TLR3 and TLR4 ligation.
Osteogenesis was chosen as a model for MSC differentiation. Osteogenic
parameters included measuring of Ca2+ deposits and alkaline phosphatase (ALP)
activity at day 7, 14 and 21 of culture in an osteogenic medium.
Our results show that WJ-MSC exhibit a much lower osteogenic potential than the
two other MSC types. However, inflammation was able to strongly increase the
osteogenic differentiation of WJ-MSC as calcification and ALP activity appeared
as early as at day 7. This latter enzymatic activity remained lower than those
disclosed by BM-MSC. TLR3 or TLR4 triggering increased the osteogenesis in ATand to lesser extend in BM-MSC.
In conclusion, WJ-MSC constitutively disclose a lower osteogenic potential as
compared to BM and AT-MSC which is not affected by TLR triggering but strongly
increased by inflammation allowing WJ-MSC to reach the level of BM-MSC. These
observations suggest that WJ-MSC constitute attractive alternative MSC type for
bone repair. Indeed, WJ is an easily accessible source of large amounts of MSC
which can efficiently differentiate into osteoblasts in an inflammatory setting.
50
MESENCHYMAL STROMAL CELLS FROM UMBILICAL CORD
BLOOD AND PLACENTA SUPPORT PROLIFERATION AND
EXPANSION OF HEMATOPOIETIC PROGENITOR CELLS.
Guadalupe Fajardo-Orduña, 1 Héctor Mayani,1 Patricia Flores-Guzmán,1 Eugenia FloresFigueroa,1 Karina Estrada-González,1 Marta Castro-Manrreza,1 Guadalupe Alarcón-Santos,2
Juan José Montesinos,1 1Oncology Research Unit, Oncology Hospital, IMSS, Mexico City,
Mexico, 2Troncoso General Hospital, IMSS, Mexico City, Mexico.
Umbilical cord blood (UCB) and placenta (PL) have been suggested as alternative
sources of mesenchymal stromal cells (MSCs) instead of bone marrow (BM)
for cellular therapy protocols. In recent years, it has been shown that BM-MSCs
accelerates hematopoiesis and enhance hematopoietic stem cells transplantation.
We have shown that UCB- and PL-MSCs possess immunophenotypic and
22
biological characteristics similar to BM-MSCs, however, their capacity to support
proliferation and expansion of Hematopoietic Progenitor Cells (HPCs) at the
same conditions that BM-MSCs, is unclear. The present study was aimed to
isolate human MSCs from BM, UCB and PL and to compare their ability to support
in vitro proliferation and expansion of HPCs. To compare the hematopoietic
supporting capacity of MSCs, CD34+CD38-Lin- from UCB were cocultured in
contact with BM- UCB- and PL-MSCs in the presence of SCF, FL, TPO and IL-6.
Suitable aliquots of cells were used to monitor cell production, clonogenic activity
(CFC), and long-term culture-initiating cells (LTC-IC) output. MSCs from the
three sources were positive for “mesenchymal” antigens and several adhesion
molecules and were negative for hematopoietic and endothelial markers. MSCs
from the three sources had the same capacity to differentiate into osteocytes and
chondrocytes, however, UCB- and PL-MSCs had a lower capacity to differentiate
into adipocytes than BM-MSCs. In the presence of MSCs from three sources,
similar proliferation of HPCs and expansion of myeloid-CFC and CD34+CD38-Lincells were observed. Furthermore, similar eritroid-CFC and LTC-IC cell expansion
were detected in all cocultures with MSCs. Our results indicate that UCB- and
PL-MSCs have the same capacity than BM-MSCs to support proliferation and
expansion of HPCs in vitro. In conclusion, UCB- and PL-MSCs may prove useful
in the development of cellular therapy protocols.
51
GMP CELL CULTURE MEDIA FOR EXPANSION OF MSCs PRIOR TO
ALLOGENEIC OR AUTOLOGOUS TRANSPLANTATION
James R Musick, Ph.D., Andrea I Hall, Vitro Biopharma, Golden, USA.
Adult stem cells known as mesenchymal stem cells (MSCs) may be derived from
numerous tissue sources including Warton’s jelly of the umbilical cord, cord
blood, bone marrow, dental pulp, etc. MSCs are showing considerable promise in
clinical applications to treat a variety of conditions including articular disease and
injury, numerous forms of organ failure including heart, kidney and lung as well
as treatment of autoimmune diseases including MS, through immunosuppressive
effects of MSCs, etc. Many of the therapeutic applications of MSCs require
significant numbers of cells to achieve therapeutic benefit and expansion of
MSCs through cell culture procedures prior to transplantation is commonly used
to produce MSCs for therapeutic applications in both animals and humans. We
develop, manufacture and distribute the MSC-Gro™ Brand of cell culture media
optimized for MSC growth and differentiation. We offer formulations of MSC-Gro™
with application to clinical studies and therapeutic uses that comprise serum-free,
chemically-defined, animal-free formulations optimized for the growth of human
MSCs. One formulation is a complete, serum-free media that produces growth
rates comparable to serum-containing media and the other formulation is intended
for further supplementation by autologous or allogeneic serum to produce high
growth rates. We further describe the regulatory status, competitive performance
and availability of dried compositions of these clinical grade media formulations
manufactured to GMP standards.
52
Will not be presented
53
IDENTITY AND PURITY CHARACTERIZATION OF MESENCHYMAL
STEM CELLS EXPANDED IN A 3L STIRRED TANK BIOREACTOR
Julie Murrell, 1 Sanhdya Punreddy,1 Manjula Aysola,1 Ellen Binder,2 Donghui Jing,1 Daniel
Kehoe,1 Neethu Sunil,1 Knut Niss,1 Martha Rook,1 1EMD Millipore Corporation, Bedford,
USA, 2Merck Millipore, Darmstadt, Germany.
Mesenchymal stem cells (MSCs) are an attractive target for clinical study as
therapeutic agents. However, current multilayer flatbed culture expansion paradigms
are cumbersome, time consuming and typically limited in the ability to monitor cell
POSTER ABSTRACTS
characteristics during the growth process. We have described a new expansion
paradigm that uses a three liter, single use, stirred tank bioreactor with a microcarrier
scaffold for suspension expansion of MSCs. The bioreactor enables sampling
throughout the expansion run enabling monitoring of the cells to give an indication
of the quality of the expanded cells and to ensure the bioreactor expands MSCs in
the desired undifferentiated manner. Here we describe implementation of a panel
of assays that characterizes the cell state and the identity and purity of the cells.
Cell state assays include viability, apoptosis and cell cycle. Identity and purity were
assessed using both the standard recommended ISCT panel of positive and negative
markers in addition to markers indicative of MSC properties. Compared to standard
flask based expansion paradigms, we see few changes. When we intentionally perturb
the system, the combination of cell state assays, ISCT panel and expanded marker
panel clearly identify a change from the expected. In addition to indicating the cells
have changed and may no longer be appropriate for further experimentation, the
cell state assays can also indicate a need for feeding or harvest decisions. Thus, the
combination of cell state and marker panels for identity and purity can be considered
a process monitoring tool for expansion of MSCs in addition to a quality control
assessment following expansion.
54
Common Expression of Stemness Molecular Markers
and Early Cardiac Transcription Factors In Wharton’s
Jelly-Derived Stem Cells and hESCs
Lian R. Gao, 1 Qing A. Ding,1 Hai Y. Chen,1 Ning . Zhang,1 Shu Jiang,2 Tian . Li,1 Yu Chen,1
Zhi G. Wang,1 Yang Ye,1 Xiang Hu,2 1Navy General Hospital, Beijing, R.P.China, 2Beike BioTechnology Company, Shenzhen, R.P.China.
At present, there are still significant problems that impede the clinical use of
hESCs and iPS cells including ethics, immunorejection, tumorigenesis from hESCs
and teratoma formation from iPS cells. It is therefore necessary to search for
alternative sources of stem cells. WJSCs originate from embryonic epiblasts and
possess properties intermediate between hESCs and adult stem cells. However,
the stemness properties of molecules in WJSCs remain unclear compared to those
of hESCs. In the present study, we isolated WJSCs by a non-enzymatic method.
Further, using microarray analysis by Affymetrix GeneChip and functional network
analyses, we determined the degree of expression of stemness genes exhibited by
the Human Stem Cell Pluripotency array. We also defined a wide range of stem cell
gene expression in the WJSCs in comparison with hESCs. At the same time, the
definitive markers of early cardiac precursor cells and more committed progenitors
were further characterized in WJSCs. Our results demonstrated for the first time
that WJSCs had significant expression of undifferentiated human embryonic stem
cell core markers, such as SOX2, NANOG, LIN28, SSEA1, SSEA2, SSEA3, SSEA4,
KLF4, c-MYC, CRIPTO, and REX1 with a relatively lower level of expression than in
hESCs. We also found WJSCs have high expression of early cardiac transcription
factors, such as filk-1, Isl-1, and Nkx2.5. Functional analysis revealed signature genes
of WJSCs with specific roles involved in immune, cytoskeletal, and chemokine
regulation, cell adhesion and cell signaling. Our study indicated that there is a
significant overlap between the stemness genes expressed by hESCs and WJSCs.
WJSCs harbor a true stem cell population and are promising cells for stem cellbased therapies.
55
Immunomodulatory effects of mesenchymal stem
cells on allogeneic lymphocytes
Linda Koutová, Daniel Lysák, Monika Holubová, Vladimír Koza, Dept. of Hematology and
Oncology, University Hospital Pilsen, Pilsen, Czech Republic.
Mesenchymal stem cells (MSC) have effective immunomodulatory properties.
They can affect the level of lymphocyte activation and proliferation. MSC represent
promising treatment option for steroid refractory graft-versus-host disease.
In our in vitro study we investigated immunosuppressive potential of MSC cocultivated with allogeneic lymphocytes.
MSC isolated from bone marrow of healthy donors were cultivated in complete
culture medium supplemented with platelet lysate until reaching 80 – 90%
confluence. MSC from 2nd to 5th passage were used for the co-cultivation
experiments. Peripheral blood lymphocytes were nonspecifically stimulated with
phytohemagglutinin and cultivated for 4 days without or with addition of MSC
(ratio 2:1). The expression of activation markers CD25, CD69 and HLA-DR was
measured using the flow cytometry. Lymphocyte proliferation was tracked with
CFSE staining.
Proliferation of stimulated lymphocytes generated up to 7 daughter populations
after 4 days of cultivation. The number of undivided lymphocytes was higher
during cultivation with MSC (51% vs. 17,4%, p<0,0001), while the numbers
of cells in daughter populations were decreased in the MSC presence in 4th
generation (10,7% vs. 32,8%, p=0,0004) and 5th generation (10,1% vs. 30,0%,
p=0,0008). Also the proliferation index was significantly reduced with MSC: 1,84
vs. 3,65 (p<0,0001). CD25 expression increased during cultivation on stimulated
CD4+ and CD8+ cells. After addition of MSC the changes in kinetics disappeared
and absolute numbers of CD25+ cells achieved lower levels. Expression of CD69
decreased irrespective of MSC presence; slighter decline was detected after MSC
co-cultivation. HLA-DR expression was also slightly decreased on lymphocytes
cultivated with MSC.
Our study confirmed immunosuppressive effect of MSC on nonspecifically
stimulated allogeneic lymphocytes. Experiments revealed changes in cell
proliferation and downregulated (CD25) or upregulated (CD69) expression
of some activation antigens. This functional test can be used as a part of
characterization of MSC, when considered for clinical application.
56
QUALITY CONTROLS OF IMMUNE REGULATORY PROPERTIES OF
EX-VIVO, GMP-GRADE EXPANDED MESENCHYMAL STROMAL CELLS
FOR CLINICAL USE (EUROPEAN MULTICENTER STUDY CASCADE)
Luciano Pacelli, 1 Cedric Mènard,2 Giulio Bassi,1 Joelle Dulong,2 Isabelle Bezier,2 Jasmina
Zanoncello,1 Francesco Bifari,1 Hubert Schrezenmeier,3 Luc Sensebé,4 Mauro Krampera,1
Karin Tarte,2 1University of Verona, Stem Cell Research Laboratory, Department of
Medicine , Verona, Italy, 2INSERM U917 Faculté de médecine, Rennes, France, 3Institute
of Transfusion Medicine, University of Ulm and German Red Cross Blood Donor Service
Baden-Württemberg – Hessen, Ulm, Germany, 4EFS Pyrénées Méditerranée - Université Paul
Sabatier UMR5273 - Inserm U1031, Toulouse, France.
CASCADE Immunological Unit set up and validate a panel of assays to fully
characterize in a standardized manner the immunomodulatory properties of
Mesenchymal Stromal Cells (MSCs) obtained inside CASCADE Units from
different sources and different expansion protocols. Primed MSCs (pMSCs) were
obtained by 48h-treatment with rh-INFγ and rh-TNFα. Cocultures were set up by
plating MSCs or pMSCs in presence of T, B, NK cells. At day 4 and 6 of coculture,
proliferation was evaluated by CFDA-SE dilution. T cells were stimulated with
αCD3 and αCD28 antibodies; B cells were activated with CD40L, its enhancer,
IL-2, CpG, and anti-IgM/IgA/IgG; NK cells were activated with 100 U/ml of rhIL-2.
Moreover, cocultures, were performed in the presence of specific inhibitors: L-1MT
(IDO inhibitor), snPP (HO-1 inhibitor), NS-398 (COX2 inhibitor), L-NMMA (iNOS
inhibitor) and anti-IFNγ antibody. For MSCs immunogenicity assay, proliferation
of allogeneic T cells was evaluated at day 5 of coculture; in addition, NK cells
were activated by 48-h stimulation with rh-IL2 and MSCs or pMSCs were used as
target cells. Priming upregulated MHC I/II, CD54, CD106, CD40, CD274, CD112,
CD155 expression. MSCs coculture inhibited T and NK cell proliferation, and
this effect was greater in presence of pMSCs. On the contrary, only pMSCs were
capable of suppressing B cell proliferation. MSCs inhibited apoptosis of T, B, and
NK cells. T cell/MSCs coculture showed that activation of IDO and HO-1 was the
main mechanism involved in MSCs immune modulation. MSCs never promoted
23
POSTER ABSTRACTS
allogeneic T cell proliferation; by contrast, NK cells efficiently recognized and killed
MSCs but pMSCs was less sensitive to NK lysis. All the experimental protocols to
assess MSCs inhibitory effects on immune effector cells have been standardized
and will be applied for the release of GMP-grade MSCs produced inside the
CASCADE consortium.
57
HUMAN UMBILICAL CORD-DEVERIED MESENCHYMAL STROMAL
CELLS RETAIN ITS PROPERTIES AFTER CRYOPRESERVATION
Ming-Foong Chai, 1 Kong-Yong Then,1 Chee-Yin Wong,2 Ghee-Chien Ooi,1 Mohd Manzor N.
Fariha,3 Kien-Hui Chua,4 Kok-Weng Yong,1 Khong-Lek Then,1 1CryoCord Sdn. Bhd., Selangor,
Malaysia, 2Cytopeutics Sdn. Bhd., Selangor, Malaysia, 3National University of Malaysia, Kuala
Lumpur, Malaysia, 4National University of Malaysia, Kuala Lumpur, Malaysia.
Introduction: Human umbilical cord-derived mesenchymal stromal cells (UCMSC) have been identified as one of the potential stem cell sources for cell-based
therapy due to its multipotency and ease of isolation. These valuable stem cells
can be stored under cryopreservation stage for many years before the stem cells
are thawed for therapeutic purposes.
Objective: This study was performed to determine the effects of cryopreservation
on the properties of UC-MSC.
Methods: UC-MSC were isolated from cord blood samples collected after
birth from consenting mothers and werecultured for three passages before
cell harvesting. Cell viability after harvesting was determined before dividing
the UC-MSC population into two groups. The stem cells in one group were
analyzed as freshly harvested UC-MSC while the stem cells in the other group
were kept cryopreserved for 3 months before thawing for analysis. Both groups
were examined by immunophenotyping, mixed-lymphocyte reaction assay and
differentiation assays.
Results: Cell recovery rate was high at 92.1% after the cryopreserved UC-MSC
were thawed. There was no significant difference in cell viability before and after 3
months of cryopreservation. Moreover, both groups showed no differences in their
expression of immunophenotypes. Both groups were also able to demonstrate its
ability to inhibit lymphocyte proliferation and to tri-differentiate.
Conclusion: Cryopreservation of 3 months does not have an adverse effect on the
properties of UC-MSC. However, it is recommended to repeat this study with UCMSC which have been cryopreserved for a longer period.
58
MEGAKARYOCYTES GENERATION FROM HUMAN EMBRYONIC
STEM CELLS
Maristela Delgado Orellana, Sr., Gil De Santis, Instituto Nacional de Ciências e Tecnologia
em Células-Tronco, INCTC- Hemocentro de Ribeirão Preto, HC-FMRP/USP, Ribeirão Preto,
Brazil.
24
Therapies involving hematopoietic cells depend on the donation of these cells from
healthy volunteers, however, the supply of transfusable red and white blood cells,
including leukocytes and platelets, is not sufficient in many countries and could
produce unpredictable adverse results according to the current transfusion therapy
system. Human embryonic stem cells (hESCs) can be propagated in vitro indefinitely,
providing a potentially unlimited and donorless source of cells for therapeutic
purposes. The objective of this work was the generation of megakaryocytes cells
from hESCs under serum and feeder-free conditions. Embryoid body were generated
from H1 hESC (WiCell) cell line, without serum in presence cytokine cocktail
subjected to attachment culture on gelatin-coated plates and grown for 10 to 30
days in the presence of a specific cytokine cocktail. Morphological characteristics
and immunophenotype were analyzed from cells released into the supernatant.
After approximately one week, a sac-like structure filled with abundant round cells
emerged at the center of flattened spheres. After this time cells actively proliferated
and floated in the supernatant or associated weakly with the adherent cells from 15
at 20 days. Weekly were collected around 1 x 106 cells. Fifteen of these cells showed
typical morphology of megakaryocytes, 50 % mononuclear, 15% polymorphonuclear
granulocytes, and 20% blastic cells. Expression of CD31, Gly A, CD-61, CD41a and
CD42a was found in 3 -7 % of cells, CD42b, CD-16, CD-71 between 8 -15 %; CD-14
and CD-38 between 15-30%; and CD45 in 20-50%.The cells formed hematopoietic
and megakaryocytic colonies in semisolid culture and to differentiated into mature
megakaryocytes by day 15 a 30 in the presence of thrombopoietin. The results show
that megakaryocytes can be generated from hESCs under serum- and feeder-free
conditions. In conclusion, the study described here represents is an important step
in developing a renewable, donorless source of transfusable platelets.
59
AMINE-SURFACE-MODIFIED SUPERPARAMAGNETIC IRON
OXIDE NANOPARTICLES INTERFERE WITH DIFFERENTIATION OF
HUMAN MESENCHYMAL STEM CELLS
You-Kang Chang1,2,3, Oscar Lee1,2,3 1Institute of Clinical Medicine, National Yang-Ming
University, Taipei, Taiwan, 2Department of Orthopaedics and Traumatology, Taipei Veterans
General Hospital, Taipei, Taiwan, 3Institute of Clinical Medicine, National Yang-Ming
University, Taipei, Taiwan.
Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used for
stem cell labeling and tracking. Surface modification has been known toimprove
biocompatibility, biodistribution,and labeling efficiency of SPIO nanoparticles.
However, the effects of amine (NH3+)-surface-modified SPIO nanoparticles on
proliferation and differentiation of human mesenchymal stem cells (hMSCs)
remain unclear. The purpose of this study is to investigate how amine-surfacemodified SPIO nanoparticles affected hMSCs. In this study, successful
intracellular uptake and the contiguous presence of amine-surface-modified SPIO
nanoparticles in hMSCs was demonstrated by Prussian blue staining, transmission
electron microscopy and magnetic resonance imaging. Moreover, accelerated cell
proliferation was found to be associated with cellular internalization of aminesurface-modified SPIO nanoparticles. However, osteogenic and chondrogenic
differentiation potentials of hMSCs were impaired, while adipogenic potential
was relatively unaffected. Altered cytokine production profile in hMSCs caused
by amine-surface-modified SPIO nanoparticles may account for the increased
proliferation and impaired differentiation potentials; concentrations of the
growth factors in the SPIO-labeled condition medium including amphiregulin,
glial cell-derived neurotrophic factor, heparin-binding EGF-like growth factor
and vascular endothelial growth factor, as well as soluble form of macrophage
colony-stimulating factor receptor and SCF receptor, were higher than in the
unlabeled-condition medium. In summary, although SPIO labeling is effective for
cell tracking, properties of hMSCs may alter as a consequence and this needs to
be taken into account when evaluating therapeutic efficacies of SPIO-labeled stem
cells in vivo.
60
PATHOGEN-FREE PLATELET LYSATE FOR THE EXPANSION OF
BONE MARROW DERIVED MESENCHYMAL STROMAL CELL
Paola Iudicone, ScBD1 Giuseppina Bonanno, ScBD2 Daniela Fioravanti, ScBD3 Claudio
Lavorino, ScBD3 Michela Miceli, MD1 Fabrizio Massarelli, Lab Tech1 Stefano Berardini, Lab
Tech1 Marianna Nuti, MD4 Luca Pierelli, MD1 1S. Camillo Hospital, Rome, Italy, 2Catholic
University Policlinico A. Gemelli, Rome, Italy, 3S.Camillo Hospital, Rome, Italy, 4La Sapienza
University, Rome, Italy.
Platelet lysate (PL), being rich of growth factors has been used for in vitro MSC
culture and several efforts aimed to standardize PL preparations to propose them
as a substitute for animal serum in culture media. Neverthless, the concentration of
soluble factors released by platelets (PLT) from individual donors is highly variable
and a multiple source of PLT is required to compensate this variability. Producing
MSC for therapeutic purposes requires strictly adhering to GMP to ensure the
delivery of a safe product. In this setting PLT pooling procedure with the additional
POSTER ABSTRACTS
step of photochemical treatment by the INTERCEPT technology was adopted to
produce a pathogen-free PL to sustain the growth of MSC. The PL preparation
was performed in a closed system and pathogen inactivation procedure included:
mixing PLT pool with psoralen compound, illumination with UVA light, removal of
residual psoralen and free photoproducts by means of an absorption device. Two
pools of six inactivated PLTs were sterile connected to obtain a pool of twelve PLTs
which underwent three freezing and thawing steps followed by centrifugations to
remove PLT bodies. Eight preparations were produced, four pathogen inactivated
(PI-PL) and four not inactivated. Each PI-PL was used in parallel with a control PL
expand MSC from bone marrow. Four primary MSC cultures were obtained within
2 weeks and MSC, identified as CD45-, CD34-, CD90+, CD105+, CD71+, CD73+,
CD166+, were cultured up to 3 passages. The concentration of PLT released growth
factors (PDGF-AB, VEGF, basic-FGF, TGF-Beta) and the ability to select and expand
MSC from different donors were comparable between inactivated and control PL.
The results suggest that pathogen inactivation procedure does not affect the PLT
growth factors release and their potential to propagate MSC giving an additional
help toward better standardized and safe MSC products.
BM
MNC x106 at
samples culture beginning
MSC-1
5
MSC-2
5
MSC-3
5
MSC-4
5
PL
PL1
PI-PL1
PL-2
PI-PL2
PL-3
PI-PL3
PL-2
PI-PL4
day
16
16
18
18
14
14
14
14
P1
MSC x 106
0.847
0.765
0.115
0.163
0.800
1.264
0.170
0.170
day
27
27
31
31
27
27
29
29
P3
MSCx 106
171
151
25
36
90
162
27
28
Fold
Expansion
202
197
217
221
112
128
158
164
61
ISOLATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELL
(HUVEC), CD117pos MATTERS OVER ENDOTHELIAL CELLS COUNT
Remy Zevallos, MT, Carlos Villa, MT, John Pando-Mayta, MT, Eva H Obregón-Zegarra, MD,
Jorge Castillo-Aguirre, MD, Tatiana Saldarriaga, MD, Antonio A Carrasco-Yalan, MD, Instituto
de Criopreservación y Terapia Celular, Lima, Peru.
HUVEC is a unique and easy source to get endothelial cell (EC) and endothelial
progenitor cells (EPC). Many procedures for isolation had been described with
different outcomes related to cell count. EPC had been defined among others by
the expression of CD34pos, CD117pos, CD133pos, CD31pos and CD45neg and long life
term in cultures; whereas EC had been defined by flow as EPC but CD133neg with
short life term in cultures.
We processed eleven umbilical cords (UC) with 0.1% Collagenase A over 10 minutes
following Jaffe protocol. One ml of collagenase was inoculated for each centimeter of
UC. Median UC length was 30 cm (range12.5-47), median total nuclear cells (TNC)
count after incubation and double wash was 23.42*106 (range 5.4-40.5*106), no
correlation was observed between UC length and TNC count (p=ns). Median CD45neg
CD31pos count was 5.5*105 (range 0.54-28.8) of which 73% were CD133pos and 27% were
CD133neg. No major percentages changes were observed with the inclusion of CD34
as fourth marker: the CD34pos CD133pos rate was 72% of the CD45neg CD31pos and the
CD34pos CD133neg rate was 25% of the CD45neg CD31pos (p=ns).
The inclusion of CD117 as a fifth marker reduced significantly the obtained EC
and EPC counts. Median EPC count defined as CD45neg CD31pos CD34pos CD117pos
CD133pos was 2.25*105 (range 0.4-20.22) which account 52% of CD45neg CD31pos
cells; whereas median EC count defined as CD45neg CD31pos CD34pos CD117pos
CD133neg was 2.15*104(range 0-10) which is only 2.6% of CD45neg CD31pos cells.
CD117 restricts much better EC from a pool of CD45neg CD31pos cells (p>0.05)
This is the first experience of HUVEC isolation in Perú. The yield of TNC count was
very good using the Jaffe protocol and the use of five markers to define EC and
EPC. We recommended the inclusion of CD117 in the flow panel.
62
GENE EXPRESSION PROFILE OF MESENCHYMAL STROMAL
CELLS DERIVED FROM HUMAN BONE MARROW CD271+
MONONUCLEAR CELLS.
Selim Kuci, MD., PhD.1 Zyrafete Kuci, MD., PhD.1 Jutta Kollet, PhD.2 Halvard Boenig, MD.3
Ulrike Koehl, MD., PhD.1 Thomas Klingebiel, MD.1 Peter Bader, MD.1 1University Children’s
Hospital III, Dept. of Hematology/Oncology, Frankfurt am Main, Germany, 2Miltenyi Biotec
, Bergisch-Gladbach, Germany, 3Institute for blood transfusion and immunohematology,
Frankfurt am Main, Germany.
To date, there are no reports on the gene expression profile of the mesenchymal
stromal cells (MSCs) derived from CD271+ BM-MNCs (CD271-MSCs). In this
study we expanded CD271-MSCs and MSCs generated through plastic adherence
(PA-MSCs) as a control group until passage 3. Both MSC types were compared
against each other in gene expression microarray experiments. Candidate genes
of differential expression between CD271-MSCs and PA-MSCs were identified by
selection for p-values ≤ 0.01 and at least 2-fold expression difference. In CD271MSCs were identified 204 genes with lower expression and 287 with higher
expression compared to PA-MSCs. Preliminary results using Functional Grouping
Analysis showed that the most prominent associations of upregulated genes were
related to immune response. The majority of associated genes with ‘T-cell immunity’
overlap with partially redundant categories of ‘Innate immunity’ (34 genes,
p=3.9e-07), ‘Response to toxins’ (52 genes, p=9.7e-06) and ‘Receptor signaling’
(52 genes, p=5.5e-05). Interestingly, many of these genes are involved in antigen
presentation and cell-mediated immune responses: HLA-Class II, CIITA, invariant
chain (CD74), alpha-2-microglobulin and genes important in peroxisome function.
In addition, genes of innate immunity were also upregulated e.g. several members
of the defensin gene family, complement factors and iNOS. Genes identified with
‘Receptor signalling’ were mainly related to developmental processes including
cell proliferation and differentiation. The Functional Grouping Analysis of genes
with lower expression in CD271-MSCs compared to PA-MSCs did not return any
significant enrichment of associated categories. Taken together, these results may
explain the genetic basis for the functional differences between CD271-MSCs and
PA-MSCs.
63
TWIST1 FUNCTIONS AS A PRO-SURVIVAL AND SELF-MAINTENANCE
FACTOR FOR HUMAN MESENCHYMAL STEM CELLS
Siddaraju V. Boregowda, 1 Donald G. Phinney, PhD2 1The Scripps Research Institute , Jupiter ,
USA, 2The Scripps Resarch Institute , Jupiter , USA.
Mesenchymal stem cell (MSCs) populations are known to be functionally
heterogeneous and this heterogeneity is poorly predicted based on analysis of
surface phenotype using established markers. Herein we demonstrate that TWIST1
and FGFR2b mRNA and protein levels vary significantly between human MSC
donor populations and are significantly correlated with growth, colony forming
unit-fibroblast (CFU-F) activity, colony initiating progenitor (CIP) frequency and
differentiation potential. Moreover, high TWIST1 expressing populations contained
fewer apoptotic cells and were more resistant to stress-induced apoptosis. Knock
down of TWIST1 via siRNA significantly inhibited cell growth, CFU-F and CIP
frequency and resulted in increased cellular apoptosis and senescence indicating
that TWIST1 functions as a pro-survival factor. Finally, clone splitting experiments
revealed that TWIST1, TWIST2 and BMI1 were expressed at significantly higher
levels in tri-potent vs. bi- or uni-potent clones and knockdown of TWIST1 resulted
in reduced BMI1 expression levels. Collectively, these data indicate that TWIST1
regulates growth, survival and multi-potency of MSCs. Consequently, TWIST1 and
FGFR2b represent novel markers to evaluate the quality of MSC populations in
research and clinical manufacturing.
25
POSTER ABSTRACTS
64
67
Will not be presented
FUNCTIONAL HETEROGENEITY OF MESENCHYMAL STROMAL
CELLS DERIVED FROM HUMAN BONE MARROW CD271+
MONONUCLEAR CELLS AT THE CLONAL LEVEL.
65
CAN Induced pluripotent stemcells CELLS REPLACE
DENTAL STEM CELL BANKING
Sunil P Mohan, MDS, PhD, FTPRM, Jaisanghar Nallusamy, professor, Rajah Mutiah Dental
College, Annamalai nagar, Chidambaram, India.
Dental stem cells have gained momentum recent days due to its easy availability,
plasticity which paved way for dental stem cells banking. This poster briefly
reviews the advantages and disadvantages of it while comparing with iPS cells.
The iPS cells are created by reprogramming the somatic cells by transfecting with
specific transcriptional factors. Many recent studies have proved dental pulp cells
can be reprogrammed efficiently than dermal fibroblasts. I t was also proved that
it can be a promising source for iPS banking. Apart from dental pulp it was also
derived from gingival fibroblasts and periodontal ligament fibroblasts. Though
iPS cells have various disadvantages like tumor and teratoma formation, newer
non integration methods can overcome those demerits. It was also proved by
various studies that the iPS cells can be generated from Mesenchymal stromal
cells (MSCs) of dental pulp multifold times when compared with the human
dermal fibroblasts. The iPS cells were also successfully isolated from third molars
which proved it can be isolated from any teeth which are discarded.
66
SCALABLE SUSPENSION-BASED DIFFERENTIATION OF HUMAN
EMBRYONIC STEM CELLS TO PANCREATIC PROGENITORS
Thomas C. Schulz, Eugene P. Brandon, Holly Y. Young, lan D. Agulnick, Rosemary M. Cesario,
Kuniko Kadoya, Jonathan Kelly, Amanda B. McLean, Mark A. Moorman, Janice K. Payne, Eric
S. Sherrer, Xuehong Song, Chad E. Green, Evert J. Kroon, Olivia G. Kelly, Kevin A. D’Amour,
Allan J. Robins, ViaCyte, Inc., San Diego, CA, USA.
Development of a replacement cell therapy for type 1 diabetes from human
embryonic stem cells (hESC) will require the translation of research processes to
bona fide reproducible, scalable, and controlled industrial cell manufacturing. We
have previously demonstrated that at relatively small scale hESC can be directed
to differentiate to pancreatic progenitors that mature into glucose-responsive
insulin producing islets following in vivo implantation. Here we describe hESC
expansion and banking methods and a suspension-based differentiation strategy
that together comprise an integrated manufacturing system for large-scale
production of pancreatic progenitor cells. Qualified large-scale GMP cell banks
of CyT49 hESC have been generated, representing a virtually unlimited supply
of manufacturing starting material. Each vial can be thawed and expanded in
adherent culture to billions of undifferentiated hESC in two weeks. The hESC
are then aggregated into cell clusters in dynamic rotational culture, and over the
subsequent two weeks are differentiated to a population enriched for pancreatic
progenitors. Upon implant into immunocompromised rodents, progenitors
mature into functional islets that can protect animals from streptozotocin lesion
of their endogenous beta cells. The integrated tractable manufacturing system is
now being qualified and documented for first-in-human testing of an hESC-based
approach to cell replacement in type 1 diabetes.
Zyrafete Kuci, MD., PhD.1 Julia Seiberth, MD. Candidate1 Stefan Stein, PhD.2 Manuel Grez,
PhD.2 Halvard Boenig, MD.3 Ulrike Koehl, MD.,PhD.1 Thomas Klingebiel, MD.1 Peter Bader,
MD.1 Selim Kuci, MD.,PhD.1 1University Children’s Hospital III, Dept. of Hematology/
Oncology, Frankfurt am Main, Germany, 2Biopharmaceutical Institute Georg-Speyer-Haus,
Frankfurt am Main, Germany, 3Institute for blood transfusion and immunohematology,
Frankfurt am Main, Germany.
In this study, we focused on the clonal analysis of mesenchymal stromal cells
derived from CD271+ human bone marrow mononuclear cells (BM-MNCs). MSCs
derived from CD271+ BM-MNCs were designated as CD271-MSCs, while MSCs
generated by plastic adherence were designated as PA-MSCs. GFP-transfected
MSCs of passage 3 and 5 were sorted as a single cell per well in a 96-well plate.
Next day, wells were controlled for the presence of the cells. Wells containing
more than one cell or no cells were excluded from the study. Preliminary results
of this study showed that 68±7.8% of CD271-MSCs were able to give rise to
colonies compared to 43.1±0.5% of PA-MSCs (P<0.04). However, both of these
MSCs showed a similar number of population doublings (PDs) and clonogenic
potential at the secondary plating. To examine the in vitro allosuppressive
potential of both types of MSCs we performed the mixed lymphocyte culture with
the blood mononuclear cells of two HLA-disparate donors in presence or absence
of single-cell derived MSCs. We found three types of MSC-clones regarding their
allosuppressive potential: non-suppressive clones (0% inhibition), low suppressive
clones (10-50% inhibition) and highly suppressive clones (50-100% inhibition).
Approximately 30% of the CD271-MSC clones studied were low-suppressive (21.4
± 3.7% allosuppression), whereas the rest of the clones were highly allosuppressive
(80 ± 3.5% allosuppression). Only a very small percentage of clones were not able to
inhibit the allogeneic reaction in vitro. Taken together, even single cell-derived MSCs
of both types revealed a functional heterogeneity concerning their clonogenic and
allosuppressive potential.
68
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69
Will not be presented
70
Will not be presented
71
IN VITRO DIFFERENTIATION OF MESENCHYMAL STROMAL CELLS
INTO MESANGIAL CELLS AFTER CO-CULTURE WITH INJURED
MESANGIAL CELLS
Chee-Yin Wong, 1 Eng-Lai Tan,2 Soon-Keng Cheong,3 1Cytopeutics Sdn. Bhd., Selangor, Malaysia,
2
International Medical University, Kuala Lumpur, Malaysia, 3Tunku Abdul Rahman University,
Selangor, Malaysia.
Mesangial cells have been recognized as one of the three major cell lines of the
kidney glomerulus and function is to provide physical support for the glomerular
capillary lumen of the kidney. Normal glomerular capillary is essential to keep efficient
ultrafiltration of plasma, loss of mesangial cells due to pathologic conditions such
as glomerulonephritis and diabetic nephropathy leads to impaired renal function.
Mesenchymal stromal cells (MSC) are an attractive candidate for renal repair therapy
as many reports have shown MSC can enhance recovery and protect against renal
failure. These reports had also shown that MSC can differentiate into mesangial in
26
POSTER ABSTRACTS
vivo.This study was carried out to further investigate the ability of MSC differentiates
into mesangial cells in vitro. MSC were co-cultured with injured mesangial cells
before observed and analyzed by flow cytometery. MSC co-cultured with injured
mesangial cells were exhibited mesangial cell-like morphology after 7 days of coculture when observed under microscope. While for flow cytometry analysis, after
MSC co-cultured with injured mesangial cells, these cells were expressed CD62E
and did not expressed CD54. It is contradict to pure MSC which are CD54 positive
and CD62E negative. In addition, these MSC co-cultured with injured mesangial cell
were contracted in response when treated with Angiotensin II. It is showed that MSC
have a capacity to differentiate into mesangial cells in vitro following co-cultured with
injured mesangial cell.
72
INTRADISCAL DELIVERY OF AUTOLOGOUS BONE MARROWDEVIRED MESENCHYMAL STROMAL CELLS: A FEASIBLE AND
SAFE APPROACH OF CELL THERAPY IN TWO PATIENTS WITH
DEGENERATIVE DISEASE
Chee-Yin Wong, 1 Sze-Piaw Chin,1 Chee-Yew Cheok,2 Mei-Theng Ng,1 1Cytopeutics Sdn. Bhd.,
Selangor, Malaysia, 2Penang Adventist Hospital, Penang, Malaysia.
Background: Degenerative disc disease (DDD) is increasingly prevalent and has
been diagnosed in increasingly younger patients. The nucleus pulposus cells do
not regenerate itself within the intervertebral disc. Therefore, any damage or trauma
is normally progressive and irreversible. Bone marrow-derived mesenchymal
stromal cells (BM-MSC) can differentiate into cartilage and appear beneficial for
the treatment of articular cartilage injuries while animal studies have demonstrated
BM-MSC capability to differentiate into nucleus pulposus-like cells. The objective of
this study is to determine the safety and feasibility of BM-MSC treatment in DDD.
Methods: Two young patients with intractable low back pain and documented DDD
on T2-weighted magnetic resonance imaging (T2-MRI) agreed to the treatment.
Bone marrow aspirate was obtained from the posterior iliac crest under local
anaesthesia, and processed to isolate and expand the BM-MSC under sterile
conditions. 1 month later, the patients underwent electrothermal ablation of the
affected discs followed by intradiscal injection of 1x106 MSC per kg body weight per
disc. T2-MRI was repeated at 6 and 12 months after treatment.
Results: MSC were isolated from the patients diagnosed with DDD. No adverse
reactions were observed or reported by both patients. Both patients also felt
significant improvements in pain relief as indicated by score reduction in Oswestry
disability index. However, MRI showed no increment of disc height.
Conclusions: Treatment with autologous BM-MSC is safe and may be a feasible
treatment option when used in conjunction with electrothermal ablation for the
treatment of degenerative disc disease.
73
EXPANSION OF MULTIPOTENT MESENCHYMAL STROMAL CELLS
FROM FETAL ADNEXA IN XENOFREE CONDITIONS
Karen L. Prata, MD, PhD1,2 Gil C. De Santis, MD, PhD1,2 Maristela D. Orellana, MSc1,2 Taisa
R. Fernandes,1 Aline C. Garcia,1 Natalia C. Gonçalves,1 Patricia V. Palma,1 Camila C. Bonaldo,1
Dalila L. Zanette,1 Virginia M. Wagatsuma,1 Simone Kashima,1,3 Dimas T. Covas, MD, PhD1,2
1
National Institute of Science and Technology in Stem Cell and Cell Therapy, Center for Cell
Therapy and Regional Blood Center, Ribeirão Preto, Brazil, 2Department of Internal Medicine,
School of Medicine, University of São Paulo, Ribeirão Preto, Brazil, 3School of Pharmacy,
University of São Paulo, Ribeirão Preto, Brazil.
Introduction: MSCs from fetal adnexa (FA) have been considered for therapeutic
use. The objectives of this study were to establish the best FA source of MSCs and
the best human protein supplement to expand MSCs. Materials and methods:
MSCs were isolated from amniotic (AM) and chorionic membranes (CM),
placental cotyledons (PC) and umbilical cord (UC), and cultured in parallel in
medium supplemented with FBS, human plasma (HP) or platelet lysate (PL).
Comparative assays with cells isolated from 4 sources and cultured in 3 different
protein supplements were performed, aiming to evaluate: a) efficiency of the
isolation process; b) cell growth kinetics; c) morphologic and imunophenotypic
characterization; d) potency to inhibit T lymphocyte proliferation; e) capacity to
differenciate into adipocyte and osteocyte. Results (Table 1) (mean±SEM): The
cells obtained showed typical MSCs morphology and imunophenotype, and were
capable of differentiating into osteocytes and adipocytes.
AM(N=15)
CM(N=15)
PC(N=15)
UC(N=15)
10%FBS(N=20)
10%HP(N=20)
5%PL(N=20)
MSCs yield/
tissue
gram*(x105)
4.0(±0.7)
4.3(±0.7)
1.5(±0.4)
5.9(±1.0)
p=0.0013
4.8(±0.9)
3.2(±0.5)
3.8(±0.7)
p=0.1084
Final cell
expansion(x)
27.4(±2.7)
29.0(±2.5)
26.9(±1.9)
42.7(±2.8)
p<0.0001
36.7(±13)
28.9(±2.6)
28.9(±2.1)
p<0.0001
Doubling time
(6th passage,
hours)
48(±5.4);N=14
54(±4.8)
73(±10.6)
35(±2.8)
p=0.0014
42(±3.8)
51(±5.8);N=19
64(±8.2)
p=0.0454
Capacity to
inhibit T cell
proliferation (%)
81.9(±2.6)
32.6(±7.0)
45.8(±6.9)
84.3(±2.7)
p<0.0001
60.2(±7.5)
65.2(±5.3)
58.1(±7.4)
p=0.3764
*after 1st passage.
It was necessary 2 times more raw material bags (platelets or plasma) to produce
the same quantity of 5%LP than 10%HP medium. Discussion: It was possible
to expand efficiently MSCs from FA in xenofree conditions maintaining their
characteristics. HP was superior to PL considering transfusional residual risk
and stock availability of raw materials necessary to obtain protein supplement.
Umbilical cord was the best source of MSCs, as they showed an ideal association
among the evaluated parameters such as cell yield, expansion potential, cell
viability, purity and potency.
74
ACUTE ADVERSE REACTIONS SECONDARY TO MESENCHYMAL
STROMAL CELL INFUSIONS
Karen L. Prata, MD, PhD1,2 Maria C. Rodrigues, MD, PhD2 Gil C. De Santis, MD, PhD1,2 Maristela
D. Orellana, MSc1,2 Taisa R. Fernandes,1 Karina S. Candido,1 Samia R. Caruso,1 Daniela A. Moraes,
MD, PhD2 Carlos E. Couri, MD, PhD2 Raphael R. Liberatore Junior, MD, PhD3 George M. Barros,
MD, MSc2 Ana Beatriz P. Stracieri, MD, PhD2 Julio C. Voltarelli, MD, PhD1,2 Dimas T. Covas, MD,
PhD1,2 1National Institute of Science and Technology in Stem Cell and Cell Therapy, Center for
Cell Therapy and Regional Blood Center, Ribeirão Preto, Brazil, 2Department of Internal Medicine,
School of Medicine, University of São Paulo, Ribeirão Preto, Brazil, 3Department of Pediatrics,
School of Medicine, University of São Paulo, Ribeirão Preto, Brazil.
Mild cough
Headache
Fever
Facial redness
“Phlegm in throat”
Skin rash
Thoracic pain (mild)
Respiratory discomfort (mild)
Nausea
Chills and tremors
Malaise
Number of patients
experiencing adverse reactions
3 (37.5%)
3 (37.5%)
2 (25.0%)
2 (25.0%)
2 (25.0%)
1 (12.5%)
1 (12.5%)
1 (12.5%)
1 (12.5%)
1 (12.5%)
1 (12.5%)
Total of adverse
events
12 (22.6%)
9 (17.0%)
4 (7.5%)
4 (7.5%)
2 (3.8%)
1 (1.9%)
5 (9.4%)
2 (3.8%)
1 (1.9%)
1 (1.9%)
4 (7.5%)
Introduction: We have been using MSC infusions to treat type 1 diabetes mellitus
(T1DM). Unlike most indications for MSC applications, T1DM patients are
immunocompetent, therefore allowing identification of subtle acute adverse effects
to cell infusions. Our objective was to evaluate the incidence of acute adverse
reactions associated with allogeneic MSC infusions in T1DM patients. Patients
and Methods: 8 T1DM patients received a total of 53 MSC infusions. Results (Table
1): 7 out of 8 (87.5%) patients presented some kind of mild adverse reaction. One
patient presented enlargement of parotid glands, confirmed by ultrasound, starting
after the third infusion and persisting to this day, 18 months after infusion. Amylase
27
POSTER ABSTRACTS
serum levels persisted elevated up to four months. Parotid gland biopsy did not
show abnormalities. One of the patients that presented cough was submitted to
spirometry, before and after the following infusion, resulting normal. Two patients
had a clinical picture suggestive of inflammatory reaction. All adverse effects resolved
spontaneously or with analgesics. Discussion: Reports of acute adverse reactions to
MSC infusions are scarce in the literature. A confounding aspect is the background
disease, which could influence the appearance or perception of acute adverse effects.
For this reason, we believe that patients with T1DM are an adequate group of patients
to study this issue, as they are relatively healthy, and acute adverse events could be
attributed exclusively to the infusion.
75
In vitro immunosuppressive potential of gammairradiated mesenchymal stromal cells
Ana Valeria Gouveia de Andrade, 1,2 Marc Schmitz,1,3 Marcus Odendahl,4 Martin Bornhäuser,1,5
Torsten Tonn,1,6,7 1Center for Regenerative Therapies Dresden, Dresden, Germany, 2German
Red Cross Blood Donor Service East, Dresden , Germany, 3Institute of Immunology, Medical
Faculty, Technical University of Dresden, Dresden, Germany, 4German Red Croos Blood
Donor Service East, Dresden, Germany, 5Department of Medicine I, University Hospital
of Dresden, Dresden, Germany, 6German Red Cross Blood Donor Service East , Dresden,
Germany, 7Experimental Transfusion Medicine, Medical Faculty, Technical University of
Dresden, Dresden, Germany.
Introduction: Mesenchymal stromal cells (MSCs) are promising candidates for the
treatment of GvHD and autoimmune diseases. However, MSC have been discussed
to harbor the risk of malignant transformation, which may limit their clinical
use and also warrants extensive quality controls for genomic stability on each
single batch. Therefore, we asked whether gamma-irradiation could inhibit MSC
proliferation and at the same time preserve their immunosuppressive function.
Methods: BM-MSCs from healthy donors were gamma-irradiated with increasing
doses of 0, 5, 10, and 30 Gy, washed and analyzed within the assays. We assessed
the inhibition of colony formation in a standardized CFU-F (Colony Formation
Unit-Fibroblasts) assay, the immunosuppressive capacity in a MLR, and apoptosis
with Annexin-V staining. To determine the kinetics of irradiated cells, BM-MSCs
from three different donors were irradiated at day -5, -4, -3, -2, - 1 and subsequently
subjected to MLR on day 0.
Results: At a plating concentration of 500 cells/dish (n=2, passage 4), a dose of
5Gy was sufficient to abrogate CFU-F when compared to the control (33,7±4,7
colonies/dish). In a larger-scale setting (1x104cells/175cm2 flasks, n=3, passage 2)
and prolonged culture duration (4 weeks), BM-MSCs irradiated with 5Gy yielded
2-10 CFU-F. However, the dose of 10 Gy completely abrogated the outgrowth of
colonies under these conditions. In addition, functional analysis revealed that
irradiated BM-MSCs (5, 10, 30Gy) preserve their immunosuppressive potential
in the MLR (62,8%±20,1% to 78,4%±4%) compared to control (82,2%±1,4%).
Nevertheless, irradiation doses were associated with significantly increased
apoptosis (4,8%±0,43%) compared to the control (1,47%±0,37%, p<0,05)
measured by Annexin-V staining.Time course experiments demonstrated that
irradiated BM-MSCs (n=3, 10Gy) yielded potent immunosuppression even at day
5 after irradiation (77,3%±13,3%).
Conclusions: Gamma irradiation at a dose of 10 Gy effectively inhibits BM-MSC
cell growth, while the immunosuppressive capacity is retained for up to 5 days
after irradiation.
76
Investigation of the effects of shear Forces on
Human Mesenchymal Stem Cells
Daniel Kehoe, Neethu Sunil, Donghui Jing, Sandhya Punreddy, Manjula Aysola, Julie Murrell,
Martha Rook, Knut Niss, EMD Millipore Corporation, Bedford, MA, United States.
With the continuing increase in number of clinical trials using human stem cells,
the interest for a scaled-up cell production process is increasing. A commonly used
vehicle for the scale-up of cell growth is a stir tank bioreactor such as Millipore’s single
use CellReady system. In these cultures, cells are generally grown on microcarriers
in order to provide a surface for cell attachment. However, when transitioning from
a flat (2D) tissue culture to a microcarrier suspension culture an increase in shear
forces experienced by the cells is unavoidable. As these forces can influence the
differentiation of stem cells as well as cause cell death we aimed to identify molecular
markers for the effects of shear on human bone marrow derived mesenchymal stem
cells (hMSCs). For this, we first subjected hMSCs to various controlled levels of shear
forces and analyzed the global expression of miRNAs and mRNAs. Furthermore, we
performed protein analysis on selected signal transduction pathways. We then used
the identified markers to analyze hMSCs grown in microcarrier cultures in the spinner
flask and Mobius CellReady 3L bioreactor with various agitation rates. This approach
allows for a tight monitoring of shear forces in bioreactor cultures which is critical for
a successful scale-up process.
77
ISOLATION AND CHARACTERIZATION OF CLONOGENIC
MULTIPOTENT STROMAL STEM CELLS (GSSCs) FROM GINGIVA
TISSUE FOR REGENERATIVE MEDICINE
Daniel Tzu-bi Shih, PhD1,2,3 Chih-Chiang Hsiao, BS1 Yu-Hung Tseng, DDS4 1Taipei Medical
University, Taipei, Taiwan, 2University Hospital of Taipei Medical University, Taipei, Taiwan,
3
Genomic Research Center, Academia Sinica, Taipei, Taiwan, 4GWOWEI TECHNOLOGY
CO.,LTD, Taipei, Taiwan.
Gingiva is a high turnover rate tissue that composed with both epithelial and
stromal fibroblast cell types. In this study, we isolated and enriched a stromal
stem/progenitor cell subpopulation with highly regenerative characteristics by
applying hemangioblast or/and stem cell marker selections.
The isolated CD34 positive gingival stromal stem cells (GSSCs) were enriched
and expanded by in vitro cultivation. In compare to the FACS-sorted CD34
negative stromal fibroblasts (GMSFs), both populations express common
mesenchymal markers (CD29, CD44, CD73, CD90, CD105, EGFR, integrin α2β1)
and negative expression of the typical epithelial marker, cytokeratin 18. The CD34+
GSSCs express strongly ESC genes (Nanog, Oct-4, Sox2, Rex-2 and stage specific
embryonic antigen sphingolipids) with co-express myovascular neurogenic
progenitor markers (aSMA, CD54, CD117, and CD133) that are significant different
from the CD34- GMSFs population. CD34+ GSSCs display fully neurogenic
terminal dopamine neuron differentiation. In cardiomyogenic differentiation,
CD34+ GSSCs express MHC+/ Troponin+ mature cardiomyocytes that are distinct
from the CD34- GMSFs. Hepatocytes elicited from CD34+ GSSCs present more
albumin expression than CD34- GMSFs. CD34+ GSSCs also exhibit superior
vascular tube forming efficiency than the CD34- GMSFs.
In summary, the above described findings suggest that gingival tissue contains
unique CD34+ angiomyoblast like progenitor cell population with multi-potent
differentiation potential as an ideal resource for cell therapies in multi-phases
regenerative medicine. A further animal in vivo preclinical evaluation of their
engraftment efficiency and safety are under studying. Keywords: CD34+, CD133+,
CD117+, SSEA+, CD14-, SMA+, MSCs, human gingival tissue, hemangioblast, cardio-myocytic,
dopamine neurogenic, and hepatic differentiation, regenerative medicine
28
POSTER ABSTRACTS
78
Will not be presented
79
Growth Kinetics of human Mesenchymal Stem Cells in
the CellReady single use bioreactor
Donghui Jing, Daniel Kehoe, Neethu Sunil, manjula Aysola, Julie Murrell, Martha Rook, Knut
Niss, EMD Millipore Copr., Bedford, MA, USA, EMD Millipore Corp., Bedford, MA, USA.
The increased knowledge of the differentiation capability as well as of the
immunologic properties of human mesenchymal stem cells (hMSCs) has
stimulated the interest in their use as therapeutic agents. To date, several clinical
trials using hMSCs are underway in a variety of indications. However, a key
hurdle in the clinical application of hMSCs is the high cost of manufacturing. In
addition, current practices using multilayer flatbed cultures do not allow a constant
monitoring of cells during the manufacturing process and introduce a high
degree of variability. Thus, flatbed cultures can be regarded as sub-optimal for the
manufacturing of clinical grade hMSCs. To overcome these deficiencies the use of
stirred tank bioreactors provides an attractive alternative. In these cultures, hMSCs
can be grown on microcarriers and samples of cells and medium can be analyzed
throughout the process. Here we show the utility of Millipore’s Mobius® CellReady
3L single use bioreactor for the expansion of hMSCs. Cells can be seeded directly
into the bioreactor without prior need to attach the cells to microcarriers. After 2
weeks of culture, cells reach densities greater than 2×105 cells/mL while maintaining
their identity as shown by the surface expression of CD105, CD90 and CD73 and
the absences of CD14, CD34 and CD45. In addition the ability to differentiate to
the adipogenic, chondrogenic, and osteogenic lineages is retained after culture.
Furthermore, the capacity to monitor the metabolism of cells facilitates a feeding
strategy to maximize cell number. Taken together, these results show the feasibility
of expanding hMSCs in the Mobius® CellReady 3L Bioreactor. This system provides
a cost-effective approach for the production of clinical grade hMSCs and allows for
a close monitoring of the process.
80
RESULTS OF RECONSTRUCTIVE SURGERY IN 6 CHILDREN WITH
CLEFT PALATE WITH THE COADJUVANT USE OF AUTOLOGOUS
UMBILICAL CORD BLOOD.
Guillermo Trigo, 1 Marcelo Ortega,1 Roman Bayo,1 Hugo Drago,1 Marina Vilacha,1 Carolina
Bentham,1 Maria Tabares,2 Claudio Chillik,2 Gustavo Moviglia,1 1CIITT - Maimonides
University, Buenos Aires, Argentina, 2Matercell, Buenos Aires, Argentina.
Reconstructive surgery associated to orthodontic treatment is a successful
approach to solve the anatomo-physiologic alterations that happens in a child
with palatine cleft congenital defect. This therapeutic program consists of three
different surgical procedures done during child development. The first two steps
procure to solve the lips and soft tissue discontinuity. The third step, performed
usually at eight years of age, is programmed to restore palatine bone tissue
using an autograft of pelvis bone. Knowing that bone marrow mesenchimal stem
cells have been successfully used to repair bone fractures that otherwise are
impossible to be solved by bone scarring, we developed under compassionate
bases a protocol to, during the second surgical time, procure the repair of
palatine bone and to enhance the chances to induce the growth of a dental piece
in the surgical suture border, favoring the social integration of the child during
elementary school period and avoiding the third surgical time.
the treatment before the age of five months achieved the expected results.
Treatment failed in children older than 5 months of age. Post-surgical controls
evidenced that, at least one child developed dental tissue in the alveolar ridge area
where tissue was implanted.
Conclusion: Single use of autologous umbilical cord blood cells seems to be
useful in children that undergo the second surgical time before the age of five
months. For older children we are developing a second protocol where immune
cells and xenogenic acellular dermal matrix are used.
81
IN VITRO RETINOBLAST DIFFERENTIATION FROM FAT MSC
Gustavo Moviglia, Oscar Zarate, Nahuel Blasetti, David Pelayes, Maria Teresita Moviglia
Brandolino, Carlos Gaeta, CIITT - Maimonides University, Buenos Aires, Argentina.
Introduction: In previous works we have showed that adult Mesenchymal Stem
Cells (MSC) differentiation into neural cells, muscle cells, cartilage cells, bone
cells, and pancreatic islet cells. To achieve these different differentiations in only
24 to 48hs, from a single born marrow or fat MSC source, we have co incubated
these cells with homologous autoaggressive cells (EC) against acellular lysate
of nerve tissue, muscle tissue, cartilage tissue, bone tissue and pancreatic islet
tissue, respectively. We also have successfully used these cells in animal and
human regenerative therapeutic approaches (Cytotherapy 2006, 8 (3) 196-201, J.
Spinal Cord, 2009; 47(6):499-503 and ISCT meeting published abstracts of 2008,
2009 and 2011). To investigate if similar approach may be used to repair different
lesions of retina we have conducted the following study.
Methods: Human Th1 cells against retina were obtained challenge blood
mononuclear cells (MNC) with a lysate of target tissue from swine origin and
them negative selecting the specific EC set using Miltenyi immunomagnetic beads.
Human fat MSC were obtained from same human donor MNC through surgery
followed by mechanical and enzymatic dissociation and purified in tissue culture.
EC and MSC were co cultured in a serum free medium without addition of any
cytokine for 0, 24, 48 and 96 hours. Them plastic attached cells were morphological
study through invert phase microscopy and characterized by immune-staining:
To identify: retinal cell linage TUBB3 and Pax 6; Retinal Pigmented Epithelium
(RPE) Bestrophin 2 (Ionic Channel) and RPE 65 (visual pigment regeneration);
kerato epithelium Kerac C, J19 and J36; rods OPN1 SW and Rhodopsin; and retinal
ganglion cells (RGC) OPN4 (melanopsin)
Results: early signs of MSC differentiation into retina precursor and early
differentiated cell lines (neurons, rods, Müller cells, RGS and RPE) were observed at
24 hours. These changes increase during the following observation points.
82
Defined Xeno-free Media Growth Supplement for
Cultivation of Pluripotent Stem Cells
Jasmeet Kaur, Shayne Boucher, PhD, Pauline Lieu, PhD, Mohan Vemuri, PhD, Life
Technologies, Frederick, USA.
Method: Concentrated and purified autologous frozen preserved Umbilical Cord
Blood cells were injected inside the two borders of the bone and soft palatine cleft
during the second surgical time.
Introduction: Rapid progress is being made in investigations of human embryonic
stem cells and human induced pluripotent stem cells technologies in translational
and clinical research. One challenging issue is the paucity of qualified xeno-free,
serum-free media products for culturing pluripotent stem cells (PSC) used in
cell-based therapies. A complete xeno-free culture workflow would minimize
exposure risks associated with non-human animal material. To address this risk,
we developed a xeno-free growth cocktail - KnockOut™ SR GF Cocktail CTS™.
Addition of growth cocktail to xeno-free media provides investigators a complete
xeno-free workflow solution for isolating, expanding and cryopreserving PSC
cultured under feeder and feeder-free conditions.
Results: Six palatine cleft non syndrome-related patients were treated using this
technique. Treatment did not present any severe side effects. Those that received
Material & Methods: PSC lines were recovered and expanded in complete xenofree media consisting of KnockOut™ DMEM CTS™, KnockOut™ SR XenoFree
29
POSTER ABSTRACTS
CTS™ (KSR XF) and KnockOut™ SR GF Cocktail CTS™ (GFC). The cells were
expanded on human feeder cells or xeno-free CELLstart™ CTS™ substrate. PSC
were passaged using recombinant enzyme TrypLE CTS™ and cryopreserved in
the xeno free system. PSC were evaluated by morphology, immunocytochemistry,
PCR, karyotype and EB formation to assess their ability to retain pluripotency and
differentiation into three germ layers. In addition, we tested the xeno-free media
system to determine if iPSC lines can be generated using CytoTune™-iPS Sendai
reprogramming system.
84
Results & Discussion: We demonstrated maintenance, proliferation, passaging
and cryopreservation of PSC using a complete xeno-free media system.
Furthermore, we were able to successfully generate iPSC lines from the xenofree culture system. All components of the complete medium are synthetic,
recombinant, or of human origin. As translational and clinical researchers move
towards clinical application of PSC, regulatory-friendly products like KSR XF, GFC
and CELLStart™ CTS™ will facilitate standardization of ex vivo tissue and cell
processing, and minimize exposure of PSC lines to non-human animal origin
material.
Our goal is to move human mesenchymal stromal cells derived from Wharton’s jelly
(WJ-MSCs) into clinical trials. One important step is to generate an SOP to produce
GMP-grade MSCs for preclinical evaluation. Here, three commercially prepared
serum-free (SF) media and a well-defined 2% serum growth medium (SGM)
were compare to determine expansion, phenotypic stability, and multipotency. SF
conditions were desired because a single medium is simpler and eliminates exposure
to animal products. SF media produced by Biological Industries (BI), Stem Cells
(SC) and Invitrogen (IV) were compared with our SGM. WJ-MSCs were cultured
at 10000 cell/cm2 in standard conditions of 5% CO2, 21% oxygen. Additionally,
three attachment solutions as recommended by the manufacturers were used for
SF conditions (note that expansion in SGM did not require an attachment solution).
WJ-MSCs were isolated, split into four media conditions and expanded till passage
5 (p5) and the following parameters were evaluated: expansion, positive and
negative surface marker expression, differentiation potential and colony forming
unit-fibroblast (CFU-F) assay. WJ-MSCs cultured in BI medium showed significantly
greater proliferation compared to other SF media and to SGM. SF expansion did not
impact expression of CD73, CD90, CD105, HLA-ABC (all positive), or CD34, CD45,
HLA-DR (all negative). WJ-MSCs differentiated efficiently after expansion in BI
medium. CFU-F assay revealed no significant difference in colony forming efficiency
between BI medium and SGM. We conclude that for expansion of WJ-MSCs in
SF conditions using BI medium and substrate provided optimal cell expansion
compared to two other SF formulations and SGM. WJ-MSCs maintained their
phenotypic surface marker profile of MSCs and their multipotency as demonstrated
by osteocytic, chondrocytic and adipocytic lineage differentiation. Once WJ-MSCs
are isolated in BI medium, preclinical validation testing will begin.
83
CXCR4 TRANSFECTION OF MESENCHYMAL STROMAL CELLS
USING CATIONIC LIPOSOME ENHANCES THEIR MIGRATION
TOWARDS SDF-1
Leah A. Marquez-Curtis, PhD1 Hilal Gul-Uludag, PhD2 Peng Xu,2 Jie Chen, PhD2 Anna JanowskaWieczorek, PhD, MD1,2 1Canadian Blood Services, Edmonton, Canada, 2University of Alberta,
Edmonton, Canada.
Mesenchymal stromal cells (MSC) hold tremendous potential for tissue regeneration;
however, in order to execute their therapeutic functions they have to be recruited to
the site of damaged tissue. Stromal-cell derived factor (SDF)-1 is highly expressed at
sites of tissue injury and attracts cells via its receptor CXCR4. Surface expression of
CXCR4 in MSC is low, leading to a decreased migration towards SDF-1. In this study,
we investigated the delivery of CXCR4 into MSC using the cationic liposome reagent
IBAfect. MSC were established from cord blood and passages 4 to 6 were used for all
the experiments, after optimizing the confluency, period of transfection and ratio of
plasmid to IBAfect. Transfection efficiency was determined by flow cytometric analysis
and CXCR4 localization visualized by confocal microscopy. We also examined whether
CXCR4 transfection affected the ability of MSC to differentiate into osteocytes and
chondrocytes. The functional response of CXCR4-transfected MSC towards an SDF-1
gradient was evaluated using a trans-Matrigel migration assay. We found that 24-h
transfection of less than 50% confluent MSC at passage 4 with a plasmid:IBAfect ratio
of 0.6 ug/3.6 uL gave an optimal transfection efficiency of up to 40%. Confocal imaging
shows CXCR4 highly expressed on the surface as well as in the cytoplasm of transfected
MSC. Transfected MSC did not lose their ability to differentiate to osteocytes and
chondrocytes as shown by calcium deposition and proteoglycan production. Most
importantly, overexpression of surface CXCR4 using IBAfect significantly increased
(over 3-fold) the number of cells migrating towards an SDF-1 gradient relative to cells
migrating towards media alone, compared to non-transfected cells (1.3-fold). Our
results suggest that IBAfect-mediated CXCR4 gene delivery on MSC is a highly efficient
technique that may be useful for enhancing the homing of systemically delivered MSC
to effect repair and recovery of function of damaged or diseased tissues.
IDENTIFICATION OF OPTIMAL CONDITIONS FOR GENERATING
MSCS FOR PRECLINICAL TESTING: COMPARISON OF THREE
COMMERCIAL SERUM-FREE MEDIA AND LOW-SERUM GROWTH
MEDIUM.
Yelica Lopez, DVM, Elizabeth Trevino, BS, Mark L. Weiss, PhD, Kansas State University,
Manhattan, KS, USA.
85
PERFORMANCE STUDY OF HUMAN PLATELET LYSATE
PREPARATION KIT FOR MESENCHYMAL STEM/STROMAL CELLS
EXPANSION
Mélanie Gadelorge, 1 Audrey Lams,2 Marilyn Gomez,1 Christine Carena,1 Aurélie Blondy,1
Cécile Coissac,2 Nicolas Tardivel,2 Bruno Delorme,2 Philippe Bourin,3 Luc Sensebe,1
1
StromaLab, Toulouse, France, 2Macopharma, Tourcoing, France, 3CSA 21, Toulouse, France.
Due to their immunosuppressive properties and their differentiation potential,
Mesenchymal Stem/Stromal Cells are of great interest for transplantation and
regenerative medicine. The production of MSC using defined, standardized and
animal-free conditions represents a major issue for their clinical uses. For this
purpose a new substitute of Foetal Bovine Serum (FBS) is increasingly used for
MSC expansion: the human Platelet Lysate (PL). The PL is rich in growth factors
and cytokines and has low immunologic and infectious risks. As there is a current
lack of standardization of PL preparation in GMP conditions, Macopharma in
collaboration with EFS has developed an innovative closed system.
This system made of 3 different products allows the standardization of the three
main steps of the process: 1. Lysis of platelets (preserved in plasma) in a specific
bag by 2 cycles of freezing/ thawing and high centrifugation. 2. Filtration of the
platelet lysate using a dedicated kit (0.65µm) 3. Sampling of filtered platelet lysate
in 9 small bags allowing storage at -80°C. In comparison with a manual process,
this innovative system (Macopharma) provides a quick handling-time, safety
conditions for PL preparation. The PL prepared according this method maintains
a good bone marrow mesenchymal stem/stromal cells expansion.
30
POSTER ABSTRACTS
This development is CE marked, validated on bone marrow mesenchymal stem/
stromal cells expansion, and ready to be used in clinical applications.
86
OPTIMISING CONCENTRATION STRATEGIES FOR
PERCUTANEOUS INJECTION OF BONE MARROW DERIVED
MULTIPOTENTIAL STROMAL CELLS
Richard J Cuthbert, Mr, Peter V Giannoudis, Prof, Hiang B Tan, Mr, Dennis G McGonagle,
Prof, Elena Jones, Dr, Leeds Institute of Molecular Medicine, Leeds, England.
The use of multipotential stromal cells (MSCs) not subject to tissue culture
expansion is gaining interest. One strategy currently undergoing trial in clinical
orthopaedics for treatment of fracture non-union is direct percutaneous injection
of autologous bone marrow (BM) derived MSCs following volume reduction (1).
Critical factors determining efficacy of this strategy include: aspirate harvesting
technique, volume of bone marrow obtained, time from harvest to injection and
efficiency of concentration procedure. Here we examine the effect of changes in
aspiration technique and the efficiency of the Biomet MarrowstimTM stem cell
concentrator.
BM aspirates were obtained from 23 patients undergoing elective surgery. 20ml
were taken from one needle position or as 4x5ml draws from separate needle
positions. For the MarrowstimTM concentrator one 60ml draw was taken from
a single position as per manufacturer’s instructions. MSCs were enumerated
using classic colony-forming unit-fibroblast (CFU-F) assay either immediately (<4
hours) or following 24 hour storage at 4oC.
CFU-F frequency obtained by one single 20ml draw: 264 colonies/ml (range: 53504) was lower compared to 4x5 ml draws (428 colonies/ml, 56-902, p=0.08). The
MarrowstimTM device was able to concentrate CFU-Fs by an average of 10-fold from
15 colonies/ml (3-53) to 146 colonies/ml (20-568) however the final MSC frequency
remained significantly below that collected using 4x5 ml draws (p=0.028). Aspirate
storage for 24 hours resulted in up to 70% reduction in CFU-Fs.
The aspiration technique employed is critical to obtaining high numbers of MSCs.
One 60ml draw prior to concentrating is disadvantageous due to dilution with
blood (2). Combination of optimal aspiration and concentration should result in
much higher MSC doses for percutaneous injection.
1. Heringou et al., Bone Joint Surg-Am Vol. 2005 Jul;87A(7):1430-7
2. Cuthbert et al., Cytotherapy. 2012 Jan 24
87
isolation and expansion of human bone marrowderived mesenchymal stem cells and human adiposederived stem cells IN a serum-free medium
Ian M Kaplan, Ph.D., Mindy D Goldsborough, Ph.D., Kirsten B Crapnell, Ph.D., Robert E
Newman, Ph.D., BD, Cockeysville, MD, USA.
fibroblast (CFU-f) assays showed that BM-MSCs and ADSCs formed colonies
in both serum and serum-free conditions demonstrating the ability of Mosaic
to support the growth of cells with high proliferative capacity in culture. Data
show greater initial numbers of BM-MSCs at P0 in Mosaic and decreased time
to 8 population doublings from P0 (12.5 days vs. 16 days, respectively). Although
the immunophenotype of Mosaic-isolated MSCs were similar to serum isolated,
Mosaic MSCs exhibited more homogeneous expression of CD44. Cells grown
in Mosaic and serum-containing media were also both able to differentiate into
adipocytes, osteocytes and chondrocytes. In addition, cells grown in Mosaic
and serum-containing media were able to suppress T-cell proliferation in CD3/
CD28 stimulated peripheral blood mononuclear cells cultures. Results from these
experiments suggest that BD Mosaic, which is a serum-free media, is a suitable
substitute for serum-containing media for both isolating and expanding BMMSCs and ADSCs.
88
NOVEL METHOD TO ISOLATE MESENCHYMAL STROMAL CELLS
FROM BONE MARROW
Satoru Otsuru, MD, PhD1,2 Ted J. Hofmann, PhD1,2 Edwin M. Horwitz, MD, PhD1,2 1The
Children’s Hospital of Philadelphia , Philadelphia, USA, 2The University of Pennsylvania
Perelman School of Medicine, Philadelphia, USA.
Mesenchymal stromal cells (MSCs) are currently being studied as cell therapy for
a wide array of indications. In the most protocols, MSCs are isolated from bone
marrow and must undergo ex vivo expansion to obtain a sufficient dose of cells for the
investigational treatment. However, extended tissue culture is theoretically fraught with
hazards, including contamination and most worrisome, malignant transformation.
Moreover, gene expression changes with prolonged culture which could alter the
therapeutic potential of the cells. Thus, increasing the recovery of MSCs from the
freshly harvested bone marrow allowing for less culture expansion would be a major
advance in MSC therapy. In this study, two independent investigators isolated MSCs
from human bone marrow (n=6) using a novel MSC separation device(KANEKA
CORPORATION, Japan) containing a nonwoven fabric filter composed of rayon and
polyethylene in comparison with the conventional MSC isolation method by density
gradient centrifugation. The recovery of mononuclear cells (MNC) was significantly
higher using the device compared to density gradient centrifugation for each of the
two investigators (p=0.0042, p<0.0001). Furthermore, using the device, up to 2.8fold more CFU-F colonies were obtained from the same volume of bone marrow and,
most importantly, the total number of isolated MSCs after the 2nd passage was up to
3.4-fold greater. Both methods generated cells that expressed CD90, CD105, and CD73
and lacked hematopoietic antigen expression. Similarly all MSCs showed trilineage
differentiation (osteoblast, adipocytes and chondrocytes). Finally, the closed system
device method has a lower risk of contamination compared to the density gradient
centrifugation, requires less technical skill and substantially less time for processing.
Thus, this novel MSC separation device is fast, efficient, and reliable system to isolate
MSCs and will greatly expedite preclinical and clinical investigations of MSC therapy.
Isolating bone marrow-derived mesenchymal stem cells (BM-MSCs), and
adipose-derived stem cells (ADSCs), under serum-free conditions has
traditionally been problematic for researchers and cell therapy companies. BD
has developed a serum-free media formulation, BD Mosaic™ hMSC SF, that
can be used to isolate and expand MSCs from bone marrow, and isolate and
expand ADSCs from fat. Mosaic consists of a base media formulation, a growth
factor supplement, and a surface coating reagent for expansion of BM-MSCs and
ADSCs. Supplementation of this expansion formulation with collagen I and IL17 creates an isolation formulation. BM-MSCs were isolated from mononuclear
cells from bone marrow aspirates of 3 donors. Serum-free isolation conditions
were tested against a traditional serum-containing media isolation using qualified
serum lots. ADSCs were isolated from fat tissue using a similar protocol and
also compared to serum-containing media. Both cell types were then expanded
in culture in both serum-free and serum-containing media. Colony forming unit-
31
POSTER ABSTRACTS
89
Comparison of therapeutic effects between human
umbilical cord derived mesenchymal stem cells and
human umbilical cord blood derived hematopoietic
stem cells on experimental heatstroke
Sheng-Hsien Chen, 1,2 Jhi-Joung Wang,3 Hsiu-Kang Chang,4 Wei-Chun Chen,5 Fong-Ming
Chang,6 Wei-Yu Lo,7 11. Department of Obstetrics and Gynecology; Department of Medical
Research, Chi Mei Medical Center, Tainan, Taiwan, 2Department of Biotechnology, Southern
Taiwan University, Tainan, Taiwan, 3Department of Medical Research, Chi Mei Medical Center
, Tainan, Taiwan, 4Stem cell Research Center, Health Banks Biotech Co., Ltd, Taipei, Taiwan,
5
Stem cell Research Center, Health Banks Biotech Co., Ltd., Taipei, Taiwan, 6Department
of Obstetrics and Gynecology, Cheng Kung University, Tainan, Taiwan, 7Stem cell Research
Center, Health Banks Biotech Co., Ltd., Tainan, Taiwan.
Background: From our previous study, it showed human umbilical cord blood
(hUCB) derived hematopoietic stem cells (hUCBHSCs) transplantation can
resuscitate heatstroke(HS) rats. However, whether resuscitating ability after
human umbilical cord–derived mesenchymal stromal cells (hUCMSCs)
transplantation is superior to hUCBHSC transplantation during experimental HS
has never been reported.
Materials and Methods: All SD rats are randomly assigned to five groups.
Herein, we will assess the effects of heat exposure (e.g. 43oC, 68min) on body
temperatures, cardiovascular responses: 1; N=6) in normothermic control rats
(kept at 24 oC environment), 2; N=6) in heatstroke controlled rats (kept at 43oC
environment for 68 min then put on 24 oC environment, 3; N=6) in heatstroke rats
treated with hUCBHSCs (5×105 /ml) immediately after 68 min heat exposure. 4;
N=6) in heatstroke rats treated with hUCMSCs (5×105 /ml) immediately after 68
min heat exposure. 5; N=6) in heatstroke rats treated with hUCMSCs (2.5×105 /ml)
combined with hUCBHSCs (2.5×105 /ml) immediately after 68 min heat exposure.
Herein, we measured the survival time (period after 68 min heat expoure to
death) Simultaneously, we will utilize the H&E (hematoxylin and eosin) staining
to assess the neuronal damage, and immunofluorescence staining to detect the
migration of human cells.
Results: We compared these five groups and found after heat stroke, hUCMSCs
have the longest survival time compared to hUCMSCs combined with hUCBHSCs
and hHUCBHSCs only. Furthermore, hUCBHSCs have a shortest survival time.
Simultaneously, we analyze neuronal damage plus scoring for organ dysfunction
(under HE stain), hUCMSCs group has the least neuronal damage. Finally,
we indicated the most human nuclear antibody positive cells detected over
hypothalamus in the hUCMSCs group.
Conclusion: We successfully demonstrated hUCMSCs group could get the most
resuscitating effects for HS among these three treating groups. However, hUCMSCs
combined with hUCBHSCs therapy was superior to hUCBHSCs groups.
90
Tracking T cell clones using high-throughput
sequencing of antigen receptor CDR3 chains
Cindy Desmarais, 1 Jessica Matthis,2 Robert Livingston,1 Ryan Emerson,1 Nisanth
Marthandan,1 Anna Sherwood,1 Jessica Andriesen,3 Helena Reijonen,2 Christopher Carlson,3
Gerold Nepom,2 Cassian Yee,3 Karen Cerosaletti,2 Harlan Robins,3 1Adaptive Biotechnologies,
Seattle, USA, 2Benaroya Research Institute, Seattle, USA, 3Fred Hutchinson Cancer Research
Center, Seattle, USA.
The cellular adaptive immune system generates a remarkable breadth of diversity
in Y-like antigen-specific T cell receptors (TCR) by combinatorial shuffling of gene
segments in somatic cells. The existence of multiple V, D, and J gene segments
in the TCR loci (TCRΒ/TCRA and TCR∆/TCRΓ) permits large combinatorial
diversity in receptor composition, and template-independent deletion or insertion
of nucleotides at the V-J, V-D, and D-J junctions further adds to the potential
diversity. Because the potential diversity of receptors is large (approximately 10
million different TCRB), it is improbable to randomly converge on the same CDR3
32
sequence, effectively making each CDR3 sequence a unique nucleotide tag for a T
cell clone. However, the diversity of possible receptors is huge, making identifying
and tracking clones difficult.
We have developed immunoSEQ a method that amplifies rearranged TCRΒ
CDR3 sequences and uses high throughput sequencing to sequence millions of
chains per sample. This technology permits “clone-tracking” by both verifying
the presence/absence of specific T cell clones and estimating the frequency
of these clones within the overall repertoire. We verified the feasibility of our
assay to quantitatively track clones of interest by sequencing the repertoire of
samples doped with T cell clones across a 5-fold range (10-1,000,000 cells). Two
independent laboratories blindly and independently sent us samples with clones
spiked into a diverse background at 3 concentrations (4 clones) or 4 concentrations
(2 clones). We used these samples to test the precision, accuracy, and sensitivity
of our assay. We found that TCRβ repertoire sequencing accurately captures the
frequencies of clones across five orders of magnitude and is sensitive down to 1 in
100,000 cells. These results indicate that T cell receptor sequencing is an accurate
method to track T cell clones of interest. Potential applications include tracking
Minimal Residual Disease and tracking Adoptive Immune Therapy.
91
IL-2 Stimulated but Not Unstimulated NK Cells Induce
Selective Disappearance of Peripheral Blood Cells:
Concomitant Results to a Phase I/II Study
Claudia Brehm, 1 Sabine Huenecke,1 Andrea Quaiser,1 Ruth Esser,1 Melanie Bremm,1 Stephan
Kloess,1 Jan Soerensen,1 Hermann Kreyenberg,1 Christian Seidl,2 Heiko Mühl,3 Thomas
Klingebiel,1 Peter Bader,1 Jakob R. Passweg,4 Dirk Schwabe,1 Ulrike Koehl,1 1Pediatric
Hematology and Oncology, Laboratory for Stem Cell Transplantation and Immunotherapy,
Johann Wolfgang Goethe-University Hospital, Frankfurt, Germany, 2Institute for Transfusion
Medicine and Immunohematology, Red Cross Blood Donor Service, Baden-WürttembergHessen, Frankfurt, Germany, 3Pharmazentrum Frankfurt, Johann Wolfgang Goethe-University
Hospital, Frankfurt, Germany, 4Division of Hematology, University Hospital, Basel,
Switzerland.
In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high
risk leukemia/tumors received highly purified donor natural killer (NK) cell
immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell
transplantation. However, literature about the influence of NK-DLI on recipient’s
immune system is scarce. Here we present concomitant results of a noninvasive
in vivo monitoring approach of recipient’s peripheral blood (PB) cells after transfer
of either unstimulated (NK-DLIunstim) or IL-2 (1000 U/ml, 10 days) activated NK
cells (NK-DLIIL-2 stim) along with their ex vivo secreted cytokines/chemokines. We
performed phenotypical and functional characterizations of the NK-DLIs, detailed
flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine
arrays before and after NK-DLI. Patients of both groups were comparable with regard
to remission status, immune reconstitution, donor chimerism, KIR mismatching,
stem cell and NK-DLI dose.
Only after NK-DLIIL-2 stim was a rapid, almost complete loss of CD56brightCD16dim/immune regulatory and CD56dimCD16+ cytotoxic NK cells, monocytes, dendritic cells
and eosinophils from PB circulation seen 10 min after infusion while neutrophils
significantly increased. The reduction of NK cells was due to both, a decrease in
patients’ own CD69-NCRlowCD62L+ NK cells as well as to a diminishing of the
transferred cells from the NK-DLIIL-2 stim with the CD56brightCD16+/-CD69+NCRhighCD62Lphenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLIIL-2 stim
translated into significantly increased levels of various cytokines/chemokines (i.e.
IFN-Γ, IL-6, MIP-1β) in patients’ PB. Those remained stable for at least 1 h, presumably
leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast,
NK-DLIunstim did not cause any of the observed effects. In conclusion, we assume that
the adoptive transfer of NK-DLIIL-2 stim under the influence of ex vivo and in vivo secreted
cytokines/chemokines may promote NK cell trafficking and therefore might enhance
efficacy of immunotherapy.
POSTER ABSTRACTS
92
Efficient induced HBV specific CTL by HFP3 mediated
core antigen loading of human dendritic cells in
chronic hepatitis B patients
Dongyun Wu1, Ke Zhang2, Ran Tao1, Jian zhou1, Wei jiang1, Shiwu Ma2, Jin Li1, Xiangjun Zhou1,
DONGYUN WU, 1,2,3 1Shenzhen YUANXING BIO-PHARM SCIENCE &TECHNOLOGY CO.,
LTD., SHENZHEN , P.R.China, 2Ke Zhang, GUANGZHOU, P.R.China, 3Ran Tao, SHENZHEN
, P.R.China.
Dendritic cells (DCs) are the most potent professional antigen producing cells
(APCs) of the human immune system and they possess a unique ability to activate
CD4+ Th and CD8+ CTL cells for a specific antigen. The therapeutic applications of
DCs are advanced from in vivo activation of DCs by vaccine to ex vivo activation via
antigen loading with cultured DCs. DCs cultured from patients’ PBMC and loaded
with the specific antigen can be given back to patients either directly or after coincubating with patients’ T cells to activate the antigen-specific CTL. To develop
the DC therapeutic methodology for chronic hepatitis B (CHB), we loaded DC with
the recombinant hepatitis B virus core antigen (HBcAg)togetherwith the help of
a small peptide of protein-transduction domains (HFP3). Wethen assessed the
efficiency of antigen loading of the cultured DCs from health volunteers and HBV
carriers, as well as the loading effect on DCs’phenotype and function. Our results
demonstrated the HFP3 was able to enhance antigen loading on DCsand the DCs
could efficiently activate the HBcAg specific cytotoxic T cells. To evaluate the safety
and efficacy of HBcAg loaded DCs, two volunteers with chronic hepatitis B were
involved and each was administered five million DCs subcutaneously and five
hundred million DC-stimulated autonomous T lymphocytes intravenously every two
weeks for a total of 6 applications.The CHB patients injected with HBcAg loaded
DCs and DC-stimulated autonomous T lymphocytes did not exhibit inflammation,
exacerbation of the liver damage, abnormal kidney function, or other side-effects.
The long term effects (6 months) of this treatment on patients’ HBV loading and
E antigen conversion are being monitored.Our study provides evidence that HFP3
mediated HBcAg loading DC therapy issafe for CHB patients. Further studies will
be conducted to better evaluate the safety and effectiveness for other CHB patients.
93
T regulatory cells enriched from G-CSF mobilised
apheresates do not survive short term culture –
Impact on adoptive immunotherapy
Edward R. Samuel, 1 Katy Newton,2 Stephen Mackinnon,1 Mark W. Lowdell,1 1University
College London, London, United Kingdom, 2Cell Medica, London, United Kingdom.
PBMC seperated from G-CSF mobilised haematopoietic progenitor cell (HPC)
aphersates and stimulated with CMV peptides leads to the activation of CMVreactive T cells (CMV-T). In contrast to non-mobilised apheresis collections, HPC
collections contain a higher proportion of CD25+ FoxP3+ T regulatory cells (Tregs).
G-CSF mobilisation has long been associated with inhibition of type 1 cytokine
production by T cells. We investigated whether the elevated Treg population is
responsible for this effect and its impact on the generation of CMV-T for adoptive
immunotherapy. G-CSF mobilised and non-mobilised PBMC apheresates were
stimulated for 16 hours with CMVpp65 peptides, prior to isolation of CMV-T
through the activation marker CD25.
CD25 expression on CD3+ T cells following stimulation showed parity between
mobilised and non-mobilised PBMC (3.28% vs. 3.80%) and following enrichment
in yield (26.12% vs. 34.75%) and purity (89.92% vs. 93.39%).
Staining for the transcription factor FoxP3 which is expressed on CD25+ Tregs,
in CD25+ enriched CMV-T demonstrated that G-CSF mobilised PBMC contained
a higher proportion of FoxP3 expression pre and post CMVpp65 stimulation
which became significant after CD25 enrichment (P=0.008) when compared to
non-mobilised PBMC (64.2% vs. 33.8%). In suppression experiments CD25+
CMV-T enriched from G-CSF mobilised PBMC showed superior suppression of
autologous PBMC in response to antigen stimulation, in terms of proliferation,
when compared to non-mobilised PBMC. However after short term culture of
CD25+ enriched cells, FoxP3 expression was not maintained and cultured cells
secreted high levels of IFN-g and TNF-a with no IL-10 and co-expressed CD69 after
re-challenge with CMVpp65 autologous PBMC. Cultured cells were predominantly
CD3+ CD8+ (<90%) and capable of lysing CMVpp65 PHA blasts, demonstrating
they are phenotypically and functionally cytotoxic in nature.
These findings represent a potential approach for manufacturing cellular
immunotherapies from an aliquot of the original stem cell graft alleviating many
problems incurred with procuring second lymphocyte donations.
94
Utility of autologous dendritic cell as a
complementary treatment for colon cancer: A case
report
Francisco S. Chung Jr., 1,2 Monique D. Barile,1 May Lanie B. Fuentes,1 Marylette Roa,1
Alexandra Lee,1 Nelia S. Tan-Liu, MD1 Sullian S. Naval, MD1 Juanito A. Rubio, MD1 Maria
Teresa A. Barzaga, MD1 1Molecular Diagnostics and Cellular Therapeutics Lab, Department
of Pathology, Lung Center of the Philippines, Quezon , Philippines, 2Department of
Biochemistry and Molecular Biology, College of Medicine, University of the Philippines,
Manila, Philippines.
Background: Cancer ranks third in the leading cause of mortality after
communicable infection and cardiovascular diseases in the Philippines. Colon
cancer is ranked 5th as the leading cause of cancer death with approximately 38
% 3-year survival, thus warranting an improvement on colon cancer patients’
survival. With this in mind, our laboratory embarked on a safety evaluation of
the autologous dendritic cell (DC) for cancer patients. The study was approved
by the technical and ethics Committee of the Lung Center of the Philippines. This
is a case report of a 51 year old male patient who was diagnosed in September
2009 with mucinous adenocarcinoma of the colon stage 2. Methods: The patient
received an adjuvant treatment of autologous DC (six doses, 2-4 weeks apart)
during his treatment with Capecitabine (Xeloda). Upon hematological and cardiac
clearances, the patient underwent apheresis procedure. The DC were obtained
from the buffy layer after density gradient separation. These cells were cultured
without employing animal-based serum and with appropriate growth factors. The
cells were monitored for sterility and viability. Circulating tumor cell antigens were
determined using qRT-PCR. Erbb2 and MAGE were detected in the blood while
the patient tested negative for both CEA and MUC1. Levels of plasma IFN-Γ were
measured in order to evaluate whether the immune response was modulated.
Results: The patient did not experience any adverse event post administration of
the primed DC. Based on the CT-scan dated January 2012, no tumor recurrence
was observed while the circulating CEA remains within the normal levels. There
was a 220 % increase of IFN-g after the sixth injection of the DC, when compared
from the baseline. Conclusions: Our data suggest that immunostimulatory effect
post DC transplantation is noted. More importantly, the study did not show any
related toxicity post administration of the DC.
95
Tregs depletion with IL-21 administration to HCC cell
total RNA-transfected DCs to boost strong specific
immune responses against HCC
Hong-Mei Zhang, 1 Li-Wang Zhang,2 1Department of Oncology,Xijing Hosptal, Xi’an, China,
2
Tang Du Hospital, Xi’an, China.
Background: Although dendritic cell (DC)- based immunotherapy appears to be
a promising modality for the treatment of hepatocellular carcinoma (HCC) in vitro
studies, no conclusions regarding the efficacy of DC therapy can be made from
the early clinical trials. The presence of CD4+CD25+ regulatory T cell (Tregs) might
account for the immunotherapeutic failure. Hence, the current strategy for HCC
33
POSTER ABSTRACTS
immunotherapy is not only to induce curative HCC specific CD8+ CTLs, but also
to reduce the immune-suppression of Tregs. Interleukin (IL)-21, a cytokine with
structural and sequence homology to IL-2, is unable to support the proliferation
of Tregs, while IL-2 efficiently enhances their proliferation. Thus IL-21 may be more
suitable to break tolerance against HCC and to induce an efficient anti-tumor
response. Therefore, according to our previous work on DC-based immunotherapy
for HCC, we proposed here a bidirectional immunotherapeutic strategy: we use HCC
cell total RNA-transfected DC to boost strong specific immune responses against
HCC; on the other hand, we simultaneously combine Tregs depletion with IL-21
administration to subvert immune- suppression, improve endogenous immunemediated tumor rejection. Methods: Mouse DC2.4 cell line was transfected with total
RNA of Hepa1-6-GFP (Hepa1-6 cell line transfected stably with plasmid pEGFP-C3).
The transfected-DCs were used to stimulate mice spleen T cells in the presence of IL21. After Tregs depletion by FACS sorting, and the resultant Ag-specific effector T cells
were analyzed by IFN-Γ ELISPOT assay and cytotoxicity capacity assay. Results: After
transfection,DC2.4 cells expressed high level of DC surface molecules. In contrast to
IL-2, IL-21 significantly increased the IFN-Γ levels in the supematants of transfected
DC2.4-activated T cells (P<0.05). Furthermore, IL-21/DC2.4- activated T cells after
Tregs depletion (eliminating CD+/CD25+ tregs by FACS sorting) lysed Hepa1-6
target cells much more effectively than IL-21/DC2.4- activated T cells did (P<0.05).
Conclusion: This attractive strategy might yield the best prospect for the treatment
of HCC.
96
PURIFICATION AND CULTIVATION OF FUNCTIONAL HUMAN
PLASMACYTOID DENDRITIC CELLS (PDC) IN A CLOSED AND
FULLY AUTOMATED SYSTEM
Jessica Blaschke, Mareke Brüning, Hermann Bohnenkamp, Volker Huppert, Stefan Miltenyi,
Gregor Winkels, Andrzej Dzionek, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany.
Clinical-scale production of dendritic cell-based vaccines includes several cell
processing steps, i.e., washing, separation, cultivation and formulation. When
performed manually, these steps make the procedure laborious and increase
the risk of contamination. We developed a cell-processing instrument, including
a closed single-use tubing system with an integrated cultivation chamber, which
performs these cell processing steps in a single automated procedure. This system
minimizes the risk of contamination, thereby helping to fulfill the increasing
regulatory requirements for the production of cell-based therapeutics.
Due to their ability to produce large amounts of type I interferon (IFN) after
TLR7/9 stimulation, PDC play a central role in antiviral immune responses.
Additionally, there is growing evidence that PDC-derived type I IFN contributes
to the break of tolerance against tumors. Here we describe the development of a
fully automated PDC separation and culture protocol using the new instrument.
PDC were enriched from leukapheresis samples by magnetic cell sorting using
CD304 (BDCA-4) MicroBeads and directly eluted into the integrated cultivation
chamber using xenofree GMP cultivation medium. The average purity was
71%±12% with a recovery of 53%±1%. The overall yield of BDCA-4+ PDC was
1.5×107±5×106 from 6.8×109±1×109 cells of a leukapheresis harvest. After overnight
cultivation using xenofree GMP cultivation medium and GMP-grade interleukin-3
cell viability was >80%. When stimulated with CpG, PDC produced high amounts
of IFN and displayed the typical activation phenotype. All results were comparable
to controls cultured in conventional cell culture plates. Cell samples could be
frozen and thawed without loss of activity, enabling preparation of cells for several
vaccination rounds.
In conclusion, the new procedure enables large-scale isolation and cultivation of
functional human PDC in a closed system.
34
97
PARTIAL T CELL DEPLETION OF MOBILIZED PERIPHERAL BLOOD
AS A STRATEGY TO REDUCE INCIDENCE AND SEVERITY OF
GRAFT VERSUS HOST DISEASE
Joscelyn Bowersock, Ayesha Patterson, William P Vaughan, MD, Lawrence Lamb, Ph.D., Bone
Marrow Transplantation and Cellular Therapy Section, Division of Hematology and Oncology,
Department of Medicine, UAB, Birmingham, USA.
Chronic graft versus host disease (cGvHD) remains a significant cause of morbidity
in allogeneic hematopoietic stem cell transplant. Recent findings have shown that
incidence and severity of cGvHD is increased with the use of peripheral blood as a
graft source (PBSC) when compared to marrow (BM-SC). PBSC grafts are easier
to obtain, associated with less donor morbidity, and require less use of hospital
resources; therefore proving to be a more cost-effective process. Evidence suggests
that this disparity is due, in part, to a higher T cell content in the PBSC graft, on
average 3x108 CD3+ cells/kg as opposed 1.5x107 for BM-SC/kg. We developed a partial
T cell depletion (TCD) strategy by which PBSC graft CD3+ content was reduced to the
average marrow content by segregation of the portion of the product that contained
a dose of 2.5 x 107 total T cells/kg and depleted T cells from the segregated product
using OKT-3 labeled ferromagnetic microspheres on a CliniMACS column using
LS type tubing and depletion protocol 2.1. The products were recombined prior to
transplant in order to deliver a maximum of 3.5 x 107 T cells/kg while retaining the
other cellular components of the original graft. Three patients have been enrolled on
the protocol. Graft analysis revealed the following:
Patient
1
2
3
CD3+/kg
2.51E+07
3.48E+07
2.00E+07
CD34/kg
1.13E+07
1.09E+07
1.90E+06
%CD34
Recovery
95.00
79.39
100.00
%CD19
Recovery
100.00
100.00
72.20
%CD56
Recovery
ND
54.87
100.00
Interim follow-up of these patients has shown all to be free of acute GvHD. One
patient expired from AML relapse. Two others are alive and disease-free without
evidence of cGvHD. These limited data suggest that a targeted T cell dose can be
delivered using this graft engineering protocol while retaining a high proportion
of hematopoietic progenitors and non-T cell graft components.
98
NK DEPLETION ENHANCES MELANOMA REJECTION BY NAIVE,
TUMOR-SPECIFIC CD4+ T CELLS
Kyle A. Wilson, Kristina M. Harris, PhD, Stephen R. Goding, PhD, Paul A. Antony, MD,
University of Maryland, School of Medicine, Baltimore, MD, USA.
According to the National Cancer Institute’s Surveillance Epidemiology and End
Results (SEER) Cancer Statistics Review, the incidence of melanoma of the skin is
increasing. Incidence rates from 2005 to 2007 suggest that 1.93% of people born
today will be diagnosed with melanoma. Adoptive cell transfer (ACT) of naïve,
tumor–specific CD4+ T cells into lymphopenic hosts has been shown to induce
regression of established melanoma in preclinical models. In determining the
mechanism of tumor treatment by cytotoxic CD4+ T cells, we found that depletion
of natural killer (NK) cells enhances therapy. NK depletion increases the number
of tumor-specific CD4+ T cells and antigen-presenting cells (APCs), as well as
the amount of surface-bound interleukin 15 (IL-15) on APCs, and the serum
concentration of pro-inflammatory cytokines. Additionally, we observe increased
autoimmune vitiligo and fewer tumor relapses after ACT when combined with NK
cell depletion. Our data suggests that NK depletion removes a cytokine sink, and
liberates IL-15 for use by melanoma-specific CD4+ T cells. This demonstrates that
CD4+ T cells can use IL-15 when no other cellular sinks are present. Understanding
this mechanism may help develop new therapies that mimic the anti-tumor benefits
of lymphopenia without having to induce it.
POSTER ABSTRACTS
99
101
Reliable assay for monitoring CMV-specific T cell
immunity following Allogeneic Hematopoietic Cell
Transplantation
CD56+human dendritic cells pulsed with tumor
antigen and/or zoledronate effectively promote the
expansion of tumor antigen-specific CD56+CD8+T cells
and /or CD56+GAMMA DELTA T cells
Liselotte Brix, 1 Dalin Pan,2 George Chen,2 Charlotte Halgreen,1 Theresa Hahn,2 Philip
McCarthy,2 Henrik Pedersen,1 Paul K. Wallace,2 1Immudex, Copenhagen, Denmark, 2Roswell
Park Cancer Institute, Buffalo, USA.
Cytomegalovirus (CMV) infects and establishes persistent lifelong infections in
50-85% of adults. Reactivation of the virus is a frequently occurring complication
of immunosuppression following transplantation and can significantly contribute
to morbidity and mortality in such patients.
Reconstitution of CMV-specific T cell immunity after allogeneic hematopoietic cell
transplantation (alloHCT) has previously been quantified using CMV tetramers,
and shown to be a valuable aid in predicting patients at risk of developing CMV
reactivation. We have developed an assay for quantifying CMV-specific CD8+ T cells
using CMV-specific Dextramers. Dextramers are MHC multimer reagents that are
used in flow cytometry to detect antigen-specific T cells in the blood. Dextramers have
much higher resolution than conventional MHC multimers like Tetramers, and thus
provide a more reliable means for identification of antigen-specific T cells.
We here show that the CMV Dextramer assay including 7 alleles (HLA-A*01,-A*02,
A*03, A*24, B*07, B*08 and B*35) has high specificity and sensitivity and accurately
enumerates CMV-specific T cells in both healthy donors and alloHCT patients, with
a lower detection limit of 0.08 cells/ul. The assay is highly reproducible with low intra
and inter assay variation.
Using the CMV Dextramer assay we were able to quantify reconstitution of CMV T cell
immunity post transplantation at day 30, 100, and 365 in 89 patients. Furthermore,
in some recipients receiving transplants from HLA-mismatched donors we could
measure CMV-specific T cells restricted by donors HLA and not recipients HLA,
indicating that adoptive transfer of CMV-specific T cells can occur with alloHCT.
This study demonstrates that CMV Dextramers are reliable reagents that can be used
to monitor reconstitution of CMV immunity post-alloHCT, and shows that adoptive
transfer of anti-CMV immunity can be quantified.
100
Mie Nieda1, Hiroshi Terunuma1, Yuuta Eiraku1, Xuewen Deng1, Andrew Nicho2 1Biotherapy
Institute of Japan, Tokyo, Japan, 2Centre for Immune and Targeted Therapy, University of
Queensland, Private Hospital.
Background and Aims: CD56+ human monocyte derived dendritic cells (MoDCs)
have recently been shown to differentiate from monocytes in response to GMCSF and IFNα in vitro (referred as IFN-MoDCs). Further, it has been shown
that CD56+CD8+T cells and CD56+gdT cells have higher effector functions
including higher expression of CD107a, perforin and IFNΓ and higher cytotoxicity
against tumor cells in comparison with each counterpart, CD56-CD8+CTLs and
CD56-VΓ9gdT cells, respectively. The aim of this study is whether IFN-MoDCs
preferentially promote the induction of CD56+effector T cells possessing high
cytolytic effector function against tumour cells.
Results and discussion: The conventional mature MoDCs (conv. mMoDC) which
were differentiated from monocytes in the presence of GM-CSF, IL-4 and TNFα and
IFN-MoDC showed an almost identical phenotypic pattern in regard to the HLAclass I and DR molecules and the costimulatory molecules such as CD40, CD80
and CD86. However, the conv. mMoDCs mainly included CD56 and CD14 double
negative MoDCs and IFN-DCs mainly included CD56 and CD14 double positive
MoDCs. In comparison with using conv. mMoDC, tumour antigen pulsed IFNMoDCs efficiently expanded CD56+antigen specific CD8+T cells and zoledronate
pulsed IFN-MoDCs efficiently promoted the induction of CD56+VΓ9gdT cells.
Copulsing tumour antigen-pulsed IFN-MoDCs with zoledronate enhanced the
expansion of tumour antigen specific CD56+CD8+CTL, in addition to the expansion
of CD56+VΓ9 gdT cells. Both　CD56+antigen-specific CD8+CTL and CD56+VΓ9gdT
cells had higher effector function in comparison with the each counterpart. In
addition, CD56+VΓ9 gdT cells expressed antigen presenting molecules, resulted
in the expansion of CD56+antigen-specific CD8+CTL. Our findings offer a
new immunotherapy using CD56+MoDCs pulsed with tumour antigen and/or
zoledroante, resulting in efficient induction of CD56+CD8+CTL and/or CD56+gdT
cells, which may result in a better clinical outcome for patients with cancer.
SYNAPSE-DIRECTED DELIVERY OF IMMUNE-STIMULANTS USING
T-CELL-CONJUGATED NANOPARTICLES
102
Matthias T. Stephan, S. Peter Bak, Sirkka B. Stephan, Jianzhu Chen, Darrell J. Irvine,
Massachusetts Institute of Technology, Cambridge, USA.
LARGE-SCALE EXPANSION OF FUNCTIONAL REGULATORY T
CELLS USING A GAS-PERMEABLE RAPID EXPANSION CULTURE
WARE (G-REX)
Regulating molecular interactions in the T cell synapse to boost anti-tumor immunity
has long been a goal in cellular immunotherapy. However, delivering therapeutically
meaningful doses of immune-modulating compounds into the synapse represents a
major challenge. Here, we report that covalent coupling of maleimide-functionlized
nanoparticles (NPs) to free thiol groups on T cell membrane proteins enables efficient
delivery of compounds into the T cell synapse. We demonstrate that surface-linked
NPs are rapidly polarized toward the nascent immunological synapse (IS) at the T
cell/APC contact zone during antigen recognition. To translate these findings into a
novel therapeutic application we tested the NP delivery of NSC-87877, a dual inhibitor
of Shp1 and Shp2, key phosphatases that downregulate T cell receptor activation in
the synapse, in the context of adoptive T cell therapy of cancer. Conjugating NSC87877-loaded NPs to the surface of tumor-specific T cells just prior to adoptive
transfer into mice with advanced prostate cancer greatly promoted T cell expansion
at the tumor site, relative to co-infusing the same drug dose systemically, leading to
enhanced survival of treated animals. Altogether, our studies support the application
of T-cell-linked synthetic NPs as efficient drug delivery vehicles into the IS, as well as
the broad applicability of this new paradigm for therapeutically modulating signaling
events at the T-cell/APC interface.
Rikhia Chakraborty, Post doctoral Associate1 Aruna Mahendravada,1 Cliona M. Rooney,1,2,3
Juan F. Vera,1,4 Barbara Savoldo,1,2 Gianpietro Dotti,1,3,4 1Center for Cell and Gene Therapy
, Baylor College of Medicine,Methodist Hospital and Texas children’s Hospital, Houston,
USA, 2Department of Pediatrics, Baylor College of Medicine, Houston, USA, 3Department of
Immunology, Baylor College of Medicine, Houston, USA, 4Department of Medicine, Baylor
College of Medicine, Houston, USA.
Adoptive transfer of naturally occurring regulatory T cells (nTregs) has potential
for clinical use. The complexity associated with manufacturing adequate numbers
of ex vivo expanded nTregs using cost effective procedure represents a serious
impediment for their broader clinical application. We have here optimized a
manufacturing protocol based on the use of a gas-permeable static culture flask
(G-Rex). nTregs were isolated from 7 buffy coats by CD25-immunomagnetic
selection and expanded in the presence of IL-2 (50 IU/ml), rapalog (1 μg/mL) and
irradiated feeder cells at 1:5 ratio. After three weeks, nTregs had expanded about 600
folds (from 3x106 to 1.8x109 ± 7.3x106), and 95% ± 3% of these expanded cells coexpressed CD25 and CD4, with 55% ± 4% retaining FoxP3 expression. Using a CSFEbased suppression assay, expanded nTregs significantly inhibited the proliferation
of CD8+ T cells from 82% ± 3% to 17% ± 2% (p<0.01). Suppressive activity was
35
POSTER ABSTRACTS
retained also after freeze-thawing [inhibitory properties before freezing (82% ± 3.2%
to 17% ± 2.5% p<0.01) vs. after thawing (85% ± 4.7% to 22% ± 3.2% p<0.01)]. Finally,
we validated in vivo the inhibitory function of expanded nTregs using a xenograft
model of GvHD. NSG mice (15 mice/group) were irradiated, received 15x106 PBMC
and 15x106 expanded nTregs or their depleted counterpart 15x106, and were then
monitored for sign of GvHD. Expanded nTregs efficiently suppressed lethal GvHD
measured as loss of body weight and as spleen and gut infiltration by CD8+ T cells.
Indeed, we found delayed (day 36) or no sign of GvHD in mice receiving expanded
nTregs as compared to mice receiving their depleted counterpart (day 9). KaplanMeier-Survival curveshowed a significant improvement in overall survival for nTregtreated mice (p<0.0001). Advantages can be envisioned, not only to prevent the
occurrence of GvHD but also to treat autoimmune diseases.
103
Therapeutic potential of EBV-specific CTLs in patients
with extranodal NK/T cell lymphoma
Seok-Goo Cho, MD, PhD, Hyun-Jung Sohn, Jung A Hong, PhD, Young Seon Hong, MD,
PhD, Suk Kyeong Lee, PhD, Tai-Gyu Kim, MD,PhD, Seoul St. Mary’s Hospital, The Catholic
University of Korea, Seoul, Korea.
Background: Extranodal NK/T-cell lymphoma (ENKTCL) is highly associated with
latent Epstein-Barr virus (EBV) infection and frequent relapse even after complete
response (CR) to intensive chemotherapy and radiotherapy. The role of the EBV
in pathogenesis of this disease and EBV proteins expressed in this lymphoma
provide targets for the adoptive immunotherapy with antigen-specific cytotoxic
T lymphocyte (CTL) and raise the possibility of EBV-specific CTL as therapeutic
strategies.
Methods: Eleven patients who were responded to chemotherapy were received
EBV-CTLs. For generation of EBV-CTLs in vitro, mature dendritic cells(DC) derived
from monocytes were pulsed with RNAs of EBV LMP1 and LMP2a antigens, and
then T cells were stimulated with DCs three times for 3 weeks in Catholic GMP
cell processing center. EBV-CTLs were cryopreserved for later usage and some
remaining cells were analyzed. Patients completed the induction treatments
including chemotherapy, radiotherapy, and/or high-dose therapy followed by
autologous peripheral blood stem cell transplantation (HDT/SCT) before the
infusion of EBV-CTLs and received 8 doses of 2 x10E7 CTLs/m2.
Results: Ten patients achieved CR and one patient achieved partial response after
induction therapy, and five patients including one patient in PR underwent HDT/
SCT. During the maintenance therapy with EBV-CTLs, one patient dropped out
of infection after 5 doses of EBV-CTLs therapy and the others completed 8 doses
of EBV-CTLs. Among eleven patients, one patient relapsed 17.3 months since
induction therapy. In overall, the 3-year overall survival, progression-free survival
since induction therapy were 85.7±13.2%, 88.9±10.5%, respectively with a median
follow-up of 25.2 months. For five patients who had HDT/SCT, DFS from SCT was
80.0±17.9% with a median follow-up of 21.0 months.
Conclusion: This pilot study could be applied to patients with ENKTCL with safety
and effectiveness. The larger prospective study is needed to define the role of
EBV-CTLs therapy to prevent unpredictable relapse in EBV-positive ENKTCL.
104
CYTOKINE-INDUCED KILLER CELLS ARE EFFECTIVE AGAINST
LOW-GRADE AND AGGRESSIVE B-CELL LYMPHOMA “IN VITRO”
Silvia Castegnaro, Cristina Zanon, Katia Chieregato, Elena Albiero, Martina Bernardi,
Domenico Madeo, Francesco Rodeghiero, Giuseppe Astori, Department of Cell Therapy and
Haematology. Laboratory of Advanced Cellular Therapies.San Bortolo Hospital, Vicenza, Italy.
36
Introduction and Aims. CIK cells, a subset of T lymphocytes with a NK phenotype
(CD3+CD56+), are capable of a MHC-unrestricted anti-tumor activity against
hematological malignancies. CIK cytotoxicity has been well investigated “in vitro”
against a Natural Killer-responsive Chronic myeloid leukemia (CML) human cell
line (K562) but few data are present on their effect on different hematological cell
lines. We tested the “in vitro” cytotoxic effect of CIK cells against acute myeloid
leukemia (AML) and B-cell lymphoma cell lines.
Methods. CIK cells (effector, E) were generated from peripheral blood nucleated
cells (n=5) stimulated with interferon-gamma (IMUKIN Boehringer-Ingelheim),
anti-CD3 monoclonal antibody (OKT3 Janssen-Cilag) and IL-2 (PROLEUKIN
Novartis Pharma). CIK cells were then co-cultured in the presence of 4 target cell
lines (T): AML line (KASUMI-1), low-grade B-cell lymphoma (DOHH-2, follicular
lymphoma) and aggressive diffuse large B-cell lymphoma (SU-DHL-4). A CML
cell line (K562) was used as control. Citotoxicity was quantified by measuring
the fluorescent calcein released by target cells after four hours of exposition to
effector cells at E:T ratios from 1:1 to 40:1 Experiments were performed under
Good Laboratory Practice (GLP).
Results. CIK cells showed a potent cytotoxicity against follicular lymphoma line
(DOHH-2), and diffuse large B-cell lymphoma (SU-DHL-4) cell lines. The CIK
performance against lymphoma cells was always higher than that measured in
CML cell line (K562), significantly (p<0.05) for DOHH-2; this effect was E:T ratio
dependent and detectable already at 10:1. KASUMI-1 cell line seemed to be less
responsive. Results were summarized in Figure 1.
Discussion. These preliminary results encourage further studies for defining the
activity of CIK cell against indolent or aggressive lymphoma cells, with the aim of
supporting the use of CIK cells for lymphoma treatment in the future.
105
Combination Cellular and IL-2 Therapy Improves
Survival of Ovarian Cancer Bearing Mice
Susan B. Ingersoll, Ph.D.1,2 Hasina C. McGann, MS1 Sarfraz Ahmad, PhD1,2,3 Ahad Ahmed, BS1
Neil J. Finkler, MD1,2,3 John R. Edwards, MD3,4 Robert W. Holloway, MD1,2,3 1Florida Hospital
Cancer Institute, Orlando, USA, 2Florida State University, College of Medicine, Orlando, USA,
3
University of Central Florida, College of Medicine, Orlando, USA, 4CTI Clinical Trials and
Consulting Services, Cincinnati, USA.
Objective: To test cellular therapy in combination with IL-2 in a xenograft mouse
model of ovarian cancer (OC).
Methods: SKOV3-AF2 (1x106) OC cells were injected intraperitoneally (IP) into female
athymic nude mice. On day+7 post tumor cell injection, mice were randomized to
treatment groups [n>10/group; PBS, IL-2 (4,000U); mononuclear cells (MC, 5x106);
or IL-2+MC;] and injected IP with IL-2 and/or MC. MC were isolated from two healthy
donors and cryopreserved until time of injection at which time cells were thawed,
washed, and re-suspended at 25x106 cells/ml. IL-2 injections were continued thrice
weekly. Mice were sacrificed when they became moribund due to tumor burden;
solid tumor and ascitic fluid (AF) were measured and collected for histopathological
analysis (H&E) and gene expression (SOCS1, CCN1, and E-cadherin).
Results: All cell and cytokine combinations were tolerated as evidenced by no
significant weight loss or other signs of distress. Harvested tumors consisted
of poorly differentiated surface epithelial carcinoma, growing in solid nests and
sheets of large cells with a modest amount of amphophilic/clear cytoplasm.
Tumor cells were focally pleomorphic and multinucleated; mitoses were frequent
POSTER ABSTRACTS
with abnormal forms present. Mice treated with MC+IL-2 (50% survival; p<0.05)
showed a significant improvement in survival at 8-weeks compared to mice
receiving IL-2 (6%) or PBS (8%). MC+IL-2 treated mice showed an increased
survival compared to mice receiving MC (20%); however, this difference was not
statistically significant. No significant difference in tumor weight (0.95-1.18 g), AF
incidence (20-40%), or AF production (0-6.8 ml) was observed between groups.
All tumors expressed SOCS1, CCN1, and E-cadherin; however, no significant
difference in gene expression was associated with any treatment.
Conclusion: We present evidence that cytokine-stimulated cellular therapy
produces an anti-tumor effect using a xenograft OC model. This data strongly
supports the development of cellular therapy as a potentially useful therapeutic
strategy for the treatment of OC.
106
Value of large-scale expansion of tumor infiltrating
lymphocytes in a compartmentalised gas-permeable
bag: interests for adoptive immunotherapy
Thomas Zuliani, 1,2 Julien David,2 Sylvain Bercegeay,1 Marie-Christine Pandolfino,1 Isabelle
Rodde-Astier,3 Amir Khammari,4 Cécile Coissac,3 Bruno Delorme,3 Soraya Saïagh,1 Brigitte
Dréno,1,2,4 1Cell and Gene Therapy Unit (UTCG): CIC biotherapy INSERM 0503 Hôtel-Dieu
University Hospital, Nantes, France, 2Immunodermatology Laboratory: CIC biotherapy
INSERM 0503 Hôtel-Dieu University Hospital, Nantes, France, 3MacoPharma, Tourcoing,
France, 4Dermatological Oncology Department: CIC biotherapy INSERM 0503 Hôtel-Dieu
University Hospital, Nantes, France.
Background: Adoptive Cell Therapy (ACT) has emerged as an effective treatment
for patients with metastatic melanoma. However, there are several logistical and
safety concerns associated with large-scale ex vivo expansion of tumour-specific
T lymphocytes for widespread availability of ACT for cancer patients. To address
these problems we developed a specific compartmentalised bag allowing efficient
expansion of tumour-specific T lymphocytes in an easy handling closed system.
Methods:Starting from lymph nodes from eight melanoma patients, we performed
a side-by-side comparison of Tumour-Infiltrating Lymphocytes (TIL) produced after
expansion in the compartmentalised bag (MacoPharma, patent Nr 07/00252)
versus TIL produced using the standard process in plates (Nantes Hospital,
patent 07/00238). Proliferation yield, viability, phenotype and IFNg secretion were
comparatively studied.
Results: We found no differences in proliferation yield and cell viability between
both TIL production systems. Moreover, each of the cell products complied with
our defined release criteria before being administered to the patient. The phenotype
analysis indicated that the compartmentalised bag favours the expansion of CD8+
cells. Finally, we found that TIL stimulated in bags were enriched in reactive CD8+ T
cells when co-cultured with the autologous melanoma cell line.
Conclusions:The stimulation of TIL with feeder cells in the specifically designed
compartmentalised bag can advantageously replace the conventional protocol
using plates. In particular, the higher expansion rate of reactive CD8+ T cells could
have a significant impact for ACT.
107
New GMP-grade, animal-component-free medium for
activation and expansion of T-cells
Ulrike Kolrep, Gerd Steffens, Nadine Mockel-Tenbrinck, Alexander Scheffold, Hermann
Bohnenkamp, Mario Assenmacher, Veit Bergendahl, Melanie Fahrendorff, Miltenyi Biotec
GmbH, Bergisch- Gladbach, Germany.
Therapeutic applications of T-cells in immunotherapy have recently gained
momentum with the promising results in adoptive transfer of antigen-specific
T-cells for infectious complications after allogeneic-stem-cell or solid-organ
transplantation or for immunotherapy of malignant diseases. Activation and
expansion of these cells for clinical application under controlled conditions require
GMP-grade reagents including appropriate antibodies, cytokines and media. For
standardized, reproducible cell cultivation and ex vivo differentiation procedures,
a new serum and animal-component-free, GMP-grade medium for clinical use
has been developed. High lot-to-lot consistency has been achieved by eliminating
protein components not relevant for T-cell expansion leaving human serum
albumin as the only protein component.
Using soluble antibodies against CD3 and CD28, more than 30%-higher expansion
rates of viable and functional T-cells after 6 days of expansion have been achieved
with the new animal-component-free medium compared with other serum-free
media. Transferring the same protocol to a high density cell culture system such as
a gas permeable rapid expansion device, high densities of T-cells with more than
1.5x10^7 cells/ mL were reached.
The generation of antigen-specific T cells using the Cytokine Capture System IFNgamma (CCS) and the serum and animal-component-free T-cell medium showed
similar results regarding purity, recovery and background stimulation compared to
the use of a standard basal-medium supplemented with 10% human AB-serum.
For the automation of such complex procedures, a new cell processing device
was developed.All steps for the CCS processing performed in this fully automated
device, in a closed system under sterile conditions.
In conclusion, the newly developed GMP-grade, serum and animal-componentfree T-cell medium demonstrated high lot-to-lot consistency and was superior
in its performance to other commercially available serum-free media. The new
medium can be used to replace human AB-serum supplementation for the clinical
manufacturing of T-cells resulting in easier handling and higher consistency.
108
Ex vivo expanded natural killer cells can possibly kill
cancer stem cells
Xuewen Deng, 1 Mie Nieda,1 Hiroshi Terunuma,1,2,3 1Biotherapy Institute of Japan, Tokyo,
Japan, 2Tokyo Clinic, Tokyo, Japan, 3Southern Tohoku General Hospital, Fukushima, Japan.
Introduction: Targeting cancer stem cells (CSCs) could be a strategy to improve
the outcome of cancer therapy. Natural Killer (NK) cells are thought to be a
suitable candidate for adoptive immunotherapy.
Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from
healthy donors (HDs) and cancer patients. Ex vivo expanded NK cells and freshly
isolated NK cells were prepared from PBMCs. CD133+ UB2MT and C1AK cancer
cell lines were established in our laboratory. Cell phenotype, surface marker
expression, and intracellular cytokine were detected using flow cytometry.
Cytotoxicity against cancer cell lines was tested using Terascan.
Results: By Day 14, the expansion of CD3-CD56+NK cells from three HDs reached 600fold, 1,100-fold, and 1,420-fold, respectively. By Day 21, this reached 2,170-fold, 5,610fold, and 12,240-fold, respectively. Moreover, NK cells were expanded 1,530±720-fold
(n=27) during an 18±2 day cultivation period in various cancer patients. NK cells from
the three HDs were analyzed. On Day 0, their NK cells comprised 17.5%, 8.7%, and
17% of PBMCs, which increased by Day 14 to 63.2%, 68.3%, and 77.3% of all expanded
cells, respectively. The expanded NK cells expressed higher levels of CD69, NKG2D,
and NCRs in comparison with the NK cells in PBMCs. Furthermore, the expanded NK
cells highly expressed IFN-Γ and TNF-α in response to target cells by comparison to
the NK cells in PBMCs. The expanded NK cells showed a significantly higher IFN-Γ,
perforin and granzyme positive cells when compared to expanded ΓδT cells and
expanded αβT cells. The expanded NK cells showed a much higher cytotoxicity against
K562 and MHC-class I+ CD133+ cell lines than freshly isolated NK cells, expanded ΓδT
cells, or expanded αβT cells.
Conclusion: The use of ex vivo expanded NK cells may represent a novel therapeutic
approach for cancer immunotherapy, possibly target CSCs.
37
POSTER ABSTRACTS
109
111
CURCUMIN HELPS IN TREATMENT OF NEURODEGENERATIVE
DISORDERS
HUMAN FETAL-DERIVED STEM CELLS CAN DECELERATE MOTOR
DETERIORATION AND WEIGHT LOSS IN A RAT MODEL OF
CEREBELLAR ATAXIA
AP Singh, D Mazumdar, S Aijaz, Jawaharlal Nehru University, New Dehli, India.
Curcumin is a polyphenol extracted from the rhizome of Curcuma longa and well
known as a multi-functional drug with antioxidative, anti-cancerous and antiinflammatory activities. Curcumin’s antiaging and neuroprotective potential is
widely reported.
Objective: To study the effect of curcumin in neurodegenerative disorders
Observations: In the present study, effect of curcumin treatment dose 30 mg
kg(-1) day(-1) was investigated against aluminium neurotoxicity in young and
old animals. Direct and indirect intakes of aluminium have been reported to be
involved in the etiology of several neurodegenerative disorders like Alzheimer’s
and Parkinson’s diseases. Long term Al was administered through drinking water
at a dose of 50 mg/kg/day for 6 months in both young (4 months) and old (18
months) male Wistar rats.
Results: Result obtained demonstrates that curcumin treatment attenuates the
Al-induced alterations at biochemical, behavioral and ultrastructural levels which
was well reflected in the electrophysiological recordings. Our results indicate that
curcumin’s ability to bind redox active metals and cross the blood-brain barrier
could be playing crucial role in preventing against Al-induced neurotoxicity.
110
THE USE OF HUMAN ALDHbright CELLS IN ATTENUATING
INTRACRANIAL INFLAMMATION DUE TO BRAIN IRRADIATION
Catherine T. Flores, PhD Henry S. Friedman, MD Tracy Gentry, Andrew Balber, Joanne
Kurtzberg, MD1 1Duke University Medical Center, Durham, North Carolina, USA, 2Aldagen,
Durham, North Carolina, USA.
1
1
2
2
Brain irradiation is used as part of the aggressive multi-modality therapy against
CNS malignancies. Studies suggest that irradiation of normal brain results in
sequelae leading to brain damage and cognitive impairments. Neuroinflammation
subsequent to brain radiation plays a vital role in the etiology of these effects.
As part of our efforts to develop adjunctive cell therapies in treatment of CNS
malignancies, we studied whether human bone marrow-derived cells that express
high aldehyde dehydrogenase activity (ALDHbri cells) attenuate intracranial
inflammation following whole brain irradiation therapy (WBRT). In a model of
WBRT, immunodeficient NOD/scid IL2r gamma-/- mice received a single dose of
2Gy WBRT followed by intravenous injection of 2x105 ALDHbri cells 30 days later.
Brains were analyzed 60 days post WBRT. Mice that received ALDHbri cells had
decreased intracranial TNFα and IFNΓ, and decreased expression of microglial
activation marker Iba1. In repeat studies, this effect was observed up to 60 days
after receiving ALDHbri cells. Moreover, we observed increases in levels of mouse
specific anti-inflammatory cytokines IL10 and IL5 in conjunction with decreases in
TNFα and IFNΓ. Interestingly, we also observed low levels of human specific IL10,
which does not have a mouse ortholog. Our study suggests that ALDHbri cells may
play a role in either attenuating intracranial inflammation, or preventing the onset of
the inflammatory process for an extended period after WBRT. Thus, treatment with
autologous ALDHbr cells shows translational promise for potentially preventing the
onset cognitive decline in patients who survived brain malignancies but suffer the
damaging effects of radiation therapy. Autologous bone marrow-derived ALDHbri cells
have been safely used in the clinic for cardiovascular diseases and are currently in a
Phase I safety trial for use in stroke patients.
38
Toni L. Uhlendorf,1 Alex O. Kopyov,2 Ruslan Nuryyev,3 Randy W. Cohen,4 Oleg Kopyov,2
1
California State University Northridge, Northridge, USA, 2Celavie Biosciences, LLC, Oxnard,
USA, 3California State University, Northridge, Northidge, USA, 4California State University,
Northidge, USA.
Hereditary ataxias are devastating neurological disorders that frequently result
from neurodegeneration of the cerebellum typically with Purkinje cell and granular
cell loss. These progressive ataxias exhibit neurological symptoms that include
gate incoordination, tremor, trunk instability, muscle wasting, hind limb rigidity,
and gaze apraxia.
Spastic Han-Wistar (sHW) mutant male rats characterized by progressive
neurodegeneration of Purkinje cells in the cerebellum were used in this study to
investigate the ability of human fetal-derived stem cells (Celavie Biosciences) to
correct both functional and structural deficits caused by this disease.
We have investigated comparative efficacy of two routes of SC Delivery: carotid
artery and stereotactic injection into hippocampus (AP -3.0; L -2.5; V 3.5) and into
cerebellum (AP -11.0; L -2.0; V 5.5) at 40 days of age. For each route, a control
group was used (injection of dead stem cells). Activity scores (open field test) and
weights of the animals were monitored over a period of 30 days since the onset of
symptoms and treatment. At the end of behavioral testing the animal were deeply
anesthetized with chloral hydrate, perfused with 4% paraformaldehyde prepared
for following immunohistochemical evaluation.
Groups with stereotactic and carotid injections demonstrated a statistically
significant deceleration of motor deterioration (p<0.05) as evidenced by activity
scores. As well, groups with stereotactic injection showed significant (p<0.05)
decrease in weight loss compared to controls. No significant difference in weight
loss was observed between the group with carotid injection of stem cells and its
control. Histology revealed fewer stem cells migrating into the brain using the
carotid artery injection procedure compared to stereotactic placement.
These data suggest that fetal derived stem cells are a viable treatment for
hereditary cerebellar ataxias and that stereotactic method of delivery is somewhat
superior to the carotid injection.
112
STEM/PRECURSOR CELLS IN MENINGES REACT TO SPINAL CORD
INJURY, MIGRATE TO THE PARENCHYMA AND CONTRIBUTE TO
GLIAL SCAR FORMATION
Francesco Bifari, MD, PhD1 Ilaria Decimo, PhD2 Francisco Rodriguez, PhD3 Giorgio Malpeli,
PhD4 Sandra Vasquez, MSc3 Marina Sciancalepore, PhD5 Alberto Montalbano, MSc5 Valeria
Berton, MSc2 Mauro Krampera, MD, PhD1 Guido Fumagalli, MD2 1Department of Medicine,
Stem Cell Research Laboratory, Section of Hematology, University of Verona, Verona, Italy,
2
Department of Public Health and Community Medicine, Section of Pharmacology, University
of Verona, Verona, Italy, 3Hospital Nacional de Parapléjicos , Toledo, Spain, 4Department of
Pathology, Section of Pathological Anatomy, University of Verona, Verona, Italia, 5Department
of Life Sciences, University of Trieste, Trieste, Italy.
We have previously described a new stem/precursor cell population with neural
differentiation potential in rat brain-meninges.
In this work, we describe a population of cells resident in adult rat spinal
cord meninges that express the neural stem/precursor markers nestin and
doublecortin. Stem/precursor cells can be extracted from meninges, cultured in
vitro as neurospheres and subsequently differentiated into functional neurons
and mature oligodendrocytes. Spinal cord injury induces activation of meningeal
stem/precursors by increasing proliferation and cell number. Furthermore, we
found that meninges-derived stem/precursor cells migrate and contribute to the
parenchymal reaction.
POSTER ABSTRACTS
Our data indicate that spinal cord meninges are potential niche harboring
injury-responsive stem/precursor cells that could be further tested for use in
regenerative medicine.
The study objective was to investigate the safety of intrathecal transplantation of
autologous BMMNC mixed with RBC in two neurodegenerative conditions i.e.
spinal cord injury (n=15), age group18- 65 and cerebral palsy (n=8), 6-12 years.
113
Methodology: Following standard BM collection procedure, 60-100ml of BM was
aspirated and processed using intra-operative point of care (POC) technology to
obtain 6-10ml bone marrow concentrate (BMC) rich in BMMNCs. BMC therapeutic
dose is routinely contaminated with a small fraction of RBCs having an average 10%
hematocrit with an absolute dose of 1.3 million + 0.7 (SD) RBCs/ml. The BMC was
transplanted intrathecally into 23 patients.
Preparation of human Schwann cells for
transplantation. Human serum albumin in final wash
steps enhances cell viability.
Gagani Athauda, MD1,2 Adriana Brooks-Perez, BSc1,2 Aisha Khan, MBA PhD1,3 Dalton Dietrich,
Phd1,2 Pat Wood, PhD1,2 James Guest, MD PhD1,2 1University of Miami Miller School of
Medicine, Miami, USA, 2The Miami Project to Cure Paralysis, Miami, USA, 3Diabetes
Research Institute, Miami, USA.
We developed an autologous human Schwann cell (hSC) product for application
in subacute spinal cord injury. Cell preparation for clinical use requires careful
specification of wash and final product components to ensure cell products are
similar from subject to subject, and satisfy regulatory requirements. Here we
report the effect of inclusion of human serum albumin (HAS) in the final rinse
steps on total cell recovery and cell stability in the final HSC product. Method:
We compared total cell counts in the hSC product when the HSA was either
present or absent in the final rinse step during product formulation. To measure
cell stability, we compared the viability of three different hSC products at time
intervals of 0, 6, 8 and 24 hours following final rinses with or without HSA, using
anautomated dye exclusion assay. Results: SC cell counts after third wash were
28.7 x 106 ± 8 without HSA compared to 33.9 x 106 ± 5 (p=0.3281) for 5% HSA.
SC viability at 0hr was 93.9±0.6 without HSA compared to 99.2 ± 0.1 for 5% HSA
(p= 0.0001). Viability at 6hr was 92.7 ± 1.2 without HSA compared to 98.7 ± 0.5
for 5% HSA (p= 0.0011). Viability at 8hr was 92.4±1.0 without HSA compared
to 98.9 ± 0.2 for 5% HSA (p= 0.0003). Viability at 24hr was 90.4 ± 2.7 without
HSA compared to 97.7 ± 0.9 for 5% HSA (p= 0.0116). Conclusions: There was no
significant difference in the final cell count of products harvested with and without
HSA. The presence of HSA in the final rinses increased the viability of the cells as
measured over a 24 hour period. These results suggest that inclusion of HSA in
the final rinses would significantly improve the viability of the hSC product at the
time of transplantation.
114
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115
Will not be presented
116
Intrathecal administration of autologous
bone marrow cells WITH 10% Hematocrit / RBCs are
clinically safe
Venkatesh Ponemone, PhD1 Roma Gulati, MBBS2 Mitchel Sivilotti, MSc2 Arun Mukurjee, MD3
Yashbir Dewan, MS, MCh4 Kenneth Harris, MS1 1TotipotentRX Corporation, Los Angeles,
USA, 2TotipotentSC, Gurgaon, India, 3Shubam Hospitals, New Delhi, India, 4Fortis Hospital,
Noida, India.
Objective: Cellular transplantation, a promising therapeutic strategy for neurological
regenerative indications; requires safe and efficient protocols of cell delivery. The
recent adoption of point-of-care devices in lieu of lab processing has changed the
cellular constituency of the transplant product. Rapid, single sitting and efficient
bone marrow mononuclear cells (BMMNC) recoveries are trading-off with high
RBC contamination.
Most common route of administration for neurological regeneration is intrathecal
i.e. lumbar puncture (LP). However, concern has been specifically expressed on
the safety of injecting a therapeutic dose of stem cells with contaminating RBCs.
Results: Intrathecal transplantation of autologous BMC with 10% hematocrit did
not result in any clinical safety concerns, and is concluded safe for treatment. The
patients were followed up for 6 months to 1 year post-transplant with no adverse
responses. Two patients out of 23 were reported to have injection-related adverse
effects- transient fever and headache which, resolved within 48h with symptomatic
treatment. No major adverse effects were reported during follow up.
Conclusion:Study demonstrated that intrathecal administration of autologous
BMMNCs with <10% hematocrit is clinically safe and feasible procedure. Further
studies with higher percentage of hematocrit are required to conclude the safety or
tolerable dose range of autologous RBCs.
117
EXPLORING THE USE OF HUMAN VERY SMALL EMBRYONIC-LIKE
STEM CELLS ISOLATED FROM ADULT PERIPHERAL BLOOD FOR
THERAPY OF DRY AGE-RELATED MACULAR DEGENERATION
Yajuan Jiang, 1 Caihui Jiang,2 Guochun Chen,2 Petr Baranov,2 Desiree Cyr,2 Elizabeth Leary,1
Gregory Yavanian,1 Sean Hall,1 Sarah Eminli,1 Wayne A. Marasco,3 David W. O’Neill,1 Anthony
Salerno,1 Kameran Lashkari,2 Michael J. Young,2 1Neostem, Inc, Cambridge, MA, USA,
2
Schepens Eye Research Institute, Massachusetts Eye & Ear, Harvard Medical School, Boston,
MA, USA, 3Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
Age-related macular degeneration (AMD) is one of the leading causes of incurable
blindness in the world. Atrophic (dry) AMD, the most prevalent form, accounts
for 90% of cases, is characterized by progressive deposition of debris under the
retinal pigment epithelium (RPE), a highly specialized tissue that supports and
protects the light sensitive photoreceptors of the outer neural retina, leading to
their degeneration. Cell transplantation (RPE, photoreceptor precursors or cells
with neuroprotective abilities) could be used to restore sight in advanced AMD by
replenishing the subretinal anatomy and re-establishing the functional relationship
between RPE and photoreceptors. Very Small Embryonic-like Stem cells (VSELs),
found in human bone marrow and in adult peripheral blood, are small (5 to 9 μm)
Lin- CD45- cells that can express CD133, CD34, CXCR4, and markers characteristic
of embryonic stem cells Oct-4 and Nanog. To explore the regenerative potential
of VSELs in the retina, we transplanted PKH26-labeled enriched human VSELs
into mouse eye - by injecting the cells into the vitreous space, and by injecting the
cells subretinally in a SCID mouse model of retinal detachment. We then assessed
the ability of human VSELs to engraft, survive and differentiate into retinal or
neuroectodermal cells in the mouse retina. At 2 and 4 weeks after transplantation,
subretinally and intravitreally injected human VSELs were able to engraft, survive
and migrate within the retina. Furthermore, immunohistochemistry analysis
revealed that subretinally transplanted cells could differentiate and express markers
of retinal stem and developing progenitor cells such as Nestin and PAX6, of neuroectodermal cells such as MAP2 and beta-3-tubulin, and the early photoreceptor
marker recoverin. These studies indicate that human VSELs can engraft, migrate
and differentiate along the retinal lineage. These observations warrant further
investigation to evaluate the potential role of VSELs to treat AMD. *Disclosure: Dr.
Marasco is a paid consultant and owns stock in NeoStem.
118
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39
POSTER ABSTRACTS
119
Will not be presented
120
Dealing with cord blood selection for allogeneic
transplantation: the number of CD34+ cells matters
Brigitte BIREBENT, 1 Isabelle KHADHER,1 Mickael LECUYER,1 Laetitia MOECKES,1 Stéphan
ROUX,1 Philippe BIERLING,2 Luc DOUAY,1,3 Hélène ROUARD,1,4 1EFS Ile-de-France, cellular
therapy department and cord blood bank, CRETEIL, FRANCE, 2EFS Ile-de-France, CRETEIL,
FRANCE, 3UPMC, PARIS, FRANCE, 4UPEC, CRETEIL, FRANCE.
Allogeneic cord blood units (CBU) has been established as an alternative source of
hematopoietic stem cells when patients lack HLA-matched donor. In order to select a
banked unrelated CBU appropriate for one patient, two main parameters are usually
screened: the number of HLA disparities between donor and recipient and the pre
freezing number of total nucleated cells (TNC). The dose of CD34+ cells is often not
considered.
As a public CBU bank, we had the opportunity to investigate with one set of in vitro
tests only, the quality of the CBU before freezing as well as the stability of the graft
at defrosting and the recovery of the hematopoietic stem cells. We had defrosted 26
CBU not suitable for transplantation. The data issued from the quality control lab had
shown that TNC recovery is 78.2±6% but falls to 39.6±8.6% when we focus on alive
nuclear cells. The CD34+ alive cells recovery was much higher : 68.3±18.9%.
FACT standards require that the viability of CD34+ cells from a segment attached
to the CBU bag will be determined before CBU release. We have defrosted attached
segment from 10 CBU not suitable for transplantation and compared the CD34+
viability from the attached segment to the CD34+ viability from the corresponding
bag. The viability of the CD34+ cells from the segment is significantly lower than the
one of the CD34+ cells from the bag (60.39±21.43% versus 86.97±5.28%; p=0.0068,
Mann-Whitney test).
In conclusion, we believe that even though these data have been obtained on few
defrosted CBU: the selection of a banked unrelated CBU should take into account the
number of CD34+ cells of the CBU before freezing since their recovery is higher. The
quality control data of the defrosted attached segment are not representative of the
quality of the CBU
121
Addition of plerixafor to mobilization regimens in
autologous peripheral blood stem cell transplants
does not affect the correlation of pre-harvest
hematopoietic precursor cell (HPC) enumeration with
first harvest CD34+ stem cell yield
Carlos Villa, MD, PhD, Tsiporah Shore, MD, Cheryl Goss, MD, Koen van Besien, MD PhD,
Melissa M Cushing, MD, Weill Cornell Medical College, New York CIty, USA.
The CXCR4 antagonist plerixafor is being increasingly utilized in the mobilization
regimens for autologous peripheral blood stem cell (PBSC) transplantation. Because
this agent may mobilize a different subset of the stem cell population than traditional
regimens such as growth factors (with and without chemotherapy), it is important to
determine whether this agent has an effect on the predictive utility of measurements
used to predict the yield of CD34+ cells. These measurements are usually either preharvest peripheral blood CD34+ enumeration by flow cytometry or hematopoietic
precursor cell (HPC) enumeration by automated hematology analyzers. Although
HPC enumeration has a weaker correlation with first harvest CD34+ cell yields, this
parameter still plays an important role in timing apheresis procedures for autologous
PBSC transplantation due to its technical simplicity and low cost. In the present study
we retrospectively examined the correlation of HPC measurements with CD34+ cell
yields in patients with multiple myeloma and lymphoma undergoing autologous
PBSC transplantation and investigated how the mobilization regimen affected these
40
results. We found that the correlation coefficients ranged from 0.5877 to 0.7668 and
were not significantly impacted by differences in diagnosis or inclusion of plerixafor in
the mobilization regimen. The predictive ability of HPC enumeration for various target
yields was also examined and receiver operating characteristic curves were generated.
Regardless of whether plerixafor is included in the mobilization regimen, an HPC
cutoff of 20 should result in adequate initial CD34+ cell yields (>2.5x106 cell/kg) in
>80% of autologous donors. This study confirms the utility of HPC enumeration
in prediction of adequate initial cell yields, and demonstrates that this utility is
maintained regardless of whether plerixafor is included in the mobilization regimen.
122
UNREPORTED CORD BLOOD (CB) CD34DIM/+ CELL SUBPOPULATIONS AND THEIR POST-PROCESSING RECOVERY
Christianna N Henderson, BSc, Salem M Akel, PhD, St Louis Cord Blood Bank @ SSM
Cardinal Glennon Children’’s Medical Center, St Louis, USA.
Enumeration of the CD45DIMCD34+ cell population is a useful quality indicator for
CB selection as a stem cell source for hematopoietic reconstitution. This traditional
fraction represented 57.9% ± 24% of total CD34DIM/+ cells enumerated (n=23). CB
collections harbor sub-populations of unreported CD34DIM/+ cells in varying amounts,
which may be clinically significant. Three distinct CD34DIM/+ sub-populations of
interest were identified based on their degree of CD45 expression: CD45NEGsub,
CD45WEAKsub, and CD45+ sub. The CD45NEGsub demonstrated consistent very small size (26um), and represented 19.5% ± 17% of total CD34DIM/+ cells. This cell sub-population
was distinctly higher (54%, 62%, 66%) in 3 out of 23 CB collections analyzed. We
have reported a similar cell fraction in G-CSF mobilized PB (m-PB) harvests, albeit
at lower percentages (8.7% ± 7.4%). Recent studies have indicated that CB contains
a very small, non-hematopoietic, endothelial and/or embryonic-like CD45NEGCD34+
population potentially contributing to tissue regeneration. Of interest, there was no
correlation between this sub-population with nucleated RBCs, platelets, immature
platelet fraction, or WBCs, thus, supporting the notion that this cell population has
independent identity. The CD45WEAKsub (predominantly 5-8 µm) and the CD45+sub (> 10
µm) were reported in CB at 11.4 + 4.7% and 32.1 + 25% respectively. The engraftment
potential of these presumably hematopoietic sub-populations is not defined.
Interestingly, percentages of CD45+sub were higher in m-PB harvests (75.7 + 20.2%),
which may suggest a possible role of this population in short-term engraftment
leading to shorter time to engraftment for m-PB products compared to CB. From
our observation, routine minimally manipulative CB processing resulted in significant
loss, in order of 20 to 85% for both CD45NEGsub and CD45WEAKsub, with minimal loss
observed in the CD45+sub. Conclusion: Recovery of total CD34DIM/+ cells including small
size sub-populations shall be considered when processing CB harvests to maintain
cells with potential clinical significance.
123
Development of quantitative model of engraftment
of NOD-scid IL2rynull mice
Angel Gu, Chy-Anh Tran, Hieu Vu, Monica Coronado, Agnes Gardner, David DiGiusto, City of
Hope National Medical Center, Duarte, USA.
The use of the NOD-scid IL2rynull (NSG) mouse model to support human
hematopoiesis is well established. In our current studies, we evaluated the
reproducibility of engraftment of adult hematopoietic stem and progenitor cells
(HSPC) in NSG mice. Growth factor (G-CSF) mobilized peripheral blood apheresis
products (HPC-A) were obtained from healthy donors (under informed consent)
and CD34+ HSPC were isolated with high purities (98±2.3% CD34+) and viability
(>99%). NSG mice were irradiated with 300cGy and then transplanted with fresh,
previously frozen or ex-vivo expanded HSPC. High levels of human CD45+ cells
were observed in bone marrow and spleen 16 weeks post transplantation (up
to 60%). The marrow contained predominantly CD19+ B-cells with evidence of
CD14+ monocytes as wells as CD34+ and CD33+ progenitors. The spleen also
POSTER ABSTRACTS
contained CD4+ and CD8+ T-cells, CD19+ B-cells and CD14+ monocytes. Our
dose response curves demonstrated that as few a 5x10e5 freshly isolated CD34+
cells were needed to engraft >63% of injected mice and that 1x10e6 freshly
isolated CD34+ cells resulted in engraftment of virtually all mice. A similar dose
response was seen using frozen CD34+ HSPC following overnight prestimulation
in cytokines (SCF, Flt-3L, TPO, IL-6). Cells expanded for 7 days in cytokines plus
Stem Regenin-1 engrafted with a slightly lower efficiency (when normalizing to
CD34 content). The average CD34 expansion (13-fold) exceeded the reduction
in efficiency of engraftment (1.5-fold) demonstrating a net increase of “NSG
engrafting units” from adult HSPC under these conditions. Taken together, these
data provide evidence that the NSG mouse model can be used as a quantitative
model of engraftment and that cells from adult growth factor-mobilized apheresis
products can be expanded to allow for transplantation of >100 mice using HSPC
from a single donor. We are currently using this model to develop stem cell based
gene therapy for HIV.
125
124
Methods: Three RBC-reduced cryopreserved cord blood units were thawed,
washed, and CD34+ enriched on a CliniMACS device prior to 14-day culture in
serum-free medium supplemented with cytokines. Harvested material was
concentrated in 200ml of Normosol®-R + 1% HSA and sampled for baseline
analysis of TNC and CD34+ number, viability, immunophenotyping, and in
one case, transplantation in NOG mice. The product was then packaged in a
NanoCool™ temperature-regulated device and shipped overnight. Products were
sampled at 24-hour intervals up to 72 hours post-harvest and data analyzed using
ANOVA with Tukey’s post-hoc test.
COMPARISON BETWEEN FICOLL DENSITY GRADIENT
CENTRIFUGATION AND PREPACYTE ® -CB FOR NUCLEATED
CELL SEPARATION FROM UMBILICAL CORD BLOOD, AND THE
EFFECT ON RECOVERY OF CD 34+ CELL SELECTION, PURITY, AND
VIABILITY.
Halah I. Alkadi, MT(ASCPi)1 Kathy Mintz, MT(ASCP), SBB2 Brett Loechelt, MD2 Ross M.
Fasano, MD2 1Catholic University of America, Washington D.C., USA, 2Children’s National
Medical Center, Washington D.C., USA.
Background: Prepacyte®-CB (BioE) has been shown to be superior to Ficoll
density gradient centrifugation for nucleated cell recovery from Umbilical Cord
Blood (UCB). However, there has been no formal evaluation of these separation
methods’ effect on CD34+ cell selection recovery, purity and viability. We intended
to compare Total Nucleated Cell (TNC) and CD34+ cell recovery between
PrepaCyte®-CB and Ficoll-based nucleated cell separation methods, and to
evaluate their effect on CD 34+ cell selection, purity and viability.
Methods: Nineteen fresh UCB were divided into two equal volume aliquots and
processed by Prepacyte®-CB and Ficoll-based separation methods. The two
methods were performed in parallel in open systems. TNC testing was performed
on Sysmex Analyser. CD 34+ cells were positively selected using auto-MACS™
(Miltenyi Biotech) and were enumerated by flow cytometry (BD FACSCanto ™ II).
Results: TNC recovery (n: 19) was 81.1 ± 5.7% with PrepaCyte®-CB versus 42.95
± 11.93% with Ficoll-treated cells (p: 0.0001). Of the 19 separation trials, 12
underwent CD34+ positive selection. Final CD 34+ cell selection recovery from
initial UCB aliquots was 60.04 ± 42.64% with PrepaCyte®-CB versus 52.5 ± 43.2%
with Ficoll-treated cells (p: 0.6711). Recovery of CD34+ cells after separation from
initial UCB aliquots (n: 19) was 96.06 ± 33.15% with PrepaCyte®-CB versus 75.6 ±
27.27% with Ficoll-treated cells (p: 0.0477). CD 34+ cell purity was 53.5 ± 24.04%
with PrepaCyte®-CB versus 60.8 ± 18.5% with Ficoll-treated cells (p: 0.416). CD
34+ cell viability loss was 21.7 ± 13.34% in PrepaCyte®-CB versus 20.5 ± 11.67%
Ficoll-treated cells (p: 0.821).
Conclusion: Despite higher TNC recovery, final CD34+ cell selection recovery, purity
and viability showed no statistically significant difference between PrepaCyte®-CB
and Ficoll-treated cells. Prepacyte®-CB may be used as an alternative for nucleated
cell separation from UCB, and does not adversely affect CD34+selection.
Validation and Implementation of Non-Cryopreserved
Transport of Ex Vivo Expanded Cord Blood
Progenitors for Clinical Application
Ian B. Nicoud, 1 Joseph M. Blake,1 Howard Voorhies,1 Daniel Weber,1 Shelly Heimfeld,1
Colleen Delaney,1,2 1Fred Hutchinson Cancer Research Center, Seattle, USA, 2University of
Washingtion, Seattle, USA.
Background: Autologous and allogeneic cell therapy products require delivery
of source material to cGMP production facilities and transport of the final
product to clinical sites. For multi-center trials, methods for product shipping
and determination of product expiration based on maintenance of viable and
functional products must be validated and FDA-approved. Herein we report the
development of a non-cryopreserved storage and shipping methodology of a
freshly harvested cell therapy for clinical application.
Results: Beginning 48 hours after harvest, the viability (Figure 1a) had significantly
declined however this effect was exclusive of the CD34+ cell population as
evidenced by total viable cell counts. There was no statistically significant
difference in the absolute number of viable CD34+ cells over the 72-hour period
(Figure 1b), while viable TNC counts were significantly lower beginning 48 hours
after harvest (Figure 1c). Furthermore, in vivo repopulating ability in NOG mice
was observed at all time points post-shipment (data not shown). Thus, SOPs
were established for overnight shipment of freshly harvested expanded cord
blood progenitors with product expiration set at 48 hours. To date, two patients
have received cells that have been locally manufactured and shipped overnight
for infusion. No infusional toxicities have been observed and both patients have
engrafted and remain disease free at last follow-up.
126
A FULLY AUTOMATED CELL PROCESSING SYSTEM FOR VARIABLE
APPLICATIONS
Iris Spiegel, Elmar Fahrendorff, Volker Huppert, Gerd Steffens, Stefan Miltenyi, Miltenyi
Biotec GmbH, Bergisch Gladbach, Germany.
There is a need for a system offering options for different automated cell washing
procedures in a closed system because manufacturing protocols for cellular
products are usually accompanied by a series of pre- and post-separation handling
steps. For this reason, we have developed a medical device which is also a platform
for liquid exchange and general cell processing.
To realize processes like density gradient centrifugation, volume reduction of cell
culture suspensions, cultivation of cells and red blood cell reduction, we developed
three functionally closed tubing sets. All tubing sets have several options for
connecting media, buffer or other supplements. The tubing sets include a novel,
single-use centrifugation chamber, enabling cell washing and density-based
41
POSTER ABSTRACTS
separations of cell suspensions. Integrated channels allow liquids to be added or
removed during centrifugation.
128
One tubing set is for the concentration of high volumes of cell culture suspensions.
Cell washing processes, density gradient centrifugation and red blood cell reduction
are possible with the second tubing set. The third tubing set has the option for
cooling, heating and gassing cells, which enables cultivation of cells in an automated
system. With these tubing sets, basic important steps in sample preparation are
feasible.
IMPROVED IMMUNOMAGNETIC ENRICHMENT OF CD34+ CELLS
FROM UMBILICAL CORD BLOOD USING THE CLINIMACS CELL
SEPARATION SYSTEM
Additionally, we are developing flexible, user-specific programs.
Background. CD34+ cell enrichment from cord blood units (CBU) is increasingly
utilized before ex-vivo manipulation for clinical applications. The only current
cGMP immunomagnetic cell selection device, the CliniMACS instrument from
Miltenyi Biotec, was originally developed for processing apheresis collections. Two
CliniMACS tubing sets are available for clinical cell processing: the standard tubing
set (TS) with a maximum Total Nucleated Cell (TNC) capacity of 60 billon cells, and
the large-scale (LS) tubing set with TNC capacity of 120 billion. Single CBU typically
contain < 2 billion cells, raising a question as to which tubing set is optimal for CBU
CD34+ enrichment. In this report we compare the performance characteristics of TS
vs. LS for CD34+ enrichment of CBU.
We have developed an automated density gradient centrifugation with Ficoll® as
density gradient media. The results were within the technical specifications of
the manufacturer. Viability was higher than 96 % and the maximum granulocyte
contamination was below 4.5 % (n=10).
For volume reduction of cell culture suspensions, we are developing a continuous
centrifugation system. With a newly designed centrifugation chamber, we observed
cell loss approximately 10 % working at a pump rate of 120 mL/minute.
127
Immune Cellular Reconstitution and Donor Cell
Chimerism after Myeloablative and Reduced Intensity
Conditioning with IV Busulfan and Allogeneic Stem
Cell Transplantation (SCT)
Jeremiah Oyer, 1 Alicja Copik, Ph.D.1 Yasser Khaled, M.D.1 Melhem Solh, M.D.1 Israel
Pacheco,2 Brian Hernandez,2 Vijay Reddy, M.D., Ph.D.1 1Florida Center for Cellular Therapy,
Orlando, United States, 2University of Central Florida, Orlando, United States.
Adequate engraftment of donor immune cells is necessary to avoid complications
of allogeneic SCT. Conditioning influences the process of immune reconstitution
as recently demonstrated by Reddy and colleagues, showing a difference in
immune cell recovery and outcomes between myeloablative and reduced
intensity conditioning (Haematologica, 2008). IV Busulfan is commonly used in
SCT conditioning. However, the immune reconstitution and clinical outcomes
between high dose and reduced intensity Busulfan is unknown. This retrospective
study compares 3.2 mg/kg of Busulfan given intravenous (IV) daily for 4 days
versus for 2 days in addition to Fludarabine (FluBu4 vs. FluBu2).
Patients: 39 patients (Ages: 26-74; median 58) undergoing allo-SCT during 20092011 were included. Out of these patients, 23 received myeloablative (FluBu4)
and 16 received reduced intensity (FluBu2) doses of IV Busulfan. Day 30 and day
100 WBC counts and donor chimerisms by short tandem repeat cell separation
methods were used for analysis. One patient died after 17 days due to infection
and was not included in analysis.
Results: All patients engrafted. At day 30, FluBu4 patients had an average donor
chimerism of 99%, 94%, and 98% for granulocyte, T cell, and B cell, respectively
in comparison to 97%, 94%, and 96% for FluBu2 patients (p>0.05). One year
overall survival was comparable for both groups (70% FluBu4 vs. 51% FluBu2,
p=0.32). However, incidence of relapse was lower in FluBu4, 13%, vs. 33% for
FluBu2 (p=0.019). Of the patients who relapsed, only 30% had full donor T cell
chimerisms (>95%) whereas 74% of non-relapsed patients achieved full donor T
cell chimerisms (>95%) at day 30 (p=0.34).
Conclusion: Patients treated with ablative and reduced intensity doses of IV
Busulfan engraft successfully and offer comparable average donor chimerisms
at day 30 and 100. The low rate of full donor T cell chimerism reconstitution in
relapsed patients grants further investigation with more patients to determine its
potential prognostic relevance.
42
Joseph M. Blake, 1 Ian B. Nicoud,1 Daniel Weber,1 Howard Voorhies,1 Shelly Heimfeld,1
Colleen Delaney,1,2 1Fred Hutchinson Cancer Research Center, Seattle, USA, 2Department of
Pediatrics, University of Washington, Seattle, USA.
Methods. 46 freshly collected CBU (< 36 hours) were processed for CD34+
enrichment; 22 consecutive units were selected using the TS and a subsequent 24
processed with the LS. Cell counts and immunophenotyping were performed preand post-selection to assess TNC, viability, and CD34+ cell content.
Results. Two-sample t-tests of mean CD34+ recovery and TNC viability revealed
significant differences favoring the LS (CD34+ recovery: LS=56%, TS=45%, p=0.003;
viability: LS=74%, TS=59%, p=0.011). Multiple linear regression, considering preprocessing unit viability, TNC, time post-collection, and CD34+ purity, demonstrated
statistically significant correlations of the tubing set used and time post-collection
on CD34+ cell recovery and viability (p=0.009 and 0.04, respectively). Interaction
tests between time post-collection and tubing set used suggested a greater effect of
the tubing set on CD34+ cell recovery for units < 20 hours.
Discussion. For CD34+ enrichment from fresh CBU, the higher capacity LS provided
improvements in post-selection viability and CD34+ recovery. In this case, the lower
maximum TNC specification for TS did not predict better performance. The same
improved performance may hold for smaller-scale enrichment of other cell types
with the CliniMACS instrument.
POSTER ABSTRACTS
129
Transplanting Autologous Peripheral Hematopoietic
Progenitor Cells Stored At 40C and Not Cryopreserved
Ahmed Albahrani, Consultant1 Ashiq Salam, Supervisor1 Dawa Aldossary, Medical Lab
Technologist1 Hani Alhashmi, Consultant2 Khalid Alanazi, Consultant2 Ahmed Alsagheir,
Consultant2 Khawaja Haque, Consultant1 1Blood Bank/Transfusion Medicine & Stem Cell
Laboratory, King Fahad Specialist Hospital, Dammam, Saudi Arabia, 2Hemato-Oncology, King
Fahad Specialist Hospital, Dammam, Saudi Arabia.
Introduction: Noncryopreserved peripheral hematopoietic progenitor cells as
collected by apheresis (HPC-A) is able to restore hematopoiesis. Processing
and freezing cost of multiple HPC-A products is higher as compared to
noncryopreservation. We performed autologous transplant in multiple myeloma
(MM) patients by infusing fresh noncryopreserved HPC-A products following high
dose chemotherapy.
Methods: 8 autologous MM (5 male, 3 female, age 16-63 yrs and weight 65-98
kg) entered in this study. GCS-F mobilizing agent used to collect HPC-A by Optia
Spectra (version 5.0). CD34+ yields enumerated pre- and post-collection. HPC-A
bag was placed in 4±20C refrigerator and HPC were mixed gently by inversion every
4 hours. Prior to infusion in autologous MM patients all HPC-A products were
stored between 24 to 72 hours
Results: 4.8-13.3L of blood volume processed for collection of HPC-A products.
5 patients required only one and 3 patients had two apheresis sessions. Median
volume of collected HPC-A product was 180 mL and median WBC count in
final HPC-A product was 159x109/L. CD34+ count were taken as a progenitor
cells indicator. Median number of transplanted CD34+ cells/kg was 5.03x106 of
recipients. HPC-A cells were fresh, viable and timely engraftment was achieved in
all autologous recipients.
Conclusions: CD34+ count prior to apheresis and processing blood volume are well
correlated with efficiency of final HPC collection. As WBC count of each autologous
HPC-A product was less than 300x109/L, addition of autologous plasma was not
indicated. All patients had mobilized sufficient number of HPC and all achieved
engraftment successfully. A coordinated and disciplined approach is important for
an efficient HPC mobilization by GCS-F, collecting HPC-A, and infusion of fresh
HPC-A products in a timely manner to achieve successful transplant. The whole
process is straightforward, reliable, minimizing cost and can be performed in
facilities with limited resources.
130
IMPROVING CORD BLOOD PROCESSING METHODS: COMPARISON
BETWEEN MANUAL PROCESSING AND AUTOMATED PROCESSING
Kong-Yong Then, 1 Noor Akmar Yusli,1 Chee-Yin Wong,2 Ghee-Chien Ooi,1 Soon-Keng
Cheong,3 1CryoCord Sdn. Bhd., Selangor, Malaysia, 2Cytopeutics Sdn. Bhd., Selangor,
Malaysia, 3Tunku Abdul Rahman University, Selangor, Malaysia.
Introduction: Cord blood contains hematopoietic stem cells which can be used to
treat blood and genetic disorders. Cord blood samples collected after birth from
consenting mothers are processed to extract the stem cells by depleting the red
blood cells (RBC) and plasma. Cord blood processing can be performed either by
manual processing or by automated processing.
samples of each cord blood processing method.
Result: There was a significant higher MNC recovery rate when cord blood
samples were processed with AXP AutoExpress Platform than with Hespan
method. Additionally, cord blood processing with AXP AutoExpress Platform
resulted in significant reduction of HcT value when compared to the HcT values
after being manually processed. However, there was no difference in the results
from CFU Assay on the samples of both processing methods.
Conclusion: Cord blood processing by automated processing method is a better
and more effective method as compared to manual processing method because
automated processing method can recover more MNC and reduce more HcT
from a cord blood sample.
131
Concentrations of selected cytokines in plasma
and bone marrow of patients with hematological
malignancies
Martin Klabusay, 1 Viera Hrabcakova,2 Jana Chovancova,2 Hana Noskova,1 Petr Coupek,1,3
1
International Clinical Research Center, INBIT, Brno, Czech republic, 2Laboratory of Flow
Cytometry, University Hospital Brno, Brno, Czech republic, 3Czech Geological Survey, Brno,
Czech republic.
Introduction: Cytokines are small cell-signaling protein molecules participating
as important mediators in immune responses. Cytokines´ actions influence
development and survival of many cells, including haematopoietic cells. They may
play critical role in the growth mechanism of neoplastic clone. Concentrations of
selected cytokines can be used as markers of oncological diseases.
Material and methods: We investigated plasma concentrations of cytokines
IL-2, IL-4,IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, TNF-α, TGF-β1, IFN-Γ, VEGF,
G-CSF, soluble cytokines´ receptors sIL-2R, sIL-6R and co-stimulatory molecules
sCD23 and sCD40L using ELISA assay. The analysis included 138 samples from
98 patients. The analyzed file consisted of 93 samples of peripheral blood
and 43 samples of bone marrow of patients with hematological malignancies,
which included lymphoma, multiple myeloma, acute lymphoblastic and myeloid
leukemia, chronic lymphocytic leukemia and other rare diseases. The patients
were grouped according to their diagnosis and activity of the disease. Control
group consisted of 30 healthy volunteers.
Results: When patients were compared according to the diagnosis, the most
significant digferences were found in cytokines concentrations of IL-4 (p=0.027),
IL-5 (p=0.038), IL-13 (p=0.002) and TNF- α (p=3.0011.10-5). Comparison of
patients in active disease and disease in remission showed significant differences
in concentrations of IL-8 (p=0.002), sCD23 (p=0.043) and sIL2R (p=0.002).
Systematic higher values of these cytokines were found in patients with active
disease. Our study did not reveal any significant differences between values in
peripheral blood and bone marrow.
Conclusions: Changes in concentrations of plasmatic cytokines compared to the
diagnosis and stage of disease may be important indicator in diagnostic process
and can help us to understand changes in immune responses in hematological
malignancies. There is no need to examine bone marrow samples, as cytokines
concentrations in peripheral blood reflect well their levels in bone marrow. This
work was supported by grant IGA NS9671-4.
Objective: This study was performed to compare the effectiveness of two types of
cord blood processing method which are Hespan method (manual processing)
and AXP AutoExpress Platform (automated processing) in terms of mononuclear
cells (MNC) recovery rate and hematocrit (HcT) reduction rate.
132
Method: Cord blood samples were collected from consenting mothers delivering
full-term infants. Collected data from the processing of cord blood samples by
Hespan method (n= 1000) and by AXP AutoExpress Platform (n= 1000) were used
in this study. Using the collected data, the MNC recovery rate and post-processing
HcT values were compared. CFU Assay was also performed on randomly selected
Mary Beth Fisk, MT (AMT) ASCP, Ray Adams, Christopher Cielak, South Texas Blood &
Tissue Center, San Antonio, USA.
COMPARISON OF UMBILICAL CORD BLOOD POST-PROCESSING
VERSUS POST-THAW VIABILITY
Background: Umbilical cord blood, an important source of hematopoietic stem
cells for transplantation, has potential uses in novel regenerative medicine
applications. To ensure units of cord blood maintain the highest quality for use in
43
POSTER ABSTRACTS
medical therapies, cells must be processed and stored under optimal conditions. A
comparison between post-processing and post-thaw viability is described.
134
Methods: Cord blood collected from 15 hospital collection sites donated for allogenic
use was processed utilizing red cell and plasma depletion (standard manual method
and the semi-automated Sepax method) methodologies. Post-processing viability
was measured using the 7AAD method for each unit. Following standard testing,
DMSO was added to each unit, frozen in a rate controlled environment, and placed
in vapor phase into traditional manual liquid nitrogen freezing chambers or the
BioArchive system, for long term storage. Units selected for confirmatory testing
(CT) by transplant centers were included in this study, with post-thaw viability
measured on each sample using the 7AAD method.
ANALYSIS OF INFUSED CD34+ DOSE AND HEMATOPOIETIC
RECOVERY IN PATIENTS UNDERGOING AUTOLOGOUS STEM
CELL TRANSPLANT (ASCT)
Results: Forty-five cord blood units with a mean storage time of 19 months were
studied. Prior to storage, the units’ mean cell viability was 95%. The mean postthaw cell viability reported was 88%. From time of processing to clinical selection
for CT there was a mean decrease in viability of 7%. The range of variation between
post-processing and post-thaw viability was 0 to 13%. The post-thaw (CT) cell
viability and the decrease in viability were not significantly correlated between
time of storage to CT.
Conclusion: Cord blood units processed and stored maintained a high cell viability
when selected for confirmatory testing and eventually shipped for transplantation.
The length of storage did not account for variability seen in post-thaw viability or the
minimal viability loss seen from time to processing for clinical use. The viability loss
may be caused by cell death from the freeze thaw cycle or due to variation in processing
methods. Units processed and stored under optimal conditions maintained cell
viability after long term storage and preparation for clinical use.
133
Cord Blood Collections: Operational Methods
Mary Beth Fisk, MT (AMT) (ASCP) CQA (ASQ), Ray Adams, Bethany Davis, South Texas
Blood & Tissue Center , San Antonio, USA.
BACKGROUND: Umbilical cord blood is a prime source of hematopoietic
stem cells for transplantation to patients with cancers and metabolic/immune
disorders. Successful cord blood transplantation is dependent on the number of
stem cells, and the immune match to the recipient.
METHODS: This public cord blood bank collects cord blood units from 15
contracted hospitals, non-fixed sites, related donor program, and private
collections. The hospital selection process is based on the number of deliveries
and patient population by ethnicity.
Only low risk deliveries are acceptable for cord blood collection, and all physicians
must successfully complete a competency assessment prior to starting any
collection and annually thereafter. Collection occurs in the third stage of labor
after delivery. The umbilical vein is disinfected and cannulated with a sterile
collection bag set. Blood fills the bag facilitated by uterine contraction. Third stage
labor collections are recommended for the following reasons:
•
•
•
Ability to collect from placentas
Fewer collections lost to clotting
Higher hematopoietic stem cell yield
RESULTS: Units meeting the quality requirements of the program are listed on the
national registry. As progress is made in the collection procedures, TNC is one
parameter which determines the efficacy of the unit for transplant. To date, the
center has collected over 40,000 units, banked over 10,000 units, and provided
150 units for transplantation worldwide.
CONCLUSIONS: Quality indicators such as deferrals due to low TNC, low
weight, and documentation deviations are used to track and provide feedback to
collectors. Bacterial contamination and clotted units may result in deferral, thus
these parameters are tracked continuously. The average number of units deferred
for low TNC is 43% and the average percentage of bacterial/fungal contamination
of units is less than two. The number of clotted units decreased by 30% with the
introduction of a new sterile collection bag/system.
44
Shaun DeJarnette, MT(ASCP), Bhaskar Gadi, MD, Dean Merkel, MT(ASCP), Omar Aljitawi, MD,
Sid Ganguly, MD, Joseph McGuirk, DO, Sunil Abhyankar, MD, The University of Kansas Cancer
Center, Kansas City, USA.
Rapid hematological recovery after ASCT has been associated with diminished
infection rates and shorter admission duration. Prior studies have shown that
CD34+ cell dose does impact time to hematopoietic recovery post ASCT. We
conducted an analysis of hematopoietic recovery following ASCT as a function
of CD34+ cell dose in patients with lymphoma and multiple myeloma (MM)
patients, admitted to the University of Kansas BMT program from January 2009 to
December 2010. A total of 206 patients, 78 lymphoma (38%), 121 myeloma (59%)
and 7 (3%) patients with other malignancies underwent stem cell mobilization
with G-CSF +/- plerixafor for ASCT. MM patients received Melphalan. Lymphoma
patients received BEAM or BEAC chemotherapy. Doses of CD34 (+) cells were
grouped as 2-2.5 × 106 cells/kg (n=16), 2.51-3 × 106 cells/kg (n=60), 3.01-4×106
cells/kg (n=78), and >4 x 106 cells/kg (n= 52). Time to absolute neutrophil count
(ANC) recovery >500/µL, platelet recovery to 20,000/µL, 50,000/µL, 100,000/µL
and absolute lymphocyte count (ALC) recovery >500/ µL were calculated. Groups
were compared using the Kruskal Wallis test. 7.8% of patients received lower dose
of CD34 (+) cells (i.e., <2.5 × 106 cells/kg), and 92.2 % received higher doses.
Median engraftment times for ANC >500/µL were 12 and 11 days respectively in the
lower dose vs. higher dose groups (p=0.0002). Median time of platelet recovery
>50,000/µL were 19 and 18 days respectively in the lower vs. higher dose groups
(p=0.01). Beyond the dose of > 2.5 × 106 cells/kg, platelet recovery to 20,000 and
>100,000/ µL did not differ across higher cell doses. ALC engraftment did not
reach statistical significance (p=0.43). CD34+ doses of >2.5 × 106 cells/kg resulted
in faster recovery of ANC and platelets >50,000/µL in patients undergoing ASCT
at our institution. Future prospective studies are needed to confirm our results.
135
IMPROVED EX VIVO EXPANSION OF HEMATOPOIETIC STEM AND
PROGENITOR CELLS FROM CORD BLOOD IN A NOVEL SERUMAND ANIMAL-COMPONENT-FREE MEDIUM
Ning Yuan, 1 Terry Thomas, PhD1 Allen Eaves, MD,PhD1,2 Bert Wognum, PhD1 1STEMCELL
Technologies Inc., Vancouver, Canada, 2Terry Fox Laboratory, BC Cancer Agency, Vancouver,
Canada.
Ex vivo culture of CD34+ cells can be useful to increase hematopoietic stem
and progenitor cell (HSPC) numbers or generate a large number of mature
blood cells to treat or prevent cytopenia in patients. We have developed an
animal-component-free (ACF) expansion medium that contains only synthetic
and recombinant components, and herein demonstrate its ability to support
expansion of CD34+ cells in culture. Cord blood CD34+ cells were purified using
EasySep immunomagnetic cell separation and plated at 10,000 cells/mL in ACF
medium and, as controls, in StemSpan-SFEM containing bovine albumin, and
StemSpan-H3000 containing human plasma derived components. The cells were
stimulated for seven days with FLT-3 Ligand, Stem Cell Factor, Interleukin (IL)-3
and IL-6, and then counted and analyzed for expression of CD34, lineage-antigens
(CD11b, CD14, Glycophorin-A, CD41), and for their content of colony-forming cells
(CFCs) by replating in semisolid medium.
Cell viability was high in all three media (range = 93-99%, n=10). Cell expansion
ranged between 14 and 76-fold for 10 different CBs with similar, on average ~40fold expansion, including myeloid cells (CD11b+, CD14+), erythroid cells (GpA+)
and megakaryocytes (CD41+) in all three media. Average CD34+ cell output was
similar in ACF and SFEM media (12% and 10% CD34+ cells, and 6 and 4-fold
expansion, respectively; p=0.05, paired t-test), but significantly higher than in
POSTER ABSTRACTS
H3000 medium (6% CD34+ cells and 3-fold expansion; p<0.01 vs. ACF; p<0.05
vs. SFEM). Similar differences were observed for the CFC content of the expanded
cells. These results suggest that ACF and SFEM media may better support self
renewal or delay differentiation of HSPCs in culture than H3000. ACF expansion
medium may be useful for developing safe and effective HSPC culture methods
directed toward accelerating hematopoietic recovery after cytoreductive therapy
or to treat acute blood loss with transfusions of culture-expanded blood cells.
136
Use of the BioSafe Sepax RM™ to Wash and Consolidate
Cryopreserved Hematopoietic Progenitor Cell,
Apheresis Products Free of DMSO for Infusion
P. Jacobson, 1 B. Bullough,1 F. Hsieh,1 Deepika KC,2 S. Shah,3 W. Gruber,3 L. Kelley,4 M. Boyer,1
1
Cell Therapy Facility, University of Utah, Salt Lake City, USA, 2Memorial Sloan-Kettering
Cancer Center, New York, USA, 3Biosafe, Houston, USA, 4Dana Farber Cancer Institute,
Boston, USA.
Transplant recipients may have limited tolerance to DMSO, a commonly used
cryoprotectant. Adverse effects of DMSO include changes in blood pressure and
nausea. Patients previously treated or collected for stem cells often mobilize
poorly having elevated white blood cell count and low CD34 percentage. This
increases the level of DMSO that is infused at transplant as total viable nucleated
cell (vTNC)/mL is routinely limited at cryopreservation and product must be
distributed to many cryobags.
The adverse side effects of DMSO can be reduced by washing the Cryopreserved
Hematopoietic Progenitor (HPC) product free of DMSO post thaw. The Sepax
RM™ Cell Separation System removes DMSO from thawed HPC(A) products in
an automated and controlled environment. Cells are washed, consolidated and
resuspended in fresh buffer, ready for transplantation. The “Smartwash” Program
was utilized to validate two Cryopreserved HPC(A) wash and consolidation
protocols; a 1-2 bag, small volume and a 3-20 bag large volume method resulting
in the following recoveries (n=7):
Gold Standard – Effect of Thaw
and Processing
% Recovery
Small vol. 61.5 +/- 6.1
Final / Pre-freeze
Large vol. 63.1 +/- 9.5
Effect of Thaw Only
% Recovery
Small vol. 71.1 +/- 13.4
Post Thaw / Pre-Freeze
Large vol. 73.7 +/- 17.1
% Recovery
Small vol. 89.2 +/- 18.3
Final / Post Thaw
Large vol. 87.7 +/- 17.9
Effect of Processing Using the
Sepax RM™ Smartwash
The recovery data indicates that vTNC loss is primarily due to the effect of
cryopreservation and thaw. Viability of final products were ≥ 70% for 24 hours
post processing. The final product is volume reduced by 40 to 60%, and free
of aggregates; therefore, 170µ filtration is rarely required. The Sepax RM™
SmartWash protocol produces a high quality, DMSO-free product for transplant
and will be established as Standard Operating Procedure in our laboratory.
137
ASSESSMENT OF LEVELS OF IGF-1, IGFBP-3 AND IGF-1/IGFBP-3
RATIOS IN UMBILICAL CORD/MATERNAL BLOOD IN RELATION
TO CELLULAR CONTENTS OF CORD BLOOD HARVESTS
Salem Akel, PhD, Amy MacRae, PhD, Melissa McKenna, Christianna Henderson, Donna
Regan, St. Louis Cord Blood Bank @ SSM Cardinal Glennon Children Hospital , St. Louis,
USA.
Quality of collected cord blood (CB) harvests varies significantly, as indicated
by wide ranges of Total Nucleated Cells (TNCs: WBCs plus n-RBCs) and CD34+
CD45dim hematopoietic progenitor cell (HPC) counts. Several studies suggest that
Insulin–like growth factor (IGF-1) is implicated in fetal development, expansion
of the stem cell pool, and risk of cancer. We have investigated the relationship
between concentrations of IGF-1, and IGF binding protein (IGFBP-3) in CB and
maternal (M) plasmas, and the various cellular contents of collected CB ( 28 CB
from full term pregnancies /healthy Caucasians donors). Results: Levels of IGF-1
and IGFPB-3 as determined by ELISAs in M and CB plasmas were comparable to
those reported by others. As expected, IGF-1 levels were positively correlated with
those of its transporter IGFBP-3 (p < 0.01). No correlation was found in levels
of IGF-1or IGFPB-3 between paired CB and M samples, supporting the idea that
both factors do not cross the placenta. Although none of the studied CB cellular
parameters significantly correlated with M-IGF-1 levels, TNCs/product, TNCs/kg
of baby weight, and CD34+ events/TNCs ratio were all positively correlated with CB
- IGF-1 levels (p < 0.05). However, CB - IGF-1 correlation with CD34+ events/TNCs
was not maintained when using counts of CD34+ CD45dim representing HPCs.
Of interest, we found that the molar ratio of CB IGF-1/ IGFBP-3 is significantly
correlated with HPCs/ml, HPCs/product, and HPCs/TNCs (r2 > 0.4; p < 0.05).
Finally, levels of CB-IGFBP-3, M- IGFBP-3, and the M-IGF-1/ IGFBP-3 ratio did not
show significant correlation with any of the CB cellular parameters. Conclusions:
our results suggest that CB IGF-1 level and molar ratio of CB IGF-1/IGFBP-3 are
potential predictors of CB quality in term of TNCs and HPCs contents respectively.
Our findings support the involvement of the CB IGF-1/IGFBP-3 gradient in fetal
HPCs development.
138
WONDER FILE: Automatic calculation of total blood
volume, circulating CD34+/kg, collection efficiency
and morE
SHANTA SHARMA, MANAL YAGHMOUR, LARA F SARHAN, FAWZI ABDEL RAHMAN,
ABDELGHANI TBAKHI, King Hussein Cancer Center, Amman, Jordan.
Successful hematopoietic stem cell engraftment requires infusing adequate
number of progenitor cells. Typically, for peripheral blood derived progenitor
cells, the patient/donor is mobilized with cytokines and/or chemotherapy
and circulating CD34+ cells are monitored to determine the peak to minimize
apheresis procedures.
The endpoint is to determine suitable time of collection and to calculate CD34+
cells/kg in the graft. Calculating total blood volume (TBV) is required. If the
patient is obese, actual weight may be converted to ideal or adjusted ideal body
weight. Formulas are gender and height/weight dependent.
Manual calculations are tedious, time consuming and prone to human error.
Therefore, we have created an excel spreadsheet, which enables us to calculate
all above information with just a few keystrokes. The same, gender-specific
spreadsheet can be used for either autologous or allogeneic donors . Additionally,
it determines the collection efficiency by comparing total number of circulating
CD34+ cells with the number of cells in collected product.
The result is a single page document, which displays all above information,
accommodates up to six collections and has additional columns which make
monitoring, tracking and record keeping very easy.
Creating this spreadsheet has taken us many years. We call it a “Wonder File”
given how, with such few keystrokes, so much information is generated, not to
mention its value as a time saving and error prevention tool.
139
INFLUENCE OF HYDROXYETHYL-STARCH (HES) ON CELL
RECOVERY IN CORD BLOOD PROCESSING
Simone Hennerbichler, PhD, Susanne Süssner, Christian Gabriel, MD, Red Cross Blood
Transfusion Service of Upper Austria, Linz, Austria.
The Cord Blood Bank of the Red Cross Blood Transfusion Service of Upper Austria
was founded in 2002 as part of the blood bank.
Initially, processing of cord blood donations was done manually according to the
45
POSTER ABSTRACTS
method by Rubinstein et al. (1998) but since 2004 cord blood units are processed
automatically on the Sepax System (Biosafe, Switzerland).
142
From 2008 to 2011 we were forced to processed cord blood donations with a
different type of HES (molecular weight 240.000). By the end of 2011 we were able
to purchase our initially used HES from Grifols (Spain) with a molecular weight
of 407.000 again.
ROBUST EX VIVO EXPANSION OF CD34+ CELLS FROM CORD
BLOOD, BONE MARROW, AND MOBILIZED PERIPHERAL BLOOD
USING NANEX NANOFIBER SCAFFOLD
Comparing these two types of HES it could clearly be demonstrated that HES
with a molecular weight of 407.000 improved the recovery of total nucleated
cells enormously from 79,50% (+/- 12,49%) to 96,58% (+/-5,86%) and red cell
sedimentation was more efficient again.
Hematopoietic stem/progenitor cells (HSPCs) from umbilical cord blood (UCB),
bone marrow (BM), and mobilized peripheral blood (MPB) are used to treat a
variety of malignant and non-malignant hematologic disorders. The success of
these therapies is dependent on both the cell dose and pluripotency of donor cells.
Because the number of HSPCs available from donors can be limited (particularly
for UCB), strategies that can increase the yield of multipotent HSPCs would have
enormous clinical impacts.
140
will not be presented
141
A NOVEL CRYOPRESERVATION SYSTEM SETTING AND METHOD
FOR FREEZING HPC-CORD BLOOD
Sreedhar Thirumala, Ph D1 Vincent Gallagher,1 William S. Goebel, MD, Ph D1,2 Erik J. Woods,
Ph D1,2 1General Biotechnology LLC, Indianapolis, USA, 2Indiana University School of
Medicine, Indianapolis, USA.
The objective of the current study was to develop an optimal cryopreservation
system setting and method for human umbilical cord blood hematopoietic
progenitor cells (HPCs) as evidenced by improved viability, total nucleated cell
count (TNC) and colony forming units (CFUs). Based on our previously published
research utilizing theoretical modeling of membrane osmometric/permeability
characteristics of HPCs and the ability of frozen-thawed HPCs to engraft in
irradiated NOD/SCID mice, we determined the optimal cryopreservation
procedure to include 0.7 molal (~5%) DMSO concentration at a cooling rate
of 4˚C/min, and a plunging temperature of -44 ˚C. For cryopreservation setting,
we utilized a recently developed Advanced Controlled Rate Freezing (ACRF)
technology from Praxair® Inc in which highly consistent and homogeneous
freezing conditions are achieved by employing forced convection cooling using
a laminar and uniform flow of cryogen along with temperature or pressure
controlled seeding capacity.
Processed cord blood was cryopreserved
as shown in Figure 1. After at least a week,
the cord blood samples were removed from
liquid nitrogen and immediately thawed in a
37 ˚C water-bath and prepared using standard
procedures for TNC, CFU quantification and
viability. Total nucleated cells were counted
using a Coulter Counter MDII. For CFU assay,
nucleated cells were cultured in triplicate
using a semisolid clonogenic assay. Cell
viability was determined using Trypan blue
by adding it to cells at 1:1 (v/v) and evaluated
using hematocytometer. All the data were
compared with the most routinely used cord
blood cryopreservation procedure involving
dumping in -86 ˚C to cool at -1 ˚C/min for 24
hour before transferring to LN2.
Our experimental setting produced significantly higher % viability, CD34+ recovery,
and total CFUs compared to dump freezing method. Therefore, we hypothesize
that the proposed new cryopreservation system setting is a better alternative to
most commonly used cord blood cryopreservation procedure.
Yiwei Ma, Jacqueline M Fonseca, Stephen E Fischer, Arteriocyte, Inc, Cleveland, USA.
Ex vivo expansion of HSPCs is one strategy that offers great promise for increasing
cell doses and improving clinical outcomes. In many systems, HSPCs are cultured in
suspension and the interaction of these cells with a substrate is ignored. However,
in the native bone marrow microenvironment, HSPCs maintain close contact with
the extracellular matrix, suggesting that cell-matrix interactions play an important
role in maintaining HSPC pluripotency. With the goal of mimicking this feature of
the bone marrow niche, Arteriocyte, Inc. has developed a 3-D nanofiber substrate
(NANEX). The NANEX substrate is designed to provide topographical and
substrate-immobilized biochemical cues that act in synergy with media additives
to enhance cell proliferation and better maintain pluripotency.
Here, we present our recent work with the NANEX platform towards achieving
a high yield expansion of CD34+ HSPCs from UCB, BM, and MPB. UCB-derived
CD34+ cells demonstrated the highest capacity for expansion on NANEX (at least
100-fold in 10-day culture), but BM- and MPB-derived CD34+ cells also showed
enhanced expansion (2- to 3-fold improvement with NANEX over conventional
plastic dishes). Additionally, all sources showed a high level of CXCR4 expression
and a significant expansion of CFUs on NANEX when compared to conventional
plastic. Our data indicates that NANEX technology provides a robust ex vivo
expansion of HSPCs and, with further development as a GMP product, offers
great potential in the clinic.
143
QUALITATIVE AND QUANTITATIVE ANALYSIS OF BONE MARROW
CONCENTRATE (BMC) PRODUCED USING RES-Q™60 BMC- A
POINT OF CARE MEDICAL DEVICE
Vijay Kumar, Ph.D.1 Nico Forraz, Ph.D.2 Gianluigi Atzeni, MSc.2 Colin P. McGuckin, Ph.D.2
1
Thermogenesis Corp., Rancho Cordova, USA, 2CTI-BIOTECH & CTI-LYON, Cell Therapy
Research Institute, Lyon, France.
Introduction: There is a growing interest in the infusion of bone marrowderived stem cells to treat a variety of acute and chronic conditions, including
cardiovascular and orthopedic. The Res-Q™ 60 BMC system is a novel point of
care medical device for efficient and reproducible preparation of a bone marrow
concentrate (BMC) from a bone marrow aspirate. The objective of this study was
to characterize the resultant BMC with respect to complete blood counts (CBC)
and flow cytometry based analysis of different cell subpopulations, using bone
marrow aspirates from normal donors.
Methods: Bone marrow aspirate (60 mL) was concentrated into a final volume
of 7 mL using the Res-Q™ 60 BMC 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) assays cells were plated at a density
of 0.25x 105/plate. Flow cytometric analysis was performed using the Becton
Dickinson FACS CANTO II machine.
Results: For this study, 10 bone marrow aspirates were processed. The cell counts
for pre- and post- Res-Q™ 60 BMC samples, cell enrichment factor and cell doses
46
POSTER ABSTRACTS
are reported in the table below. The Res-Q™ 60 BMC system removed greater
than 90% of the RBCs resulting in a mean Hct of 11.1 % ± 2.4 in the final BMC
product. The cell viability for the post sample was 99.6% ± 0.01. The Res-Q™
60 BMC system maintained the sterility of the final product as evidenced by the
BioMerieux BacT/ALERT testing system.
Conclusion: The results demonstrated an average 6.5 times cell enrichment and
422 million cells dose for mononucleated cells in the final BMC product. The
Res-Q™ 60 BMC system demonstrated an efficient, rapid and easy method for
preparing BMC from human bone marrow in an intra-operative setting and at the
point of care.
CD73 positive while negative for CD34 and CD45. The cells were also induced into
adipocytes, osteocytes and chondrocytes stained positively with oil red O, alizarin
red S and alcian blue respectively.
Conclusion: Results showed that adenovirus transduction of IL-6 shRNA
significantly reduced IL-6 in MSC without affecting their biological properties.
The potential of MSC for stable gene suppression using adenovirus-based shRNA
transduction should be further investigated as a potential application in cancer
therapy setting.
146
Will not be presented
147
Optimized manufacture of CTLs with anti-virus and
anti-tumor specificity for neuroblastoma patients
post stem cell transplantation
Jiali Sun, 1,2,3 Eric Yvon,1,2,3 Ann M. Leen,1,2,3 Juan F. Vera,1,2,3 G Doug Myers,4 Malcolm K.
Brenner,1,2,3 Cliona M. Rooney,1,2,3 1Center for Cell and Gene Therapy, Houston, USA, 2Baylor
College of Medicine, Houston, USA, 3Texas Children’s Hospital , Houston, USA, 4Children’s
Mercy Hospital, Kansas City, USA.
144
Will not be presented
145
SUPPRESSION OF INTERLEUKIN-6 IN HUMAN MESENCHYMAL
STEM CELLS BY ADENOVIRUS-BASED SHORT HAIRPIN RNA
TRANSDUCTION
Hoon Koon Teoh, 1,2 Pei Pei Chong,2 Maha Abdullah,2 Zamberi Sekawi,2 Chooi Fun Leong,3
Soon Keng Cheong,4 1PPUKM-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.
Background: Mesenchymal stem cells (MSC) are used to deliver therapeutic
cytokines directly to target area due to their unique ability to migrate and engraft
at injury or tumour sites. However, usage of MSC for stable gene suppression
mediated by vector-based small hairpin RNA (shRNA) remains largely unexplored.
In this study, we evaluated the suppression of interleukin-6 (IL-6) post adenovirusbased shRNA transduction in human bone marrow-derived MSC and their post
transduction biological properties comprising viability, immunophenotype and
differentiation capacities.
Methods: IL-6 shRNA adenovirus vector (pAd-BLOCK-iT/IL6) was transfected
into 293A cells using Lipofectamine 2000. Plaque assay was carried out to
determine pAd-BLOCK-iT/IL6 titer by lysing 293A cells using 3 freeze/thaw cycles.
6x103 MSC were transduced with pAd-BLOCK-iT/IL6 at Multiplicity of Infection
(MOI) from 0-50. Supernatant from transduced MSC were collected 120h post
transduction and IL-6 level was determined using ELISA assay. Transduced MSC
were subjected to viability assay, immunophenotyping analysis and differentiation
study. Untransduced MSC were used as control.
Results: pAd-BLOCK-iT/IL6 titer in 293A cells was determined to be 1x108 pfu/
ml. At 120h post transduction, IL-6 was suppressed to 21.8% at MOI=20 when
compared to control MSC (100%). MSC viability was at 86.8% when compared to
control MSC (100%). Immunophenotyping analysis showed that transduced and
control MSC displayed typical MSC surface markers of CD90, CD44, CD105 and
As an NHLBI-PACT production, we were charged with production of trivirus (EBV,
CMV and adenovirus)-specific T cells expressing a chimeric antigen receptor
(CAR) specific for the disialoganglioside, GD2, for the treatment of patients with
relapsed neuroblastoma after allogeneic HSCT. Adoptively transferred T cells
specific for endogenous viruses like CMV and EBV expand massively after HSCT
due to the presence of viral antigens and lymphopenic environment. This approach
therefore provides a means to increase the numbers and persistence of GD2.CAR+
expressing T cells. Limitations of our current protocol for retroviral transduction
of virus-specific T cells include late transduction (day 19) leading to delayed T cell
expansion, differentiated effector phenotype and unstable transgene expression. To
shorten production and ensure transduction of T cells specific for all three viruses
we evaluated different combinations of antigen presenting cells, cytokines and days
of transduction.
In our optimized protocol, PBMCs stimulated with autologous EBV-transformed
B cell lines (LCLs) transduced with an adenoviral vector expressing pp65 of CMV
(Ad5f35pp65) in the presence of IL-4 and IL-7 were transduced with the GD2.CAR
vector 2 days later. On day 9, T cells were restimulated with Ad5f35pp65-transduced
LCLs and expanded in a GRex flask. Starting with 107 PBMC, we produced 80 to 100
x 107 T cells comprising GD2.CAR-transduced CMV, EBV and adenovirus-specific T
cells by day 16. Transduction efficiency was higher than with day 19-transduced cells
and remained stable after multiple stimulations (60% to 70% after the 5th or 6th
stimulation vs 10% to 30% if transduced on day 19). Sufficient T cells for clinical use
could be obtained by day 16 compared to a minimum of day 30 with the old protocol.
This approach should produce a virus and tumor-specific T cell product with high
potential for in vivo proliferation and long-term persistence.
148
Genetically Modified Donor Derived EBV-Specific T cells
for the Treatment of Pediatric Relapsed CD19+ ALL post
allo-HSCT
Kevin J. Curran, M.D., Nancy A. Kernan, MD, Xiuyan Wang, PhD, Clare Taylor, MSc, Ekaterina
Doubrovina, MD, PhD, Shirley Bartido, PhD, Kirsten Fuller, Heather Reel, Jolanta Stefanski,
Oriana Borquez-Ojeda, Teresa Wasielewska, Olszewska Malgorzata, Jinrong Qu, Michel
Sadelain, MD, PhD, Richard J. O’Reilly, MD, Renier J. Brentjens, MD, PhD, Isabelle Riviere,
PhD, Memorial Sloan-Kettering Cancer Center, New York, USA.
Human T-cells can be genetically modified to target tumor antigens through the
expression of a chimeric antigen receptor (CAR). CD19 targeted CAR modified
T cells provide a novel therapeutic approach for patients with relapsed B-ALL
47
POSTER ABSTRACTS
following allo-HSCT. We have previously demonstrated our ability to generate
donor derived Epstein-Barr virus specific cytotoxic T cells (EBV-CTLs) modified by
retroviral gene transfer to express a CD19 specific CAR (19-28z). 19-28z+ EBV-CTLs
exhibit cytotoxicity against CD19+ targets, retain EBV-specificity without allogeneic
cytotoxicity, and eradicate systemic Burkitt lymphoma in a murine model. Based
on our preclinical studies, we have initiated a phase I dose escalation clinical
trial (NCT01430390) in pediatric patients with relapsed CD19+ B-ALL following
allo-HSCT utilizing donor 19-28z+ EBV-CTLs. Two patients received conditioning
with fludarabine (25mg/m2 x 5 days) followed by infusion of 1x106 EBV-CTLs/kg
modified to express 19-28z CAR (CAR+ T cell dose 1.4 - 3x105 cells/kg). On day +3
post-infusion patient 1 developed fever and biopsy proven grade II skin graft vs
host disease (GVHD) that resolved with topical steroids. Fever resolved 4 days
after onset while on broad spectrum antibiotics with a negative infection workup.
Modified T cells were detected by RT-PCR in the peripheral blood and bone marrow
up to 3 weeks after infusion. Following evidence of disease progression patient 1
was taken off study 42 days after receiving modified T cells. Patient 2 has recently
been treated and will be monitored for toxicity, persistence of modified T cells,
and disease response. Modified T cells have been generated for a third patient
who is scheduled for infusion. These early results demonstrate the feasibility of
1928z+ EBV-CTL infusion without the development of significant GVHD post alloHSCT. Subsequent cohorts of patients will be evaluated for safety and persistence
of modified T cells following conditioning chemotherapy with escalating doses of
19-28z+ EBV-CTLs.
149
CONCOMITANT OVEREXPRESSION OF IGF1 AND BMP2 IN
MESENCHYMAL STEM CELLS MEDIATES CYTOPROTECTION
THROUGH BOTH AUTOCRINE AND PARACRINE ACTIVATION OF
AKT, ERK1/2 AND SMAD1/5/8 PATHWAYS.
Manuela Mura, PhD1 Federica Pisano, Bs1 Federica Longo,2 Patrizia Danieli, PhD1 Elisabetta
Cervio, PhD1 Massimiliano Gnecchi, MD, PhD2,3 1Fondazione IRCCS Policlinico San Matteo ,
Pavia, Italy, 2University of Pavia, Pavia, Italy, 3Fondazione IRCCS Policlinico San Matteo, Pavia,
Italy.
Background. Bone marrow mesenchymal stem cells (BM-MSC) are valuable tools
for cardiac repair, acting mainly through release of paracrine factors. However, the
effects of BM-MSC are limited by poor engraftment and low rate of differentiation
events. To overcome these limitations, we genetically engineered BM-MSC with a
novel bicistronic lentivirus co-expressing IGF1 and BMP2 (IB), two factors known to
be involved in both cardiac differentiation and cytoprotection.
Methods. Rat BM-MSC were transduced with a control virus (GFP-MSC) or IB virus
(IB-MSC). Autocrine and paracrine cytoprotection was evaluated in transduced
MSC or in H9c2 cells treated with unconditioned (CTRL-M) or conditioned media
(GFP-CM or IB-CM), after 24h of hypoxia. Cell viability was measured by MTS assay.
Apoptosis was evaluated through caspase-3 activation.
Transcriptional levels of pro and anti-apoptotic genes in H9c2 were measured by
RT-PCR. Activation of IGF1 and BMP2 pro-survival pathways (Akt, ERK1/2, and
SMAD1/5/8) in both MSC and H9c2 were assessed by western blot.
Results. IB-MSC showed a marked reduction of apoptosis (-50% p<0.001) vs
GFP-MSC after 24h of hypoxia. IB-CM increased H9c2 viability (+32,1% p<0.001)
compared with CTRL-M, while GFP-CM had no effect. Caspase-3 activation was
reduced in the presence of IB-CM of 63,9% vs CTRL-M (p<0.001) and of 49,7 % vs
GFP-MSC (p<0.05). H9c2 treated with IB-CM showed enhanced expression of Bcl-2
and Stat3 pro-survival genes, and inhibition of FasL and TNFα pro-apoptotic genes.
Both IB-MSC or IB-CM treated-H9c2 showed a strong activation of Akt, ERK1/2 and
SMAD1/5/8 pathways, confirming that IGF1 and BMP2 transgenes are acting both
in autocrine and paracrine manner.
Conclusions. IGF1 and BMP2 transgene overexpression in MSC increases cell
survival and cytoprotective paracrine properties. In particular, these effects are
48
mediated by the activation of pathways known to be involved in cell survival.
150
Will not be presented
151
LIPOXYGENASE INHIBITOR ENHANCES THERAPEUTIC EFFECT
OF THE TREATMENT OF TRAIL-SECRETING MESENCHYMAL STEM
CELLS IN GLIOMA
Seong Muk Kim, Ji Sun Woo, Chang Hyun Jeong, Sin-Soo Jeun, The Catholic University of
Korea, Seoul, South Korea.
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the
most promising candidates for cancer gene therapy. However, many types of cancer,
including gliomas, are resistant to TRAIL-induced apoptosis and the highly invasive
nature of glioma cells is the major obstacle to a cure. Here, we describe the enhanced
therapeutic potential of combining the lipoxygenase inhibitor, MK886, and TRAILsecreting human mesenchymal stem cells (MSC-TRAIL) that provide targeted and
prolonged delivery of TRAIL both in vitro and in orthotopic mouse models of glioma.
An in vitro efficacy experiment using the combination of MK886 and MSC-TRAIL to
treat either TRAIL-sensitive or -resistant human glioma cells resulted in significantly
enhanced apoptosis compared with that observed for treatment with each agent
alone. We verified that MK886 effectively increased the sensitivity to TRAIL-induced
apoptosis via upregulation of the death receptor 5 (DR5) and downregulation of the
antiapoptotic protein survivin in human glioma cell lines and in primary glioma cells,
which was confirmed using siRNA inhibition. This regulation was accompanied by a
substantial increase in caspase activation after combined treatment. Furthermore,
in vivo survival experiments and imaging analysis in orthotopic xenografted mice
showed that MSC-based TRAIL gene delivery combined with treatment with MK886
into the tumors had greater therapeutic efficacy than that observed for single-agent
treatment. These results indicate that the combined treatment using MK886 and
MSC-based TRAIL gene delivery may represent a novel strategy for improving the
treatment of malignant gliomas.
152
Will not be presented
153
Will not be presented
154
PROVINCE WIDE ELECTRONIC NOTIFICATION OF TRANSFUSION
REQUIREMENTS FOR ALLOGENEIC TRANSPLANT PATIENTS
ENSURES SAFE TRANSFUSION PRACTICES
April M. Hillman, BSc/MLT1 Susan M. Berrigan, MLT III1 Debbie L. Kury, MLT II1 Hana
Stastna, MLS1 Alli K. Kornylo, BSc/MLT1 Nicole L. Prokopishyn, PhD1,2 1Calgary Laboratory
Services, Calgary, Canada, 2University of Calgary, Calgary, Canada.
ABO/Rh allogeneic blood and marrow transplants are routinely performed due to
the absence of ABO/Rh antigens on the hematopoetic progenitor cells. However,
these “incompatible” transplants create unique situations in transfusion support as
it is necessary to ensure compatible blood and blood products are provided to the
patient post-transplant. Internal systems have been in place for several years that alert
front-line transfusion medicine staff as to appropriate products for patients posttransplant. This includes information in an internal laboratory information system
(LIS) regarding donor and recipient blood group and recommended transfusion
products. However, the increase in patient portability over a large geographic area
(Alberta, Canada) requires external notification systems of these unique transfusion
requirements. Previously, a card and letter was provided to transplant patients as
POSTER ABSTRACTS
a method of informing future caregivers and medical staff of blood requirements.
However, the onus was on the patient to make this information known, which is not
practical in many situations, especially emergency and urgent care situations. Our
centre has developed an electronic notification for transfusion requirements that
is available electronically to all health care providers in the entire province via the
province wide accessible database of patient information (NetCare). These safety
precautions ensure patients that are post-allogeneic transplant receive transfusion
support that is appropriate for them, regardless of care facility. An overview of the
system and preliminary results since implementation of this new safety procedure
will be presented.
155
POST-THAW ASSESSMENT OF CD34+ CELL RECOVERY AND
VIABILITY IN ALLOGENEIC CELLULAR THERAPY PRODUCTS
SUGGESTS FRESH IS BETTER THAN FROZEN
April M. Hillman, BSc/MLT1 Susan M. Berrigan, MLT III1 Debbie L. Kury, MLT II1 Hana Stastna,
MLS1 Alli K. Kornylo, BSc/MLT1 Nicole L. Prokopishyn, PhD1,2 1Calgary Laboratory Services,
Calgary, Canada, 2University of Calgary, Calgary, Canada.
Typically our centre utilizes fresh infusion for allogeneic cellular therapy products
(CTPs). Occasionally, the logistics of donor availability, courier transport, and
patient delays dictate cryopreservation of products until transplant can occur.
With increased utilization of matched unrelated donors (MUDs) from around
the world, cryopreservation of allogeneic products is becoming more common.
Although extensive studies have demonstrated excellent post-thaw recovery of
CD34+ cells following cryopreservation in CTPs collected from autologous sources,
similar studies are not available for CTPs used in allogeneic transplants. Using
our previously described dilution method for post-thaw assessment of CD34+ cell
recovery and viability, we compared cryopreserved allogeneic products (n=8) to
cryopreserved autologous products (n=31) to examine if there was a difference
in recovery of cells in allogeneic vs autologous cryopreserved CTPs. CD34+ cell
recovery averaged 31.1% lower in the allogeneic CTPs as compared to autologous
CTPs despite use of the same cryopreservation method. Viability of the allogeneic
cryopreserved CD34+ cells was also slightly lower than with autologous (95.8%
vs 98.7%). As expected, post-thaw WBC recovery was low in both allogeneic
and autologous CTPs. Not only is there an increased chance of adverse infusion
reaction due to presence of DMSO and cellular debris in cryopreserved CTPs but
there appears to be a decreased survival of CD34+ cells in the allogeneic CTPs.
Interestingly, engraftment data available for the frozen related donor transplants
(n=4) as compared to an equivalent population receiving fresh CTPs indicated
a longer engraftment period with frozen products (19 days platelets vs 17 days).
These initial findings suggest that utilization of fresh allogeneic CTPs for the
majority of patients is ideal.
156
VACUUM FAILURE WITH IMPLOSION: A RARE BUT POTENTIALLY
CATASTROPHIC RISK FOR AGING OR DAMAGED LIQUID
NITROGEN FREEZERS
D A. Wall, 1,2 A Giftakis,1 M Tulloch,1 J Madden,3 O Sturtevant,4 D Hearsey,4 D Schott,4 L Kelley,4
1
Cancer Care Manitoba, Winnipeg, Canada, 2University of Manitoba, Winnipeg, Canada, 3Praxair
Inc., Winnipeg, Canada, 4Connell O’Reilly Cell Manipulation Core Facility, Dana Farber Cancer
Institute, Boston, United States.
Introduction: Stored hematopoietic (and other) stem/progenitor (HSPC)
products in liquid nitrogen (LN2) can be endangered by freezer failure. We are
reporting two sudden freezer failures with implosion of the tank.
disaster plan. Following evaporation of the residual LN2 the lid would not open
easily due to the tank having imploded – mangling the stainless steel lining of the
tank. 2) LN2 tank (MVE 1411F, manufactured in 2003) alarmed due to failure to fill
with LN2. The problem could not be resolved so the products were removed from
the freezer. Within hours of removing products the lining of the freezer imploded
and the lid cracked in half. Implosion was determined the result of loss of vacuum.
Conclusion: This is the first report of vacuum failure with implosion in an LN2
storage tank. It is presumed that a microscopic breach in the lining of the tanks
led to leakage of small amounts of LN2 into the vacuum space. As the tanks
warmed above the boiling point of LN2 (-190º C) the resultant rapid 700 fold
expansion of the LN2 in the closed vacuum space generated sufficient pressure
to implode a stainless steel-lined tank. The setting where this is at greatest risk
is during transport of tanks or planned evaporation for cleaning (recommended
q2-5 years by manufacturer). In the least this risk underscores the importance of
monitoring LN2 usage and having a contingency plan for product storage in case
of loss of a freezer.
157
SUPPLIER AND SUPPLY QUALIFICATION – SINGLE CENTER
EXPERIENCE WITH ISLET ISOLATION SUPPLIES
Dave Krugh, MT(ASCP)SBB, CLS, CLCP(NCA)1 Hillary Bradbury, MT(ASCP)1 Steven Devine,
MD2 Lynn O’Donnell, PhD1 1OSU-CCC The James Cancer Hospital and Solove Research
Institute, Columbus, US, 2OSU-CCC Division of Hematology, Columbus, US.
Our Cell Therapy Laboratory (CTL) supply qualification process (QP) requests
suppliers to complete CTL qualification forms (QF) summarizing demographics,
regulatory, quality, source material and manufacturing information. Only 50% of
islet suppliers completed CTL QFs, citing number of requests, workload, legal
review and potential legal implications as reasons. Remaining suppliers were
qualified using phone interviews or website information to complete the CFs. The
QP was laborious and time consuming, requiring multiple contact attempts over
several months to complete. We were able to obtain a Certificate of Analysis (COA)
for 50% of supplies. Certificates of Compliance (COC), which may or may not be
based on testing to industry standards, were available for another 36% of supplies,
all of which were disposables. 5 supplies contained materials of animal origin, but 4
had either a Certificate of Origin or were Certified TSE-free.
Qualification Process
Suppliers (n=14)
Supplies (n=36)
Number of
Animal or
Attempts
ISO
FDA
COA
COC, COQ Manufactured
Bacterial
to Obtain Certified Registered Obtained Obtained GMP or USP
Origin
Information
Initial Contact
to Qualification
(Days)
5–343
(mean 97,
median 40)
1–9
(mean 5,
median 4)
93%
50%
50%
36%
78%
19%
A risk assessment (RA) was required as defined by the CTL QP and was performed
based on information on the QFs and COAs for 61% of supplies. RA consists of a
safety, reliability and supply chain matrix with 2-3 risk levels and a maximum score
of 300. Scores ranged from 3-111 (mean 21; median 16), but did not appreciably
change how the supplies were controlled. Based on our experience, suppliers are
unprepared to meet the CTL supplier qualification processes, using this single
site model where site visits are not feasible and paper audits are the main data
source. Furthermore, the information obtained was low reward and did not
contribute to new risk management strategies. There is an unquestionable need
in the cell therapy community for the development of a uniform and centralized
supplier qualification process.
Case Reports: 1) LN2 tank (MVE XLC 1200 – manufactured in 1999) historically
evaporated 1 inch/day of LN2. From Sept-Nov 2011 daily monitoring identified an
increasing LN2 loss to 3 inches/day. Visual inspection did not reveal frosting and
lid fit well. On percussion the tank sounded duller compared to the other storage
vessels. HSPC products were transferred to another tank, as per institutional
49
POSTER ABSTRACTS
158
DETECTION OF BACTERIAL CONTAMINATION IN CORD BLOOD:
A MATTER OF SAMPLING VOLUME.
Elisabeth Semple, 1 Sandra Ramirez-Arcos,2 Eva Friedman,1 Qi-Long Yi,2 Michael Virro,1 1Cells
for Life Cord Blood Institute, Toronto, Canada, 2Canadian Blood Services, Ottawa, Canada.
Introduction: Cord blood collected and processed for long-term storage must be
evaluated for bacterial contamination as presence of bacteria can cause harm
if not known at time of transplant. Detection of bacteria is typically done by
inoculating paediatric blood culture bottles with 0.5 – 1 ml of the processed cord
blood, with or without addition of plasma from the same unit. The aim of this
investigation was to determine the impact of sampling volume on the detection
rate of bacterial contamination.
Materials and methods: Cord blood (n=104) was collected in-utero, transferred to
Top/Bottom processing bags (VRT 0000XU, MacoPharma) and spun at 3000g for
12 min. A buffy coat of 21 ml was obtained using the Optipress (Fenwal). After
addition of 5 ml DMSO/Dextran40, 25 ml was cryopreserved and the residual buffy
coat was diluted with plasma from the same unit. All manipulation of the unit was
done either in a closed system or a biological safety cabinet. Culture bottles were
inoculated as follows:
Paediatric bottle
(BacT/ALERT PF)
Paediatric bottle
(BacT/ALERT PF)
Adult bottles
(aerob BacT/ALERT SA; anaerob
BacT/ALERT SN)
1 ml
4 ml
8+8 ml
The bottles were incubated for 5 days in the Bact/ALERT system (BioMerieux).
In case of bacterial contamination, the type of bacteria present was determined.
Results: The concurrence between methods, positive or negative, was 88%.
The bacterial contamination rate was 4%, 6% and 16% (paediatric 1 and 4 ml,
and aerob/anareob combo, respectively). Bacteria that were not detected with
the paediatric methods included Escherichia coli, lactobacilli, coagulase negative
Staphylococcus, Gardenella vaginalis, Peptostreptococcus and diphtheroid bacilli.
Conclusion: This study showed that the detection rate of bacterial contamination
in cord blood was proportional to the sampling volume. To better assess
contamination rates, cord blood banks need to take this into consideration when
evaluating and validating methods for bacterial detection.
159
EVALUATION OF GRANULOCYTES IN ASSESSMENT OF HPC,A
QUALITY
Erica L Fitzgerald, Robert Taylor, MD, Jonathan Treisman, MD, Kate Dennert, Jaime Kroll,
Angela Timler, Nina Garlie, PhD, Aurora St. Luke’’s Medical Center, Milwaukee, USA.
Post-thaw assessment of cryopreserved Hematopoetic Progenitor Cells, Apheresis
(HPC,A) is vital to cell processing laboratories: to fulfill requirements of regulatory
agencies, ensure quality of the HPC,A for clinical purposes, and provide data for
process analysis. One method to determine the quality of HPC,A is mononuclear
cell (MNC) recovery. In our lab, MNC recovery has been determined using a postthaw cell count and manual viability, based on the expectation that granulocytes
will die during the cryopreservation process and remaining viable cells will be
MNC. This method has been found to be unreliable, as a population of surviving
granulocytes often skews data and results in artificially high MNC recoveries.
To improve accuracy of post-thaw MNC assessment, we clearly defined remaining
granulocytes using flow cytometry. Cryopreserved HPC,A were thawed and analyzed
with lymphocyte marker CD45, granulocyte marker CD66, and viability dye 7AAD. By
defining MNC as CD45+/CD66-, the percent of viable MNC was determined and used
to calculate the MNC recovery. This method resulted in an MNC recovery free from
granulocyte contamination. Preliminary data shows that the flow cytometery based
50
assessment results in lower MNC recoveries compared to those determined by cell
count and manual viability alone (60% ± 20% and 135% ± 59%, n=15, respectively).
The difference between the two methods is greater in products with a high granulocyte
population (r = -0.94, p>0.001, n=15), therefore HPC,A with low MNC require flow
cytometry to attain accurate post-thaw recovery data.
A granulocyte exclusion flow cytometry antibody panel is a relatively quick and
inexpensive HPC,A post-thaw assessment method. By obtaining quality data on
HPC,A, we are better able to evaluate cell products for clinical purposes, and to
more accurately correlate the recovery of MNC and granulocyte populations to
variables of interest when tracking processing changes and investigating potential
process improvements.
160
HUMAN BLOOD-DERIVED RAW MATERIAL: ENABLING
CONTROLLED, CONSISTENT COLLECTION
Gaytha McPherson, 1 Pete van der Wal,1 Scott R. Burger, MD2 Anna Stock,1 1HemaCare
Corporation, Van Nuys, USA, 2Advanced Cell & Gene Therapy, LLC, Chapel Hill, USA.
Human cells and tissue are critical raw material for cell therapy and tissueengineered products, as well ex vivo gene therapy products. Quality of this living
cellular raw material is a major determinant of final product characteristics, but
inter-individual variability and biological heterogeneity are complicating factors.
Controlled, qualified cell collection minimizes operational sources of variability,
increasing likelihood of successful manufacturing. HemaCare, a long-standing
supplier of human-derived blood components for novel biologic therapies
and assays, medical devices, and clinical applications, established a program
for controlling and qualifying its apheresis procedures and collection sites.
This includes comprehensive staff training and qualification, documentation
supporting cGMP operations, equipment and procedure validation, and
monitoring in accordance with a rigorous quality system. Donor recruitment,
screening and IRB-approved consents follow GTP requirements. The quality
program tracks and investigates deviations from process, complaints, and error
rates in specific categories, representing donor screening and testing, collection,
product preparation, testing, labeling, distribution, and GMP/GTP systems. As
part of continuous process improvement, in 2011 HemaCare validated or revalidated 14 apheresis instruments for mononuclear cell (MNC) collections
at 3 collection sites, using GMP validation standards established for platelet
collection, and validated a new automated cell analyzer, the Horiba Pentra XL80.
HemaCare performed 14,439 apheresis procedures in 2011, including collection
of patient and normal-donor peripheral blood MNCs, G-CSF-mobilized peripheral
blood progenitor cells, plateletpheresis products, and therapeutic apheresis,
supporting commercial cell therapy and clinical trials, preclinical research, and
validation studies. Unmobilized apheresis products contained an average of 13.1
x 109 nucleated cells, and were appropriately MNC-rich with an overall average of
77.7% MNC ± 8.0% (mean ± 1 SD). Rigorous quality systems enable controlled,
consistent cell collection, a critical factor for development and commercialization
of novel cell-based products and technology.
161
VALIDATION OF BACT/ALERT FAN BOTTLES USED FOR
CONTAMINATION TESTING OF HAEMATOPOIETIC PROGENITOR
CELLS
Kerry Varettas, Senior Scientist1 Craig Wright, Quality manager2 Kon Zarkos, Senior Scientist3
1
, South Eastern Area Laboratory Services, St George Hospital, , Sydney, Australia, 2Royal
Prince Alfred Hospital , Sydney, Australia, 3Royal Prince Alfred Hospital, Sydney, Australia.
The SEALS Central Microbiology department based at St George Hospital,
Kogarah, uses a continuous blood culture instrument, the BacT/Alert 3D, with FAN
aerobic, anaerobic and paediatric bottles. The current method of contamination
testing of haematopoietic progenitor cells (HPCs) using the BacT/Alert 3D and
POSTER ABSTRACTS
FAN bottles was validated.
A group of nine bacteria and fungi were used during validation. The organisms
were inoculated into FAN aerobic, anaerobic and paediatric bottles in a seeded
bottle trial. The bottles contained human plasma and processed cells collected
from patients at the Apheresis Unit and processed by Cell and Molecular
Therapies Laboratory at RPAH, Sydney. The organisms were also trialled against
the cryoprotectant DMSO and DMSO+Albumex® (4% human albumin),
routinely used in the cryopreservation of processed cells. Two sets of FAN bottles
were inoculated for each organism – one set was incubated in the BacT/Alert
immediately and the other set had a delayed loading of 24-hours.
In the seeded trial and in the bottles containing patient plasma, all 9 organisms
were isolated within the incubation period from one, two or all of the FAN
aerobic, anaerobic and paediatric bottles loaded immediately after inoculation
and with a 24-hour delay. All expected aerobic organisms were isolated from
the FAN paediatric bottles containing DMSO or DMSO+ Albumex® and loaded
immediately after inoculation and with a delay of 24-hours. Only one organism
did not grow as expected in the FAN paediatric bottle containing processed cells
after immediate loading and with a delay of 24-hours.
This validation confirms that the current method using the BacT/Alert 3D
instrument and FAN bottles is able to detect a range of organisms from patients
undergoing treatment regimes that include chemotherapy and/or mobilisation
regimes (e.g. granulocyte colony stimulating factor), and from processed cells for
cryopreservation with the addition of DMSO or DMSO+ Albumex®.
162
EVENT REPORTING AS A TOOL FOR ASSESSING STAFF
ACCEPTANCE AND COMPLIANCE WITH REGULATORY
REQUIREMENTS FOR THE MANUFACTURE OF CELLULAR
THERAPIES.
Pamela G Dyson, Ms, Kerry A Munro, Ms, Ian D Lewis, Dr, SA Pathology, Adelaide, Australia.
The aim of Good Manufacturing Practice (GMP) is to ensure that products are
consistently produced to quality standards appropriate for their intended use.
In 2000 we constructed a facility to process a range of human blood and tissue
products in compliance with the Australian Code of GMP and developed a quality
system which incorporates the elements required by the TGA, NATA, and ISO 9002.
Staff are trained in GMP, cleanroom practice, and in technical protocols. Products
manufactured under GMP include haemopoietic progenitor cells, mesenchymal
stromal cells, autologous chondrocytes, serum eye drops and keratinocytes.
Since 2000 there have been many changes to manufacturing processes to meet
regulatory requirements. The greatest changes required have been in staff training
and culture. After more than ten years of operation, a review was performed to assess
the effectiveness of the quality system and staff acceptance and compliance.
Reporting of all incidents likely to impact on manufacture and products is a key
element of our quality system. Events are categorised as either occurrences –no
perceived negative impact – or deviations – negative impact. We utilised this reporting
system to assess staff acceptance, competence and compliance with regulatory
requirements.
There has been a steady increase in the number of reports since 2000. Deviation
reports outnumber occurrence reports around six fold indicating staff training
is needed. We found a high level of subjectivity, and variability with time, in
categorisation of reports by staff. From 2004 to 2010 major deviations increased
from 3% to 60% while minor deviations) decreased from 80% to 5%.
Trends in event reporting indicated further work is required to increase the level
of acceptance and compliance by staff. Compliance could be improved by further
development of a “no blame” culture. A substantial number of repeat deviation
reports indicate that staff require training in root cause analysis.
163
USE OF UNRELATED DONOR COLLECTION CENTER CD34+
COUNTS DECREASES TIME TO PRODUCT INFUSION WITHOUT
COMPROMISING PATIENT OUTCOMES
Rebekah Knight, 1,2,3 Kelsea Shoop,1,2,3 Sara Murray,1,2,3 Laura Newell,1,2,3 Richard T. Maziarz,1,2,3
1
Hematopoietic Cell Processing Laboratory, Center for Hematologic Malignancies, Portland,
USA, 2Knight Cancer Institute, Portland, USA, 3Oregon Health and Science University,
Portland, USA.
BACKGROUND: In the setting of unrelated donor (URD) peripheral blood stem
cell transplants (PBSCT), outside collection centers are increasingly reporting
stem cell product analysis results prior to the transplant center’s receipt of these
products. In 2010, as a pilot analysis, transplantation procedures for patients
were based on collection center counts to expedite the time to infusion. OHSU
confirmed collection center counts for all patient infusions.
METHODS: A retrospective analysis of CD34+ cell doses between collection center
and OHSU was performed using a two sample t-test. Patient outcomes analyzed
included incidence of infusion toxicities, and time to engraftment of absolute
neutrophil count (ANC) >0.5x109/L and platelet count >20x109/L.
RESULTS: 42 patients were evaluable for analysis. Mean total CD34+ doses
reported by collection centers (8.76x106/kg) versus OHSU confirmed results
(8.59x106/kg) were not significantly different (p-value=0.40). Mean infused CD34+
doses reported by collection centers (6.00x106/kg) versus OHSU confirmed
results (5.73x106/kg) were not significantly different (p-value=0.06). Median time
to engraftment for these patients was Day +13 for ANC >0.5x109/L and Day +20
for platelets >20x109/L. Using collection center counts decreased the time from
receipt of stem cell product to patient infusion by approximately 2 hours (1hour
versus 3 hours).
CONCLUSION: Time to patient infusion is enhanced by using flow cytometric
CD34+ results from collection centers. Advantages include decreasing overtime
and late night efforts for laboratory and medical staff that monitor infusions.
There were no statistical differences in mean total and infused CD34+ dose. There
were no infusion related toxicities and time to engraftment met OHSU acceptable
parameters.
164
Building a Good Manufacturing Practice (GMP) facility
within the public healthcare system
Janet L Macpherson, PhD, Craig Wright, Angel Jaramillo, John EJ Rasko, Royal Prince Alfred
Hospital , Sydney, Australia.
In Australia, the Therapeutic Goods Administration requires that beyond Phase I
trials, cell therapy products for clinical use are manufactured in a licensed facility.
We designed and built a state of the art facility at a leading publically-funded
hospital in metropolitan Sydney. Commissioning and certification of the facility
is tracked and documented according to a Validation Master Plan. The Quality
Management System for the new facility is building on a TGA license to manufacture
haematopoietic progenitor cells that has been successfully maintained since 2007.
The cGMP facility was designed to meet both ISO 14644 clean room standards,
51
POSTER ABSTRACTS
and the physical containment class 2 (PC2) requirements of the Office of the Gene
Technology Regulator to accommodate genetically modified cells. The reconciliation
of potentially-conflicting requirements for containment versus sterility required
special attention.A Project Users Requirements Specification (PURS) was developed
based on detailed Room Data Sheets. Integral to the design is the concept of
flexibility with real-time monitoring of environmental parameters. The PURS was
expanded into tender specifications for electrical, mechanical and environmental
monitoring services, and contractors engaged. Hospital engineering services were
responsible for co-ordinating construction, fit out and finish. Contract service
providers were required to provide documentary evidence of conformance with the
specification and instructions for on-going maintenance of installed services.
Construction of the suite of laboratories proceeded, documentation to certify that
the facility was built to specification requested, and consultant engineers engaged
to facilitate handover. Some interpretations of the PURS were at variance with the
intention of the laboratory-based designers. Flooring, mechanical and electrical
services all required remedial work, resulting in substantial delays and both direct
and indirect budget implications. Commissioning of a PC2 cGMP clean room facility
is more complex and specialised than building other areas within the Healthcare
Service. Mutli-discipline teamwork is essential for execution.
165
Current Status of Cellular Therapy in the Asia-Pacific
Region
Janet L Macpherson, John EJ Rasko, Royal Prince Alfred Hospital, Sydney, Australia.
The Asia-Pacific region offers a diverse range in quality of healthcare services.
Almost every country in the region offers at least some cellular therapies, from the
highly regulated countries like Japan, Korea and Australia, through to countries
where regulatory oversight is less formal. The key healthcare drivers for this sector
are the ageing population, obesity epidemic, organ donation statistics and the
emergence of personalised medicine.
The future of cell therapy lies in the advancement of treatment options based on
sound clinical data, both in terms of clinical outcomes, and supported by robust
laboratory data. The emergence of clinics in the Asia-Pacific region offering onesize-fits-all stem cell injections as treatment for diverse indications is of concern
for the long term future of the cell therapy industry. Clinics that operate outside the
rigorous controls imposed by the regulatory authorities in countries like Australia,
USA and Europe, often target vulnerable patients desperately seeking a cure for
debilitating and chronic illnesses such as cerebral palsy, multiple sclerosis and
diabetes. Australia and selected other countries in the Asia-Pacific region offer a
regulated and controlled environment to ensure the safety and conformance of cell
and tissue based products. ( Macpherson JL, Rasko JE. Cellular therapy in the AsiaPacific region. A guide for the future pathologist. Pathology. 2011; 43: 616-26)
166
MANAGING A TGA GMP LICENCE FOR HPCS WITHIN NSW
PUBLIC HEALTH
Craig S. Wright, Quality manager1 Ross Brown, Production manager2 Kon Zarkos,3 Stephen
Larsen, Dr4 John Rasko, Proffessor4 John Gibson, Professor4 1Royal Prince Alfred Hospital, ,
Camperdown NSW , Australia, 2Royal Prince Alfred Hospital, Camperdown NSW , Australia,
3
Royal Prince Alfred Hospital , Camperdown NSW , Australia, 4Royal Prince Alfred Hospital ,
Sydney, Australia.
Introduction: The CMT team at Royal Prince Alfred Hospital (RPAH) obtained a
GMP licence from the TGA in 2007. The licence is to manufacture autologous and
allogeneic haematopoietic progenitor cells (HPCs) at RPAH Laboratory, collected
from RPAH and Concord Hospital (CRGH).
52
Aim: To maintain a GMP licence for manufacture of HPCs for clinical use in NSW,
Australia. We have built a quality system that involves clinicians, scientists, nurses
and NSW health infrastructure working in a co-ordinated quality environment. This
supports a flexible hospital based GMP licence and future aims to process other cell
therapies.
Method: The apheresis transplant service facility was designed to meet TGA
requirements. External contracted service providers are required to provide testing that
conforms to the TGA cGMP. A TMF was submitted with HPC product specifications.
Document controlled procedures (SOPs) & records; product validations; Training,
monitoring systems; equipment, material, change process control; auditing and nonconformance reporting; transport and management review are GMP requirements.
Result: GMP licensure maintained since 2007. Rated good TGA compliance level for
2011. Neutrophil and platelet engraftment reviewed six monthly, to monitor success
rates of HPC engraftment. Over four years, results demonstrate process remains
controlled despite wide range of patient disease variability. Internal and external audit
citations, and non-conformances, have diminished. Indicating system improvement
of quality and operations.
Conclusion: Maintaining a cGMP TGA compliant facility for HPCs has provided
product improvement for beneficial patients outcomes. Process requires co-operation
of multi-skilled group of dedicated health workers including hospital cleaners, nurses,
transplants scientists and medical officers. Hospital staff recognise the compliance
needs for TGA GMP requirements in the service.. Quality systems will be used to
develop other cell therapies. No conflict of interest to disclose
167
The Impact of Market Volatility on Cell Therapy
Industry Growth
David A. Brindley, 1,2,3 Brock C. Reeve,2 William A. Sahlman,3 Greg A. Bonfiglio,4,5 Natasha L.
Davie,1,2,6 Emily J. Culme-Seymour,7 Chris Mason,1,7 1University College London, London,
UK, 2Harvard Stem Cell Institute, Cambridge, MA, USA, 3Harvard Business School,
Boston, MA, USA, 4Proteus Venture Partners, Portola Valley, CA, USA, 5Canadian Centre
for Commercialisation of Regenerative Medicine, Toronto, ON, 6Harvard Medical School,
Boston, MA, USA, 7London Regenerative Medicine Network, London, UK.
Stock market volatility in the cell therapy industry (CTI) has greatly hindered the
investment necessary to fund the translation of life saving therapies from lab bench
to marketplace. Here, provide a quantitative review of the financial performance
of leading CTI companies, utilizing the Cell Therapy Industry Cumulative Growth
Index, between the period September 2006–September 2010.
The index suggests that that CTI has grown strongly over the five year period
studied, by an average of 141.89% compared with a an -8.13% loss experienced by
the NASDAQ. While some volatility remains, we suggest that a distinct industry
is maturing to a point at which the volatility should subside, providing a more
attractive environment for future growth. In particular, we highlight the vital role
of CTI translation centres and industry organisations including the Bio Industry
Association (BIA), the Alliance for Regenerative Medicine (ARM), International
Society for Cellular Therapy (ISCT) and the International Society for Stem Cell
Research (ISSCR).
168
OPPORTUNITIES FOR ADVANCED PRODUCT DEVELOPMENT AT
THE BIOMEDICAL ADVANCED RESEARCH AND DEVELOPMENT
AUTHORITY
Mary J. Homer, PhD, Brian Tse, PhD, Ronald G. Manning, PhD, Biomedical Advanced
Research and Development Authority/US Department of Health and Human Services,
Washington, DC, USA.
As part of its mission, the Biomedical Advanced Research and Development
Authority (BARDA), within the Office of the Assistant Secretary for Preparedness
and Response (ASPR) in the U.S. Department of Health and Human Services
(HHS), provides an integrated, systematic approach to the development and
purchase of the necessary drugs and diagnostic tools for chemical, radiological
and nuclear public health medical emergencies. Using its advanced research and
POSTER ABSTRACTS
development authority, BARDA strengthens HHS efforts to bridge the valley of
death funding gap that exists between the early stages of product development
and the acquisition of approved or approvable medical countermeasures for the
Strategic National Stockpile (SNS). BARDA advanced development programs
support efforts to develop therapeutics to treat sub-syndromes of acute radiation
syndrome as well as decorporation agents. More recently, BARDA is beginning
a new initiative to support development of blood products and blood product
platform technologies, including but not limited to cellular therapies, plasma
products, and platelet products, to bolster existing preparedness and response
resources and tools. BARDA is also developing biodosimetry tools, both devices
and assays.
169
QUALITY CULTURE AND TOOLS: SYNERGY FOR COMPLIANCE
AND ACCREDITATION
Nancy M Messino, Aimee E Stewart, Sally Anderson, Royal Children’’s Hospital, Melbourne,
Australia.
In accordance with best practice and international standards, the Haemopoietic
Stem Cell Transplant (HSCT) Programme of the Children’s Cancer Centre (CCC)
at the Royal Children’s Hospital (RCH), Melbourne, Australia, decided to obtain
FACT Accreditation. Aims of accreditation include;
•A Quality Management Plan with functional robust quality systems providing a
framework for clinical, collection and laboratory services provided by the HSCT
Programme.
•Compliance with both international and legislated national benchmarks.
While in general the RCH participates in the ACHS (Australian Council of HealthCare
Standards) accreditation system to ensure quality of clinical care, the CCC made the
strategic decision that a higher level of quality management was required to support
the HSCT Programme. A position was created to develop, implement and monitor
quality management systems for all HSCT services and attain FACT accreditation.
In collaboration with the managers of individual services, a comprehensive review
and gap analysis was undertaken across the HSCT Programme. While the laboratory
service was at an advanced level of compliance with national regulatory requirements
as assessed by NATA (National Association of Testing Authorities), the clinical and
collection services were commencing the quality journey.
Following an assessment of the available quality management tools, it was decided
to implement Q-Pulse software to support the introduction of a more stringent
quality culture and provide mechanisms for capturing data about all quality functions
being performed. The software’s flexibility facilitated the introduction, and allowed
for customization, of a compliant quality management system. Individual software
modules were tailored for document control, opportunities for improvement, nonconformance reporting, audit, and supplier and equipment management.
The implementation of Q-Pulse software has proven to be an effective quality
management strategy, providing the delivery of compliance and attainment of
accreditation by governing bodies. Therefore this solution could be utilized to solve
similar challenges in other facilities.
170
FROM REACTIVE TO PROACTIVE: THE QUALITY MANAGEMENT
MATURITY MODEL
Nicole Bleasdale, Maria Maroulis, Patrick Coghlan, Dr, Australian Red Cross Blood Service,
Melbourne, Australia.
Since 1999 the Australian Red Cross Blood Service has assisted biological
manufacturing organisations with the implementation and improvement of
their quality management systems in the fields of blood and blood component
manufacture, tissue banking, cellular therapies and clinical trials management.
More recently (since 2008), the Blood Service has applied its expertise towards
assisting 9 institutes across Australia engaged in early phase clinical cellular
therapies/regenerative medicine projects to develop and implement quality
management systems and achieve cGMP compliance.
Through our experience we have observed a correlation between the maturity of
an organisation and its approach to quality management. Organisations in their
early stages of development, such as research groups entering the translational
research phase, are commonly unaware of the requirements and benefits of
quality management and tend to take a reactive approach to quality issues. In
contrast, mature organisations take a proactive approach and integrate quality
management into all their activities.
A Risk Management Maturity Model (RMMM) developed in 2002 through a
formal collaboration between the International Council on Systems Engineering
Risk Management Working Group, the Project Management Institute Risk
Management Specific Interest Group, and the UK Association for Project
Management Risk Specific Interest Group defined four distinct levels of maturity:
Ad Hoc, Initial, Repeatable and Managed. Each level includes recommended
actions needed to move to the next level of maturity.
We recognise that this RMMM framework is readily applicable to the
implementation of quality management. As such we have adapted the RMMM to
develop a Quality Management Maturity Model (QMMM). The QMMM enables
us to categorise each client’s approach to quality management and identify the
actions necessary to achieve the next maturity level. The overall intent of this
model is to provide a tool to enable a considered, step-wise and timely approach
to implementing quality management.
171
POLICIES REGARDING HUMAN GENETIC MODIFICATION
TECHNOLOGIES IN MEXICO
Isaac O. Osadolor, MD; M.Sc; PhD; D.Sc.1,2 Magdalena Guadalupe S. Rodriguez, PhD3,4
1
Instituto de Ciencias Jurídicas de Puebla A.C., Puebla, Puebla., Mexico, 2Universidad
Popular Autónoma del Estado de Puebla, Puebla, Mexico, 3Universidad Autónoma de
Tlaxcala Facultad de Ciencias de la Salud Licenciatura en Médico Cirujano., Tlaxcala, Mexico,
4
Instituto Tlaxcalteca de Asistencia Especializada a la Salud , Tlaxcala, Mexico.
Mexican federal legislation does not regulate explicitly human genetic modification.
The General Health Law (GHL) and its Regulation on Scientific Research (RSR)
and Regulation on the Sanitary Control of Organs, Tissues and Human Cadavers
(RCOTHC), have been interpreted as implicitly prohibiting human germline
modification. Research on embryos can only be conducted for the benefit of
the embryo and then only when their “security/integrity is guaranteed,” (Art.
56). For the purpose of this law, an embryo is “the product of conception from
fertilization to the end of the week of gestation” (Art. 314). This definition is
consistent with the Regulation of the General Health Law on Scientific Research.
The aforementioned legislation regulates the use of human cells and embryos.
The only legal norm contemplating human genetic modification is the Penal Code
for the Mexican Federal District (CPDF). According to article 154 of the CPDF, the
crime of “genetic manipulation” is committed when a person (i) “with a purpose
other than eliminating or diminishing serious diseases or disorders manipulates
human genes in such a way that alters a genotype; (ii) fertilizes a human egg with a
different purpose than human procreation; or (iii) creates human beings by cloning
or performs genetic engineering with illicit purposes” (Art. 154). The disposition of
germinal cells is considered to be illicit when it is performed against the purposes
for which the donor(s) has granted authorization (Art. 149). Furthermore, Art.
155 establishes that if a child is born in contravention of the above mentioned
provisions, the compensation of damages includes the payment of child support
to the mother. There is no specific federal or local legislation dealing with somatic
human modification or gene therapy; hence it is governed by the general provisions
related to human experimentation and scientific research (Arts. 100 and 465GHL,
Arts. 13 and 14RSR).
53
POSTER ABSTRACTS
172
for the enzyme preparation used, all steps were similar. Isolations using Liberase HI
were from 2005 to 2006 compared to Liberase MTF from 2009 to current.
EFFECTS OF SILDENAFIL ON TESTICULAR TORSION/DETORSION
(TD) DAMAGE: AN EXPERIMENTAL STUDY IN A RAT MODEL
Results: No significant differences in donor age, cold ischemia time, digestion
time, or weight of the pancreases were observed between the two groups. All
examined islet cell product parameters were significantly improved with Liberase
MTF compared to Liberase HI. Islet yield before purification was 457±184×103 islet
equivalents (IEQ) for Liberase MTF compared to 373±187×103 IEQ for Liberase
HI (p=0.018). Post purification IEQ were 361±179×103 Liberase MTF versus
290±181×103 with Liberase HI (p=0.038). The post purification purity was 74±17
% when Liberase MTF was used, compared to 51±19 % for Liberase HI (p< 0.001).
Finally, insulin stimulation index of islet cell products obtained using Liberase
MTF were significantly higher compared to the Liberase HI (3.6±3.3 vs. 2.14±1.9,
respectively; p=0.009).
Isaac O. Osadolor, MD; M.Sc; PhD; D.Sc.1,2 Alfredo P. Adan, MD2,3 Magdalena Guadalupe S.
Rodriguez, Phd2,3 Agustin R. Galvan, MD2,3 Adriana S. Carmona, MD2,3 Fransisco M. Romano,
MD2,3 Lorena R. Garcia, MD2,3 1Universidad Popular Autónoma del Estado de Puebla. Dept. of
Biomedical Engineering , Puebla, Puebla., Mexico, 2Universidad Autónoma de Tlaxcala Facultad
de Ciencias de la Salud Licenciatura en Médico Cirujano., Tlaxcala, Mexico, 3Instituto Tlaxcalteca
de Asistencia Especializada a la Salud , Tlaxcala, Mexico.
BACKGROUND: Testicular torsion is a serious condition that necessitates rapid
surgical intervention to save the affected testicle. Testicular torsion is a pathologic
condition that renders the testis ischemic and surgical intervention is usually
required to reestablish blood flow. The complication of testicular torsion is ischemic
necrosis and the main pathophysiology of torsion-detorsion (TD) is associated
with ischemia-reperfusion injury in the testes caused by the twisted spermatic
cord, although apoptosis and necrosis can occur in pathophysiological settings.
Sildenafil is a potent and selective inhibitor of cGMP-specific phosphodiesterase
type 5 (PDE5) has powerful effects against ischemia-reperfusion injury.
AIM: The study was performed to throw light on the ecographic, histologic
and apoptotic changes that might occur with the effect of Sildenafil on cellular
damage induced by testicular torsion/detorsion in a rat model. We assessed the
effectiveness of sildenafil administration during ischemic period in a rat model of
testicular torsion/detorsion.
MATERIAL AND METHOD: Twenty-four male Sprague-Dawley rats were divided
equally into 4 groups (n = 6). A sham operation was performed in group A as a
control. Testicular torsion was realized in group B. In group C torsion/detorsión
and while in group D after performing the same surgical procedures as in group
C, a dose of 0.6 mg/kg of sildenafil was given intraperitoneally. In all experimental
rats, ipsilateral orchidectomies were performed for ecographic, histological
examination and apoptosis assays.
RESULTS: Our data indicate that vascular perfusion, was lower in group D
compared with group C. Histopathologically, in the group D rats, inflammation
and necrosis of the germinal cells were predominant features in sections,
apoptosis was lower in group C compared with group D injury. We obtained the
same results by tunnel.
CONCLUSION: The current findings demonstrate that the Sildenafil protects
against testes ischemia/reperfusion injury in rats. Sildenafil administration
during testicular torsion decreased ischemia/reperfusion cellular damage.
173
Improved Human Islet Isolation Outcomes Using a
Mammalian Tissue-Free Enzyme Blend
Aisha Khan, MBA, PhD, Alejandro Alvarez, MD, Armando Mendez, PhD, Luca Inverardi, MD,
Rodolfo Alejandro, MD, Camillo Ricordi, MD, Diabetes Research Institute, Miller School of
Medicine, University of Miami , Miami, USA.
Lot to lot variability and changes in enzyme blends performance over time have
traditionally imposed challenges to standardization of islet isolation methods,
reproducibility and consistency in islet isolation outcomes. In addition, the use of
mammalian derived reagents in the enzyme manufacturing process introduced
regulatory concerns for their use in clinical trials. To address these challenges, a
novel mammalian tissue-free collagenase blend product was developed (Liberase
MTF, Roche, Indianapolis, IN). Human islet isolation outcomes using this novel
enzyme blend were compared to those obtained using the previously developed
enzyme blend, Liberase HI.
Methods: Islets were isolated from pancreata using MTF enzyme (n=46) and
Liberase HI enzyme (n=75). Deceased human donor pancreata were processed
using a modified automated method, following established protocols and except
54
Conclusion: We conclude that the performance of Liberase MTF was significantly
better compared to Liberase HI. This improved enzyme blend can therefore be
effectively utilized for experimental and clinical islet cell processing.
174
will not be presented
175
An ex vivo Platform for Predicting Renal Safety and
Physiologic Mechanisms for New Chemical Entities
Andrew Bruce, 1 Tim Bertram,1 Jonathan Sackner-Bernstein,2 Rusty Kelley,1 1Tengion,
Winston-Salem, USA, 2ExVivos, New York, USA.
There is limited availability of ex vivo cell- or tissue-based platforms that reliably
predict in vivo safety and function, though developments in regenerative
medicine suggest promise. Tengion’s selected renal cells (SRC) technology
exemplifies the opportunity, with demonstrated success isolating, expanding and
cryopreserving functional SRC from normal and diseased human donor kidneys.
When implanted into diseased kidneys, SRC stabilizes renal function and extends
survival in multiple models of chronic kidney disease (CKD). SRC isolated from a
human CKD patient improves kidney function in the immuno-compromised nude
rat following ischemic/reperfusion and gentamycin injury. In the present study,
we show that well known nephrotoxic compounds reproducibly and robustly
inhibited primary human SRC functional activity in a dose-responsive manner
using two- (2D) and three-dimensional (3D) human renal cell constructs that
simulate human tissue physiology ex vivo.
Morphological, genotypic, phenotypic and functional changes to 2D cultured SRC
were monitored to 72hrs following exposure to escalating doses of the well known
nephrotoxin, Amphotericin B (Amp B). A semi-logrithmic plot of the inhibitory
effects of Amp B on the SRC-mediated brush border-based enzymatic activity,
Γ-glutamyl transpeptidase (GGT), provided a sigmoidal curve with an IC50 of
32.33µM. The AmpB IC50 strongly correlated to cell viability using Presto Blue and
apoptotic-based caspase detection. To generate SRC functional units, 3D spheroid
cultures were established in non-adherent vessels to simulate kidney tubular
structure and function prior to cisplatin exposure. Similar to AmpB, cisplatin was
shown to have a dose response inhibitory effect on SRC spheroid-mediated GGT-1
function and spheroid cell viability after 48 hrs of exposure.
That the functional properties of 2-D and 3-D SRC cultures, with established
in vivo regenerative properties, provide a platform for ascertaining drug safety,
efficacy and possibly even pharmacokinetic outcomes for new chemical entities,
is highly relevant towards reliably and reproducibly predicting clinical outcomes
in support of their regulatory review.
176
will not be presented
POSTER ABSTRACTS
177
USE OF PLATELET LYSATE FROM OUTDATED PLATELET
CONCENTRATES IN MESENCHYMAL STROMAL CELL CUTLURE
Carla Kreissig, Irene Rehfeld, Jürgen Bux, Prof. Dr., DRK-BSD-West, Ratingen-Breitscheid,
Germany.
Introduction: Since bovine spongiform encephalopathy was recognised as a
serious health treat, use of fetal calf serum in cell culture sytems became unusual.
But MSC in culture systems need cytokines, which are known to be contained in
FCS and so far unidentified, so we can not adjoin them to culture media in perfect
combination and concentration.
preparation. Samples were taken, sterile filtrated and frozen up to -80°C before
testing. Then we pooled 15 ml of four platelet concentrates. Preparation was done
in the same way as described above.
Results: For single platelet concentrates we messured following cytokine
concentrations:
PDGF: 9616 -131.000 pg/ml
VEGF: 194 -648 pg/ml
EGF: 856 -1558 pg/ml
FGF: 57 – 124 pg/ml
For pools of four platelet concentrates we messured following cytokine
concentrations:
Many experts switched to platelet lysate as MSC culture supplement. Until now
it is unknown which platelet preparation is the best: pooled or apheresis platelet
concentrates, fresh or older ones. In several experimental studies we tried to
answer some questions, which preparation is the best.
PDGF: 46.282 - 159.340 pg/ml
VEGF: 219 – 642 pg/ml
EGF: 554 – 1624 pg/ml
FGF: 64 – 77 pg/ml
Material and Method: We tested cytokine concentration in an enzyme linked
immunosorbent assay.
Discussion: Cytokine concentrations of VEGF, FGF, EGF and PGGF between
platelet preparations from different individuals diversify especially for EGF and
PDGF.
We performed ELISA for PDGF, VEGF, FGF and EGF.
We tried to answer the question, how old platelet concentrates should and could
be to guarantee best culture conditions. Therefore we tested eight specimens on
day 6 till 12 after preparation.
Results: PDGF concentration remained constant until day seven after preparation
and afterwards decreased clearly. So PDGF is the most susceptible cytokine we
tested.
VEGF concentration rised until day eight after preparation and then decreased. FGF
concentration rised until day seven after preparation and afterwards decreased
clearly. EGF concentration slightly increased until day eight after preparation and
then remained constant. So EGF is the most stable cytokine we tested.
Discussion: Cytokines are glycoproteins, which are contained in plasma or
intracellular in platelets. For PDGF, VEGF, FGF and EGF nutritional effect in
mesenchymal stromal cell culture is well known. For these cytokines we could
observe a stable level or an increase of concentration until day seven after
preparation. So we postulate, that outdated platelet preparations, which are
tested for HIV, HCV and HBV genom and other infectious agents regarding to
German drug law and transfusion guidelines, could be used for MSC culture.
178
USE OF PLATELET LYSATE IN MESENCHYMAL STROMAL CELL
CULTURE –CAN WE EXPECT A STANDARDISED CELL CULTURE
SUPPLEMENT?
Carla Kreissig, Irene Rehfeld, Dr., Jürgen Bux, Prof. Dr., DRK-BSD-West, Ratingen-Breitscheid,
Germany.
Introduction: Since bovine spongiform encephalopathy was recognised as a
serious health treat, use of fetal calf serum in cell cultures became unusual. But
MSC in culture systems need cytokines, which are contained in FCS. So far most
of them are unidentified, so we can not adjoin them to culture media in perfect
combination and concentration.
Many experts switched to platelet lysate as MSC culture supplement. Mostly they use
autologous platelet preparations or single donor apheresis platelet concentrates.
In several experimental studies we tried to discover, how different or equal
cytokine concentration in miscellaneous individuals is. Can we get standardised
culture conditions, if we use these platelet preparations?
Material and Method: We tested cytokine concentration in an enzyme linked
immunosorbent assay. We performed ELISA for PDGF, VEGF, FGF and EGF.
At first we tested specimen from eight platelet concentrates on day 5 after
That’s why we pooled 20 platelet concentrates in order to minimise differences.
Unfortunately ranges for PDGF and EGF levels remained great.
So we postulate that pooling of high numbers (up to 50) of preparation is
necessary to obtain standardised MSC culture conditions. A defined thrombocyte
count (1.200/µl) in products should be maintained.
179
BIOREACTOR CULTURE OF CD34+ CELLS FOR PLATELET
PRODUCTION; NEW SCAFFOLDS TO SUPPORT MEGAKARYOCYTE
DEVELOPMENT.
Pankaj Godara,2 Toby Brown,3 Eun-ju Lee,2 Kellie Cartledge,2 Andrew Vinson,2 Cheang Ly Be,2
Dietmar Hutmacher,3 David Haylock, 1 1CSIRO, Melbourne, Australia, 2CSIRO, Clayton,
Victoria, Australia, 3Queensland University of Technology, Brisbane, Australia.
Our two-chamber perfusion based microbioreactor for megakaryocyte
differentiation and platelet production is inspired by and mimics the critical
interaction between mature megakaryocytes and endothelial cells within the bone
marrow. The microbioreactor, constructed from polydimethylsiloxane aims to
replicate these features. Two chambers are separated by a membrane or scaffold
and independently supplied with media via syringe pumps. Cells are inoculated
into the upper chamber and depending on membrane properties are either
restricted to this space or able to extend proplatelets into the lower chamber.
Growth factor-dependent haemopoietic cells grow to a density of 1 x 10e8/mL
when supported by polyurethane and polyethylene terephthalate membranes.
To mimic the endothelial layer that supports megakaryocyte maturation
and proplatelet formation in vivo we tested a range of electrospun and melt
electrospun scaffolds fabricated from nylon, polycaprolactone and polystyrene.
Melt electrospinning is an excellent method as it provides control over fibre
size, inter-fibre distance and the architecture and topology of the final mesh. A
range of topologies are being evaluated for their ability to support megakaryocyte
maturation and platelet release. In addition, we have developed the ability to
covalently immobilise haemopoietic cytokines to these meshes through a variety
of chemistries. In this respect, c-terminal FGE-tagged stem cell factor has been
purified and immobilised via oxyamine-aldehyde chemistry. Prior to commencing
bioreactor culture of cord blood CD34+ cells, a robust static culture system for
megakaryocyte production using serum deprived media supplemented with SCF,
TPO, IL-6 and IL-9 was established. Over 14 days culture a 107-fold increase in
cell numbers with 87% being CD41a+ (80.3-95.7%, n=15) was observed. Hoechst
staining demonstrated that CD41+ cells were 4N, 8N, 16N and occasionally 32N.
Initial microbioreactor experiments with melt electrospun scaffolds have just
55
POSTER ABSTRACTS
commenced with cord blood CD34+ cells. We believe this culture approach will
lead to generation of large numbers of megakaryocytes and functional platelets.
182
180
IMPLICATIONS OF LOCAL WOUND ENVIRONMENT FOR CELLBASED THERAPY
Synthetic Surface for Culture of Human Keratinocytes
in Defined Xeno-free Medium
E. Ludwig, 1 N. Porterfield,1 J. Forsberg,1,2 E. Elster,1,2 D. Tadaki,1 T. S. Brown,1 1Naval Medical
Research Center, Silver Spring, MD, USA, 2Walter Reed National Military Medical Center,
Bethesda, MD, USA.
Kerry Thompson, Jeff Partridge, Elizabeth Abraham, Paula Flaherty, Susan Qian, Deepa
Saxena, BD Biosciences, Bedford, USA.
Keratinocytes are the most common cell type of the skin constituting ~95%
of the cells. The most important application of keratinocytes is in creating
epithelial sheets for skin grafting on to patients suffering with wounds and
burns. Keratinocyte accessibility, proliferation potential and ease of culture
has enabled use of these cells in regenerative medicine. Ex vivo expansion of
keratinocytes requires either coating of the culture vessel with human or animalderived extracellular matrix protein or a growth medium with bovine serum or
animal-derived components. Growing concerns about introducing human and
animal-derived pathogens into the culture necessitate the need for an animal free
(defined as xeno-free and human origin components-free) culture environment.
Also, media components and coating matrices of biological origin may have
batch to batch variability and can be undefined. Self-coating requires additional
time resulting in coated vessels having a limited shelf-life. Here, we report BD
PureCoatTM Collagen I peptide surface, a synthetic peptide coated surface for
culture of cell types that require Collagen I coating. BD PureCoat Collagen I peptide
surface is a pre-coated, synthetic, xeno-free, human origin components-free, and
room temperature stable surface. In this study human keratinocyte cells were
cultured on the peptide surface for multiple passages in a defined and xeno-free
media. Cell growth and morphology were comparable to cells grown on Collagen
I coated surface. Functionality of the cells was tested following multiple passages
using an in vitro wound healing model. Keratinocytes were division arrested and
a scratch was made on the monolayer. Cells migrated to the scratched region,
healing the wound. BD PureCoat Collagen I peptide surface provides a ready
to use alternative to Collagen I coating for cell culture with comparable cell
attachment and functionality.
181
OPTIMISATION STRATEGIES OF STEM CELL “NICHE” USING
SYNTHETIC BIO-POLYMERS AND HYDROGELS FOR TISSUE
ENGINEERING
Lakshmi Kiran Chelluri, Tanya Debnath, Research Assistant, Usha P, Research Assistant,
Subbaiah GPV, Chief Spine Surgeon, Ravindranath K, Chief Surgical Gastroenterologist,
Ratnakar S K, Director,Research, Global Hospitals, Hyderabad, India.
Synthetic bio-polymers are an emerging and alternative source to xenogenic-free
derived ancillary materials for tissue engineering. It is critical to evaluate these
polymers in a simulated stem cell microenvironment for optimal benefit and use
in translating the tissue constructs in routine clinical practice. The study addresses
the effects of these synthetic polymers and hydrogels on stem cell niche in terms
of genotoxicity, viability, functionality, growth, and apoptosis. Cumulative data
suggests that these synthetic biomaterials are well tolerated and has subliminal
impact on the study parameters. Hence,during the study period, it can concluded
that it is safe to utilize these materials in conjunction with stem cells in designing
new engineered tissue constructs.
56
Mesenchymal stem cells (MSC) are the progenitor cells of osteoblasts,
chondrocytes, and adipocytes and have been demonstrated to differentiate
into myocytes and neurons. Thus, MSC have been a focus of cell therapies for
wound healing. However, research in this field has commonly been conducted
with sterile, surgical wounds in the absence of a systemic inflammatory response.
Traumatic wounds with a concomitant systemic inflammatory response, such as
those resulting from explosions, often exhibit additional complications, such as
bacterial colonization, presence of foreign bodies, multiple organ involvement,
or traumatic limb amputation. Such wounds of combat casualties have exhibited
a high rate of bone development in soft tissues (heterotopic ossification; HO)
leading to a reduction in patients’ quality of life. Our research investigates the
microenvironment of traumatic wounds associated with HO development in a
background of a systemic inflammatory response.
Operation Iraqi Freedom and Operation Enduring Freedom service members with
traumatic extremity injuries were treated with negative pressure wound therapy
(NPWT). Effluent collected from the NPWT canister was analyzed for 32 cytokines
and chemokines (multiplex bead-based assay) at each surgical debridement.
Additionally, tissue biopsies obtained from the wound bed were analyzed for 190
inflammatory and wound-healing genes (qPCR).
Genes with a substantial differential expression between wounds that developed
HO and those that did not included bone morphogenic proteins (GDF5 (p=0.007),
BMP3, BMP8B), fibroblast growth factors (FGF5 (p=0.041)), cytokines (CCL17,
CXCL10, CSF2), and matrix metalloproteinases (MMP1, MMP12).
Although the specific signaling pathway underlying the formation of heterotopic
ossification is presently unknown, we have further refined the activation of bonegrowth associated pathways, in the background of an inflammatory response
profile, within the local wound environment. Caution should be taken when
developing cell-based therapies utilizing MSCs. Cytokine expression within
traumatic injuries may direct multipotent progenitor-cell differentiation in
unintended ways, not anticipated from sterile surgical models.
183
Microporous Biphasic Calcium Phosphate granules
(MBCP+®) retain immunological properties of bone
marrow-derived mesenchymal stromal cells and
promote osteoblastic differentiation
Giulio Bassi, 1 Fabien Guilloton,2 Luciano Pacelli,1 Roberta Carusone,1 Cedric Ménard,3
Francesco Bifari,1 Luc Sensebé,2 Frederic Deschaseaux,2 Serge Baroth,4 Hubert
Schrezenmeier,5 Pierre Layrolle,6 Karin Tarte,3 Mauro Krampera,1 1Stem Cell Research
Laboratory, Section of Hematology, Department of Medicine, University of Verona, Verona,
Italy, 2EFS Pyrénées Méditerranée - Université Paul Sabatier UMR5273 - Inserm U1031,
Toulouse, France, 3INSERM U917 Faculté de médecine, Rennes, France, 4Biomatlante SA,
Vigneux de Bretagne, France, 5Institute of Transfusion Medicine, University of Ulm and
German Red Cross Blood Donor Service Baden-Württemberg, Hessen, Ulm, Germany,
6
Inserm U957, LPRO, Faculté de Médecine, Nantes, France.
Bone is among the most frequently transplanted tissue with about 1 million
procedures annually in Europe. Despite their considerable disadvantages, allografts
and autografts account for more than 80% of total graft volume. Significant growth
opportunities exist for synthetic bone grafts in association with mesenchymal
stromal cells (MSC) from autologous or allogeneic sources as alternatives to
biological bone grafts in orthopaedic and maxillofacial surgery. The objective of
REBORNE is to perform five phase I clinical trials using advanced biomaterials and
cells triggering bone healing in patients. REBORNE immunological unit assess the
POSTER ABSTRACTS
MSC immunomodulatory properties in the presence of the biomaterial (MBCP+®,
Biomatlante) used as scaffold for MSC delivery.
We found that primed MSC, pre-treated with the inflammatory cytokines IFNγ and
TNFα, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression
of HLA-DR, both in standard culture conditions and in association with MBCP+®.
Immune effector cells could be cultured and collected even in presence of the
biomaterial and no significant differences were found between standard coculture
conditions and 3D-coculture conditions, in terms of proliferation of immune
effector cells. In particular, MSCs suppressed T and NK cell proliferation, even in
presence of MBCP+®, and this effect was stronger after priming with inflammatory
cytokines. In contrast, B cell proliferation was inhibited only in coculture with
primed MSCs, with slight differences related to the culture system.
BMP4 was better capable than dexamethasone to induce MSC differentiation into
osteoblast-like cells, as confirmed by RT-qPCR analysis. Moreover, MBCP+® was
more efficient in increasing osteoblastic differention as compared to standard
culture conditions, demonstrated by the higher expression of Osteocalcin and
Osterix.
These data show that the association of MBCP+® and MSC does not affect MSC
properties and suggest that it could be a treatment of choice for bone regeneration
instead of allograft and autograft transplantation.
184
Will not be presented
185
Standardized processing of platelet lysate for use in
cell culture
Karin Plöderl, 1 Katharina Höller,1 Anja Peterbauer-Scherb,1 Simone HennerbichlerLugscheider,1 Christian Gabriel,1 1Red Cross Blood Transfusion Service for Upper Austria,
Linz, Austria.
Background: Consistent and reproducible production is one important issue
among many others required by law. In cell cultures, especially for cell therapy
and tissue engineering, a major component is fetal calf serum (FCS). Due to
its animal origin and ethical concerns about its collection, FCS shall be avoided
in general. Additionally, the exact content of FCS is not really known and there
are big batch to batch variations concerning its quality. Several alternatives
are already known and mentioned in literature. Among these alternatives, one
can find human platelet lysate (hPL). hPL is obtained from whole blood and is
known for its high growth factor concentrations. Since 2007 the Red Cross Blood
Transfusion Service for Upper Austria is concerned with the production of hPL.
To guarantee GMP conformity and a standardized production process of hPL,
several studies were already performed.
Methods: A pool of six buffy coats is centrifuged leading to a separation of
platelet rich plasma and erythrocytes as well as volume reduction for increased
platelet concentrations. In order to enable a standardized processing, the manual
procedure of fractionation was compared to the Compomat G4 (Fresenius,
Austria). For comparison, platelet concentration and platelet activation were
determined. Additionally, growth factor analysis was performed.
Results: A slight decrease in platelet activation was observed in the samples taken
from the products processed using the automated procedure. Higher yields in
platelet numbers could be achieved in comparison to the manual processing.
Further data from residual white and red blood cell counts and results obtained
by growth factor analysis will be presented.
186
ROLE OF THYMOSIN β4 ON SKELETAL MYOBLAST MIGRATION,
PROLIFERATION, AND SURVIVAL
Lei Ye, MD, PhD1 Li-Ping Su,1 Wei-Feng Pi, MD, PhD2 Peter K. Law, PhD3,4 1National
University of Singapore, Singapore, Singapore, 2Xinhua Hospital, Shanghai, China,
3
Huazhong University of Science & Technology, WuHan, China, 4Cell Therapy Institute,
WuHan, China.
Background and Aims: Massive cell death associated with poor donor cell
survival was a limiting factor in the success of myoblast transfer therapy (MTT).
The current studyaimed to determine the role of thymosin β4 on human skeletal
myoblasts (hSkMs) migration, proliferation and survival under hypoxia.
Methods: hSkM was cultured in basal medium (BM, M199 medium with 10%
fetal bovine serum) supplemented with various concentrations of thymosin β4.
Supernatant was collected to test the toxicity of thymosin β4 towards hSkM. Cell
number was quantified using CyQuant cell proliferation assay kit. Cell viability was
determined by calculating the lactate dehydrogenase (LDH) in the supernatant.
hSkM migration was determined using cell culture insert.
Results: No significant cyto-toxicity of thymosin β4 towards hSkM was found when
thymosin β4 was increased up to 600 ng/ml in cell culture medium. Thymosin
β4 increased hSkM proliferation rate by 35.4±13.4% at 600 ng/ml concentration.
It enhanced hSkM viability (cell injury =10.9±1.7%) as compared with control
(cell injury =24.6%, p<0.05) under hypoxia (5% CO2+ 94% N2+1% O2) for 48
hours. Increased hKsM migration rate (164.5±15/well, p<0.05 vs control) was
achieved with thymosin β4 at 100 ng/ml concentration. Thymosin β4 increased
the activities of PI3K and AKT, and reduced the activities of caspases -3 and -8 of
hSkMs.
Conclusions: We conclude that thymosin β4 increased hSkM migration and
proliferation. It enhanced hSkM viability under hypoxia. The current study
provides a new strategy to enhance hSkM viability and improve survival duing
myoblast transfer therapy.
187
ISOLATION OF MESENCHYMAL STEM CELLS FROM UMBILICAL
CORD TISSUE (TC-MSC) PRIOR TO CRYOPRESERVATION
RESULTS IN AN 8 FOLD INCREASE IN AVERAGE VIABLE TC-MSC
RECOVERED WHEN COMPARED WITH THE ISOLATION AND
RECOVERY OF TC-MSC AFTER THAWING CRYOPRESERVED
UMBILICAL CORD TISSUE SEGMENTS
Robert Briddell, 1 Frank Litkenhaus,1 Gail Foertsch,1 Angela Fuhrmann,1 Karen Foster,1 Kate
Falcon Gerard,2 Bruce Fiscus,1 Ann Boehm,1 Mystie Pettit,1 Asimena Rigas Bridges,1 Karen
Nichols,2 William Fodor,3 Morey Kraus,2 1ViaCord LLC, a PerkinElmer Company, Hebron,
KY, USA, 2ViaCord LLC, a PerkinElmer Company, Cambridge, MA, USA, 3Cell Therapy Group,
Madison, CT, USA.
Background. The human umbilical cord (hUC) is comprised of gelatinous
connective tissue that contains mesenchymal stem cells (TC-MSC), proteoglycans
and collagen. Isolation and cryopreservation of TC-MSC for potential therapeutic
use is an active area of research in the stem cell banking industry. We have
compared two methods of cryopreservation to determine the optimal method for
storing TC-MSC: (1) cryopreservation of TC-MSC isolated from fresh hUC tissue;
and, (2) cryopreservation of intact hUC tissue followed by TC-MSC isolation.
Methods. Parallel hUC tissue segments from the same hUC were processed by
one of the two methods. Method 1: TC-MSCs were isolated from fresh hUC tissue
by overnight collagenese digestion (2.5mg/mL-g), filtration and centrifugation.
Isolated TC-MSCs from this process were cryopreserved in 11% DMSO, 1%
Dextran 40, 4% HSA in 0.9% NaCl, frozen and stored at -80oC for < 30 days.
Cells were thawed at 37oC, washed and resuspended in D-PBS for flow cytometric
analysis. Method 2: hUC tissue was cryopreserved, frozen and stored in the same
manner as in Method 1. Frozen hUC tissue was thawed at 37oC and processed as
57
POSTER ABSTRACTS
described above to isolate the TC-MSC. TC-MSC recovered from each procedure
were enumerated and phenotyped by flow cytometry. Results. The recovery of
viable CD45-/CD90+ and CD45-/CD105+ TC-MSC (after thawing) from fresh hUC
tissue (Method 1), on average, resulted in 4.61x105 and 1.33x105 cells/gram tissue
respectively (n=15), whereas the yield of CD45-/CD90+ and CD45-/CD105+ TCMSC from cryopreserved/thawed hUC tissue (Method 2), on average, resulted in
7.00x104 and 2.40x104 cells/gram of tissue respectively (n=15). Cell viabilities from
both processes were comparable. Conclusions. Access to cell therapies using TCMSC will depend greatly on the availability of high quality TC-MSC preparations
that contain maximal cell numbers in ready to use pharmaceutical compositions.
With this study we demonstrate significantly higher (p<0.001) viable cell
recoveries when TC-MSC were harvested prior to cryopreservation.
Stem Cells 2010). Our group developed the first off-the-shelf (OTS) bank where
CBMSC are produced in GMP conditions, and now available as “a drug”, to be
distributed for patient doses, much like an off-the-shelf pharmaceutical product.
CBMSC were manufactured through a multi-step controlled, well-validated
process. A quality control approach has been designed to ensure that controls
are completed satisfactorily during manufacturing operations. In fact, CBMSC
were qualified according to pre-established criteria to ensure a consistent, wellcharacterized product. The same MSC batches can be provided for pre-clinical
testing and for further validations to prepare the ad-hoc investigational medicinal
product dossier (IMPD). Now these GMP batches of CBMSC, together with GMP
adipose and bone marrow MSC are available in our OTS bank in different cell
doses to comply any kind of clinical needs.
188
190
ENGINEERED COMPOSITE SKIN TO TREAT BURNS: A PILOT STUDY
Michelle Paul, Pritinder Kaur, Heather Cleland, Shiva Akbarzadeh,
Alfred Health,
Melbourne, Australia, 2Monash University, Melbourne, Australia, 3Peter MacCallum Cancer
Centre, Melbourne, Australia.
1
3
1
1,2
1
The main practice for treating patients with extensive burns in our unit is a two
step grafting procedure with cadaver skin (allograft) to replace the injured dermis,
and secondary grafting with thin split thickness autografts. Cultured keratinocytes
have the potential to replace autologous split skin grafts; however they are fragile
and susceptible to infection when used in contaminated wound beds. Allografts
are often in short supply and may expose the patient to added risks such as
disease transmission. As cultured keratinocytes require signals from the dermis
to proliferate and make functional skin, our goal is to develop a technique for
growing patients’ keratinocytes on a carrier that supports their growth when
grafted and can be used in a one-stage autografting.
Here we will present our preliminary data on adult skin organotypic cultures by
comparing the capacity of commercially available dermal substitutes (Integra®,
Pelnac® and Allografts) to take up and expand epithelial cells. Dermal fibroblasts
are seeded onto the matrix to support the epithelial cells growth in culture. The
skin morphology of the organotypic cultures is analysed histologically on days
7-10. Previous studies have tested seeding the artificial skin substitutes or deepidermised acellular dermis with epithelial cells and/or dermal fibroblasts before
grafting into animal models. However, these studies have used very limited number
of animals for each experimental group of seeded vs. un-seeded controls (i.e. n=2),
or they have only looked at short-term regeneration capability of the graft (i.e. 2
weeks). None has proceeded to the clinics.
The organotypic cultures that have better normal skin morphology will be grafted
on nude/SCID mice for functional studies. For a graft to maintain its long term
regeneration capability requires an adequate number of stem cells. Skin stem
cells can be identified using cell surface markers. The number of stem cells in
grafts will be compared to normal skin using these markers.
189
OFF-THE-SHELF CORD BLOOD, ADIPOSE AND BONE MARROW
MESENCHYMAL STEM CELL BANK
Tiziana Montemurro, Elisa Montelatici, Cristiana Lavazza, Barbara Baluce, Mariele Vigano’,
Valentina Parazzi, Enrico Ragni, Mario Barilani, Valentina Boldrin, Silvia Budelli, Gabriella
Spaltro, Paolo Rebulla, Rosaria Giordano, Lorenza Lazzari, Cell Factory, Fondazione IRCCS
Ca’ Granda Ospedale Maggiore Policlinico, Milano, Italy.
Our hospital-based GMP facility, the first one authorized in Italy by the National
Drug Agency in 2007, has pioneered innovative technologies in order to supply
clinical programs. Cord blood (CB) mesenchymal stem cells (MSC) have been
already tested both in vitro and in pre-clinical animal models, demonstrating that
CBMSC are safe, effective and the best candidate product for several clinical trials
including neurologic (Zanier EM, CritCareMed. 2011) and renal repair (Morigi M,
58
USE OF A BONE SCAFFOLD COMBINED WITH MESENCHYMAL
STEM CELLS FOR THE TREATMENT OF VERTEBRAL BODY
DEFECTS
Vaclav Vanecek, 1 Karel Klima,2 Ales Kohout,3 Ondrej Jirousek,4 Lubica Stankova,1 Jan Stulik,5
Eva Sykova,1 1Institute of Experimental Medicine, Academy of Sciences of the Czech
Republic, Prague, Czech Republic, 2Division of Oral and Maxillofacial Surgery, Department of
Stomatology, First Faculty of Medicine, Charles University and General University Hospital,
Prague, Czech Republic, 3The Fingerland Department of Pathology, Charles University
Medical Faculty and Teaching Hospital in Hradec Kralove, Hradec Kralove, Czech Republic,
4
Institute of Theoretical and Applied Mechanics, Academy of Sciences of the Czech Republic,
Prague, Czech Republic, 5Center for Spinal Surgery, Charles University, Second Medical
Faculty and University Hospital Motol, Prague, Czech Republic.
Introduction: Vertebral body fractures are one of the most common orthopedic
disorders. Effective permanent repair of the vertebral body can be achieved using
an autologous bone graft, the amount of which is usually limited for transplantation
and the harvest is associated with donor site morbidity. Mesenchymal stem
cells (MSC) combined with hydroxyapatite scaffolds were shown to promote
osteoinductivity and bone healing in various experimental bone defects. However,
the specific biomechanical properties of vertebrae do not allow generalizing the
results from other types of bones to those of vertebral body defects treated with
tissue-engineered stem cell-based grafts. Therefore, we evaluated the safety
and efficacy of a hydroxyapatite scaffold combined with human MSC in a rat
model of vertebral body defects. Methods: MSC were isolated from the bone
marrow of healthy donors, then cultivated, expanded and characterized under
Good Manufacturing Practice guidelines. Vertebral body defects (1.5x5x1.5mm)
were created in 32 Wistar rats and either left empty (control) or transplanted
with hydroxyapatite scaffold (CEM-OSTETIC®), alone or loaded with 0.5 million
or 5 million MSC. After 8 weeks the animals were sacrificed and the vertebrae
dissected and analyzed. Results: The cells’ viability and attachment to the
scaffold material in-vitro were confirmed by fluorescent microscopy. Cells were
able to differentiate into adipocytes, chondrocytes and osteocytes. Histological
analysis and histomorphometry showed significant bone formation in the group
transplanted with 5 million MSC in comparison to a scaffold alone or loaded
with 0.5 million MSC. There was no sign of inflammation or tumor formation
in any group. Micro-CT analysis did not reveal any substantial deformation of
the vertebral body. Conclusions: Our results show that a hydroxyapatite scaffold
loaded with MSC may represent a safe and effective alternative for vertebral body
repair. Acknowledgement: Supported by P304/10/0320, AVOZ 503905703.
POSTER ABSTRACTS
191
Autologous bone marrow derived stem cell graft
facilitates remodeling of non union fractures
Venkatesh Ponemone, PhD1 Roma Gulati, MBBS2 Jyotsna Sharma, MSc3 Mitchel Sivilotti,
MSc2 Amar Sarin, MS4 Tankeswar Boruaht, MS5 Mandeep Singh, MS5 Rakesh Mattoo,6 Alok
Sharma, MS, MCh6 Gurinder Bedi, MS, FRCS5 Nitiraj Oberoi, MS, FRCS5 Harshvardhan
K. Hegde, MS5 Kenneth Harris, MS1 1TotipotentRX Corporation, Los Angeles, USA,
2
TotipotentSC, Gurgaon, India, 3TotipotentRX Centre for Cellular Medicine, Gurgaon, India,
4
BL Kapur Hospital, New Delhi, India, 5Fortis Flt. Lt. Rajan Dhall Hospital, New Delhi, India,
6
Fortis Hospital, Noida, India.
Objective: Bone marrow derived mononuclear cells (BMMNCs) have the advantage
of promoting both angiogenesis and osteogenesis. Bone marrow progenitor
cells are used to obtain bone healing of non-unions by 3 basic mechanisms of
bone regeneration i.e. osteogenesis, osteoinduction and osteoconduction. The
objective of the study is to evaluate the bone remodeling capability of autologous
BMMNCs for the treatment of non-unions.
Methodology: There were 19 patients, of which 2 got dropped out due to non
availability for the follow up. 17 patients with 17 atrophic non-unions (6 femur, 6
tibia, 2 humerus, 2 radius ulna and 1 scaphoid) available for study. The patients
included 9 males and 8 females with an average age of 43 years, . Patients who
had failed to unite following prior primary fixation were selected for the study.
Following standard BM collection procedure using heparin as the anti-coagulant
from posterior or anterior superior iliac crest, 60 ml of BM was aspirated and
processed using a U.S. FDA and India Drug Controller General approved intraoperative point of care (POC) technology to obtain 6 ml bone marrow concentrate
(BMC) rich in BMMNCs. In all cases BMC containing progenitor cells were
preconditioned with beta-tricalcium phosphate before implanting at the fracture
site during ORIF procedure except a single case of percutaneous injection.
Results: The aspirate contained an average BMMNC cell dose of 1.3 x 108 + 0.15
SEM. Confirmation of bone union was obtained in 12 patients with a union rate
of 71% between 16-18 wks time interval. The time for callus formation ranged
between 12-16 wks.
Conclusion: Transplantation of BMMNC may be a beneficial treatment for bone
repair in non-union fractures. Administration of BMMNCs may offer the surgeons
and patients a new therapeutic option when treating troublesome non-union
fractures.
192
Therapeutic angiogenesis - Pre-clinical and
manufacturing aspects
Alex Slobodianski, 1 Martin Hildebrandt,1 Astrid Kathöfer,2 Jessica Frenz,2 Kathrin Klutz,1
Hans-Günther Machens,1 1Klinikum rechts der Isar, Technische Universität München,
München, Germany, 2University Hospital of Schleswig-Holstein, Lübeck, Germany.
Background: Advanced Therapy Medicinal Products (ATMP) are recognized as a
rapidly growing area in translational research, with the aim of providing complex
medicines for complex diseases. The manufacture of ATMPs, i.e. cell-based
medicinal products, tissue-engineered products and gene transfer medicinal
products, challenges conventional concepts of drug manufacture for clinical use
and necessitates novel technological, methodological and regulatory approaches
to comply with standards of Good Manufacturing Practice (GMP), especially in
Academia where GMP knowledge is traditionally scarce.
angiogenesis” as a basis. As an approach to “therapeutic angiogenesis”, we
developed a cell-based, non-viral gene-transfer medicinal product using fibroblasts
to produce bFGF and VEGF165 temporarily, as a form of pharmacological local
preconditioning before tissue ischemia occurs.
We considered different essential aspects for production and purification of the
cell-based gene transfer medicinal product, and we identified critical elements for
technology transfer to a GMP Facility. We specified risk factors of manufacturing
of gene therapeutics and discussed them with public authorities and regulatory
experts.
Conclusions: The development of the GTMP described here may be understood as
aa conceptual framework for technology transfer of gene therapeutics production
to a GMP Facility and for the performance of pre-clinical trials, providing useful
information for other research groups Furthermore, we established techniques
that allow to recognize potential risks of gene therapeutics and to predict potential
adverse effects of Therapeutic angiogenesis.
193
Standardization of Immunosuppressive potency tests
for mesenchymal stromal cell - based therapies
Ana Valeria Gouveia de Andrade, 1,2 Marc Schmitz,1,3 Marcus Odendahl,4 Martin Bornhäuser,1,5
Torsten Tonn,1,4,6 1Center for Regenerative Therapies Dresden, Dresden, Germany, 2German
Red Cross Blood Donor Service East, Dresden , Germany, 3Institute of Immunology, Medical
Faculty, Technical University of Dresden, Dresden , Germany, 4German Red Cross Blood
Donor Service East, Dresden, Germany, 5Department of Medicine I, University Hospital of
Dresden, Dresden, Germany, 6Experimental Transfusion Medicine, Medical Faculty, Technical
University of Dresden, Dresden, Germany.
Introduction: Mesenchymal stromal cells (MSC) are emerging as novel treatment
modality for a variety of diseases, among them GvHD and autoimmune diseases.
Validated potency tests to assess immunosuppressive functionality to define
release criteria are warranted. However, results of the mixed lymphocyte reactions
(MLR), when using different donor sources of stimulator and/or responder cells,
may mount heterogeneous results. Therefore, we aimed to standardize the MLRbased assay using a pool of stimulator cells and a large aliquot of responder cells,
as well as time point and stimulator/responder ratio to allow reproducible results.
Methods: Bone marrow-derived MSCs (BM-MSCs) were isolated from healthy
donors after informed consent. To analyze the immunosuppressive potential of
BM-MSCs, a lymphocyte proliferation assay (LPA) with anti-CD3/CD28 beads as
stimulators and the MLR (1:1 ratio, in 96-well plates), using a pool of stimulator
cells (n=9) and responder cells (n=1) were studied. Proliferation of responder
cells was measured by 3H-Thymidine incorporation (18hrs) on days 5, 6, 7 (MLR)
and on day 6 (LPA), respectively.
Results: Among all variables tested, the MLR mounted the highest proliferation
on day 6 at a stimulator/responder ratio of (105/105) with a mean of 37,5 x103
(±8,1x103) corrected counts per minute (CCPM). The LPA was significantly more
potent, mounting a mean of 105,3 x103(±3,4x103) CCPM (105/well). As few as 5000
BM-MSCs/well were sufficient to significantly inhibit proliferation of responder
cells in either system with a mean inhibition of 77,7% (±3,3%) for the MLR (n=7)
compared to 70,2% (±8,4%) for the LPA (n=6), which was statistically significant
(p<0.0001).
Conclusions: Standardization of MLR and LPA is able to yield reproducible results
of the immunosuppressive functionality of MSC. Whereas the LPA assay may be
preferable for its ease of usage, the MLR appears to be more sensitive.
We describe our approach taken for the development of a gene transfer medicinal
product (GTMP), in an attempt to provide insight in the developmental process,
especially the pre-clinical studies, the interaction with the regulatory authorities and
the implementation of the process in a GMP environment. In addition, we discuss
the steps taken to embed the GTMP into the framework of a Clinical trial.
Results: In the project presented here, we used the method of “therapeutic
59
POSTER ABSTRACTS
194
Development of a new device for point-of-care
thawing and dosing of cell therapy products
Arnaud Foussat, 1 Nathalie Belmonte,1 Virginie Neveu,1 Gael Peron,2 Dominic Clarke,2 1TxCell,
Valbonne Sophia-Antipolis, France, 2Charter Medical Ltd, Winston-Salem, NC, USA.
Ovasave is a cell-based product consisting of autologous antigen-specific
regulatory T cells. A phase I/II clinical trial has been performed in Crohn’s Disease
and showed good tolerability and positive signs of efficacy. Due to limited shelf
life of T-lymphocytes in classical infusion buffers, Ovasave was frozen and needs
to be thawed, washed from cryoprotectants and adjusted at the required cell
doses for different patients cohorts. Due to regulatory constraints and to the fact
that these procedures included open steps, they were performed in accredited Cell
Therapy Units (CTU) distant from the point-of-care. For an open, uncontrolled
phase I/II study with 6 French centers, this continuation of events was acceptable
but for further clinical development, a different method for cell procurement to
the patient seems mandatory.
Bearing in mind the constraints of clinical development (keeping the blind for
physicians and patients as well as for the sponsor/manufacturer), we developed
a new device for on-site Ovasave thawing and dosing. This device is a sterile,
disposable, full closed system composed of two bags, prefilled with dilution/
infusion buffer that are connected with a syringe (see figure) (Engineered and
manufactured by Charter Medical, Ltd). Immediately after thawing, Ovasave
cells are harvested in the first bag allowing dilution of cryoprotectant. Then, the
required number of cells for patient administration is transferred from the first
to the second bag using the syringe. The second bag can be disconnected from
the kit and transported to patient’s bed for infusion. Importantly, performance
studies have shown that Ovasave stability and product characteristics were not
impacted by the whole procedure.
This device, beyond the use in Ovasave clinical development, is suitable for rapid
(less than 10 minutes), sterile and on-site dosing of any thawed cell therapy
products and provides a bench to bedside solution for clinical phase of cell
therapy development.
to visualize administration of human HSC into rodent model via two routes of
administration. Intramuscular injection of a cell suspension resulted in a rapid
dispersal of cells, resulting in a negligible signal at the injection site, accounting
for less than 5% of the administered cells. Following delivery of CD34+ HSC within
a scaffold, MR imaging revealed the retention of cells at the site of administration.
The non-invasive visual specificity of PFC labeled cells provided by MRI, paired with
the safety of the reagent in maintaining the cells’ therapeutic efficacy, as measured
through in vitro and in vivo correlates, demonstrate the potential of PFC technology
in cellular therapy and the study of cellular persistence.
196
CHARACTERIZATION OF PERIPHERAL BLOOD MONONUCLEAR
CELLS USED AS FEEDER CELLS IN EX-VIVO CLINICAL T CELL
REPLICATIONS
C. S. Abdul-Alim, Ph.D., Christopher E Shellooe, Jason N Carstens, Ph.D., Shelly Heimfeld,
Ph.D., Fred Hutcinson Cancer Research Center, Seattle, United States.
In recent years there has been a sharp increase in the number of clinical trials
involving adoptive T cell therapy for cancer. Adoptive T cell therapy involves
isolating tumor-specific T cells from the blood of cancer patients, expanding the
T cells ex-vivo and then infusing those cells back into the patients. The expansion
of T cells in culture- up to 10x109 for a single patient- is a time and labor intensive
process involving many steps and several poorly defined reagents. This process
frequently requires the use of so-called feeder cells, which generally consist of
irradiated peripheral blood mononuclear cells (PBMC)- generally obtained from
a third party donor- and lymphoblastoid cell lines (LCL). These cells allow for the
activation of the tumor-specific T cells through T cell receptor cross-linking and
CD28 co-stimulation, and presumably by providing cytokines and growth factors
that facilitate T cell replication. Indeed, the specific function of feeder cells remains
poorly understood. In this study we analyze the efficacy of feeder PBMC from 5
different donors for expanding clonal T cells ex-vivo. We find that PBMC efficacy
can vary widely between different donor lots, with some donor lots generating
up to 6-fold more tumor-specific T cell clones than other lots in a single Rapid
Expansion Procedure (REP). To better understand what cytokines are contributed
to REP cultures by the feeder cells, we tested for a host of cytokines using Luminex
bead technology. Here we find that the irradiated feeders introduce significant
quantities of IFN-g, IL-4, IL-5, IL-6, IL-10 and TNF-α. This suggests that different
sub-populations within the feeder cells play different roles in T cell expansion.
These results offer implications for further optimizing feeder cell layers used in
T cell REPs to improve the quality and quantity of tumor-specific T cells used in
adoptive cell therapy clinical trials.
197
195
HEMATOPOIETIC STEM CELL CHARACTERIZATION WITH 19F
TRACER AGENT; THE ABILITY TO EVALUATE CELLULAR PERSISTENCE
Brooke M. Helfer, 1 Anthony Balducci,2 Zhina Sadeghi,3 Adonis Hijaz,3 Chris A. Flask,3 Amy
Wesa,2 1Celsense, Inc, Pittsburgh PA , USA, 2Celsense, Inc, Pittsburgh PA, USA, 3Case
Western Reserve University, Cleveland OH, USA.
60
Hematopoietic stem cells (HSC) have numerous applications including
immune reconstitution, enzyme replacement, regenerative medicine and
immunomodulation. The trafficking and persistence of these cells after
administration is a question fundamental to the therapeutic applications of HSC.
Here we describe the labeling of human CD34+ HSC with a novel self-delivering
perfluorocarbon (PFC) emulsion. Due to minimal fluorine content in mammalian
tissues, administration of a fluorine label creates a precise image of the cells in
their anatomical context by pairing of the 19F MR image with conventional 1H MRI,
taken during the same MRI session. In a pilot xenograft study, 19F MRI was used
DEVELOPMENT TOWARDS THE VALIDATION OF A MULTICOLOR
FLOW CYTOMETRY ASSAY FOR CELLULAR PRODUCT RELEASE
Chris Wiwi, PhD, Kruti Shah, MS, Michelle McLaughlin, MS, Brian Murphy, PhD, Greg
Russotti, PhD, Celgene Cellular Therapeutics, Warren, New Jersey, USA.
Flow cytometry has broad utility in the discovery and development of cellular
therapeutics. The ability to simultaneously monitor the expression of several
cell-surface markers is a powerful tool in the characterization and manufacture
of cell therapy products. In contrast to more traditional analytical methods such
as HPLC or ELISA, unique challenges exist in the development and validation of
robust flow cytometric methods suitable for lot release. Among these obstacles is
inherent variability in cellular material, lack of true reference standards, complexity
of reagents and instrumentation, and operator subjectivity during data analysis.
Although there are few guidance documents available to assist scientists in the
development of flow cytometry methods, regulatory authorities advocate a fit-forpurpose approach in the validation of complex analytical methods. Toward this
goal, we have developed a multi-color flow cytometric method appropriate for use
POSTER ABSTRACTS
in lot release testing and as an in-process characterization tool for a novel placentalderived cellular therapy candidate. To minimize operator bias in data analysis,
software generated gating was selected in favor of manual gates. A combination
of cell and bead controls was used to demonstrate assay range, linearity, precision
and specificity. Risk-assessment exercises were carried out to select potential critical
robustness factors for further characterization. Subsequently, design of experiment
(DOE) exercises were performed to assist in assay optimization and to determine
operational ranges for critical assay robustness factors. Application of a fit-forpurpose approach was shown to be useful in the development of multi-color flow
cytometry methods. Further, a thorough evaluation of assay variables using DOE
facilitated assay optimization and identified appropriate assay acceptance criteria
for validation.
198
GMP IN A BOX: MANUFACTURE OF CELL THERAPY PRODUCTS
USING A BARRIER ISOLATOR FOR CLINICAL INVESTIGATIONS
Nicole Powell, Christopher Choi, Roswell Park Cancer Institute, Buffalo, USA.
Background aims. Manufacturing therapeutic cell products under cGMP
regulations for Phase I and II clinical trials is a challenge for the cell therapy
community. Currently, to perform Phase I and II clinical trials, a cleanroom
facility is recommended to ensure an aseptic manufacturing environment.
The high cost of building a fully operational GMP compliant cleanroom facility
makes performing Phase I and II clinical cell therapy trials cost prohibitive to
many institutes. Methods. To be able to manufacture cell therapy products in
a cost effective manner, we investigated and evaluated several Cell Processing
Barrier Isolator (CPBI) technologies as an alternative to the standard cleanroom
environment. To support our needs, we used multiple criteria such as cost,
adaptability, regulatory compliance and ability to integrate with common lab
equipment and 3rd party monitoring systems. After selecting a system, we
performed several cell manufacturing runs in support of our pre-clinical and
clinical cell-production activities. Results. The CPBI was used successfully to
manufacture high quality cell products in an aseptic manner, meeting set quality
control measures. Conclusions. The CPBI has met compliance requirements for
cell therapy manufacture in support of Phase I and II clinical trial activities for our
laboratory and can meet the needs of similar laboratories.
to increase culture capacity while minimizing volume. Culture conditions were
monitored using a blood gas analyzer and through spent media analysis in an effort
to optimize the manufacturing process.
200
TRACKING OF REPLICATIVE SENESCENCE BY EPIGENETIC
MODIFICATIONS at SPECIFIC CGP SITES
CM Koch, 1 S Joussen,1 K Shao,2 A Schellenberg,1 Q Lin,1 M Zenke,1 T Saric,2 W Wagner,1
1
Helmholtz-Institute for Biomedical Engineering, Stem Cell Biology and Cellular Engineering,
RWTH, Aachen University Medical School, Aachen, Germany, 2Institute for Neurophysiology,
Medical Center, University of Cologne, Cologne, Germany.
Cells in culture can only be expanded for a limited number of passages until
they enter senescence and stop proliferation. Furthermore, cellular aging has
fundamental implications on cell morphology, proliferation and differentiation
potential. Thus, it is crucial to take replicative senescence into account for quality
control of cellular therapeutics.
Here we describe a simple method to track cellular aging upon long-term
culture based on continuous DNA-methylation changes at six specific CpG
sites. This epigenetic signature can be used to predict the state of senescence
with respect to the passage number, population doublings or days of in vitro
culture. The average difference between real and predicted passage number was
3 passages in independent fibroblast and MSC samples. In analogy, cumulative
population doublings (cPD) and days in culture could be estimated by using the
corresponding linear regression analyses. We tested if the epigenetic senescence
signature is also applicable for various cell types and tissues: despite a wide range
of tissues, our method could clearly separate primary cells and cultured cell lines.
Subsequently, we analyzed the epigenetic senescence-signature with embryonic
stem cells (ESC) and induced pluripotent stem cells (iPS) which do not reveal any
signs of replicative senescence during culture expansion. Predictions for passage,
cPD or culture time of pluripotent cells were always close to freshly isolated cells.
Overall, long-term culture associated epigenetic modifications are specifically
reversed by reprogramming into a pluripotent state.
Our signature based on six CpG sites is universally applicable as biomarker for
cellular senescence in different tissues and can be used for quality control of
therapeutic cell preparations.
199
201
CHARACTERIZATION OF T-CELL GROWTH IN STATIC VS. AGITATED
AND FED-BATCH VS. PERFUSION CULTURE CONDITIONS
OKT3 AND CD3 PURE ANTIBODIES ARE BIOEQUIVALENT FOR THE
“EX-VIVO” GENERATION OF CYTOKINE-INDUCED KILLER CELLS
(CIK) FOR CLINICAL USE
Christopher E Shellooe, Jason N. Carstens, PhD., C. S. Abdul-Alim, PhD., Shelly Heimfeld,
PhD., Fred Hutchinson Cancer Research Center, Seattle, USA.
Adoptive T-cell immunotherapy products can be manufactured using a variety of
different cell culture systems, each of which has advantages and disadvantages.
Stationary culture conditions in flasks or bags provide some degree of
manufacturing simplicity, but quickly present fluid handling challenges upon scaleup to larger volumes or scale-out to expand cells for more patients in parallel. In
addition, these systems can require open manipulations that increase the potential
for contamination, as well as can present challenges with oxygen/carbon dioxide
transport. Agitated vessels can be more amenable to scale-up/out and will permit
a more homogenous environment with enhanced gas transport, at the expense of
more sophisticated processing equipment, potential damaging shear stress to the
cells, and the complication of still having to provide sufficient synaptic stimulation
through cell-to-cell contact or with CD3/CD28 antibodies. With these issues in
mind, T-cell growth performance was characterized using a variety of different
culture modes including static flasks and bags (T-flasks, Wilson-Wolf G-Rex flasks,
Baxter Lifecell bags, American Fluoroseal VueLife bags), and agitated vessels
(shaker flasks, WAVE bags, microbioreactors). Fed-batch and perfusion systems
were utilized to address nutrient limitation and waste metabolite accumulation
Cristina Zanon, Silvia Castegnaro, Katia Chieregato, Elena Albiero, Martina Bernardi,
Domenico Madeo, Francesco Rodeghiero, Giuseppe Astori, Department of Cell Therapy and
Haematology. Laboratory of Advanced Cellular Therapies.San Bortolo Hospital, Vicenza, Italy.
Introduction and Aims. CIK cells, a population of cytotoxic CD3+/CD56+ T
lymphocytes, have demonstrated a tumour response and/or a reduction of
relapse in patients with haematological and solid tumours both in vitro and in
clinical trials.CIK cells are generated stimulating nucleated cells (MNC) with IL2, interferon-gamma and anti-CD3-monoclonal-antibody.Good-ManufacturingPractice expansion requires the use of clinical grade reagents: the full process
must be validated ad specified in the Investigational-Medicinal-Product-Dossier
that is the basis for approval of clinical trials in the EU.
The ORTHOCLONE anti-CD3-monoclonal-antibody (OKT3.Janssen-Cilag) has
been widely employed for the generation of CIK cells in clinical trials. Recently it
has been withdrawn from the market forcing manufacturers to find a substitute
and to revalidate the CIK production process.We have identified the CD3-PURE
(Miltenyi-Biotec_Germany) as an alternative compound to be compared in term of
bioequivalence.Experiments were performed under Good-Laboratory-Practice.
Methods. MNC cells isolated from peripheral blood (n=3) were placed at 5x106
61
POSTER ABSTRACTS
cells/ml in X-VIVO 10. On day 0, cells were stimulated with 1.000U/ml of
interferon-gamma (IMUKIN.Boehringer-Ingelheim), on day 1 with 50ng/mL OKT3
or CD3-PURE and 500U/ml of IL-2 (PROLEUKIN.Novartis Pharma).Every 3 days,
cells were diluted at 1x106 cells/ml, fed with fresh medium and 500U/mL IL-2 until
day 21.At each passage, immunophenotypic analysis was performed staining cells
against CD4-FITC,CD56-PE,CD8-APC and CD3-PeCy7.The Population Doubling
(PD) and cumulative PD (cPD) were calculated.
Results. CIK cells were successfully obtained in both culture conditions and no
significant difference was observed in terms of expansion potential as evidenced
by cPD (Figure1A). Furthermore, the percentages of CD56+, expressed on cells
responsible for cytotoxic effect (NK and CIK), showed a similar trend in the
presence of OKT3 and CD3-PURE (Figure1B).
Discussion. The data presented here demonstrate the bioequivalence in vitro of
the two reagents, suggesting that CD3-PURE could replace OKT3 in the generation
of CIK cells for clinical use.
202
SUCCESSFUL TRANSITION FROM ISOLEX TO CLINIMACS DURING
A PHASE I CLINICAL TRIAL
Daniel Weber, 1 Ian B. Nicoud,1 Joseph M. Blake,1 Howard Voorhies,1 Colleen M. Delaney,1,2
1
Fred Hutchinson Cancer Research Center, Seattle, USA, 2University of Washington, Seattle,
USA.
Background: In early 2010, Baxter Healthcare’s discontinuation of reagents for
the only FDA-approved immunomagnetic selection device, the Isolex 300i, forced
those actively involved in clinical trials with this device to validate alternatives
or terminate their trials. Although not FDA-approved, the Miltenyi CliniMACS
is a clinical-grade immunomagnetic selection device available under an IND/
IDE. Herein we report our experience transitioning from the Isolex 300i to the
CliniMACS for selection of CD34+ cells from thawed cord blood units (CBU) for
ex vivo expansion, which we performed in the middle of a phase I clinical trial.
Methods: Previously frozen RBC-depleted CBU were thawed in a 37° water bath,
washed with 10% Dextran-40 + 5% HSA and exchanged into CliniMACS buffer
supplemented with MgCl2, Pulmozme™ and IVIG. Following incubation with
Miltenyi paramagnetic CD34 beads, CBU underwent CD34 selection on the
CliniMACS using large-scale (LS) tubing sets and the “CD34 selection 1” program.
Post-selection sampling was performed to evaluate CD34 purity by flow cytometry
and TNC/viability by manual trypan blue count. Cells were then cultured in serumfree medium supplemented with cytokines for 16 days.
Results/Conclusions: Results from 11 historical clinical products selected using
Isolex 300i demonstrated mean (±sem) CD34+ cell recovery of 32.6 ± 4.2%
with a corresponding purity of 51.6 ±3.9%. The results of 2 full-scale validation
runs and 4 clinical products using the CliniMACS system demonstrated a mean
CD34 recovery and purity of 49.7±4.9% and 82± 3.1%, respectively. Our results
demonstrate a statistically significant improvement in CD34+ cell recovery
(p=0.0233) and purity (p=0.0001) as compared to the Isolex 300i. The CliniMACS
device enabled continuation of our phase I clinical trial with equivalent or better
results for CD34 selection from cryopreserved CBU.
62
203
Human Mesenchymal Stem Cells: Identifying Aassays to
Predict Potency for Therapeutic Selection
Desirae Deskins, Pampee Young, MD, PhD, vanderbilt university, Nashville, USA.
Multipotent stromal cells (MSCs) have the potential to repair and regenerate
damaged tissues, making them attractive candidates for cell-based therapies for
multiple diseases and injuries. To maximize treatments using MSCs, prediction
of their therapeutic abilities must be made so that only the most efficient cells
will be employed. In this study we generated cell lines from ten normal human
bone marrow samples and used the ISCT’s minimal criteria to define them
as MSCs. Each MSC line was further characterized by its growth curve and
proliferation potential as determined by two independent evaluations (BrdU
incorporation assay and measurement of adenosine tri-phosphate levels). We
found no correlation between neither the age nor gender of the donors and the
MSC lines’ performance in the tests. To determine the efficacy of these tests to
predict the MSCs therapeutic aptitude, several lines were implanted in vivo to
examine their capacity to engraft and form granulation tissue in a well established
murine wound model using polyvinyl alcohol sponges. Long-term engraftment of
MSCs in the sponges was quantified through the presence of the human specific
Alu gene using real time PCR. In vivo success was also measured by histology
for the number of proliferating cells, amount of vascularization, and total
granulation tissue present in the sponge area. We found that high performance in
a combination of any two in vitro tests accurately predicted which lines functioned
well in vivo. These findings suggest that reliable and reproducible in vitro assays
may be used to measure the functional potential of MSCs. This may be of great
value in setting new guidelines and standards for determining lines of MSCs that
are optimal for therapeutic use.
204
DEVELOPMENT OF A CELLULAR THERAPY LABORATORY
MANUFACTURING PLATFORM FOR ADIPOSE DERIVED
MESENCHYMAL STEM CELLS
Douglas J Padley, Greg W Butler, Jarett M Anderson, Dennis A Gastineau, Allan B Dietz,
Mayo Clinic, Rochester, MN, USA.
MSCs are a promising treatment for many diseases. From a manufacturing
perspective, a process applicable across multiple protocols and diseases is
desirable. We have translated our processes for the growth of adipose tissue
(AT) derived mesenchymal stem cells (adMSC) from a pilot, single patient study
into a robust, laboratory manufacturing platform that is the basis of several
active and planned clinical trials. Initially, the protocol used AT obtained during
routine bariatric surgeries to isolate and grow adMSC in media containing fetal
bovine serum (FBS). We compared AT from surgery, abdominal needle biopsy,
and abdominal incision and found similar growth characteristics and phenotype.
To remove FBS from our platform we developed and commercialized a method
to obtain culture media supplement from human platelets. (PLTMax, Mill Creek
Lifesciences, Rochester, MN). We successfully substituted PLTMax for FBS in
adMSC culture and found this culture method equivalent in regards to MSC
phenotype but with improved growth kinetics and improved genetic stability1.
We have used this protocol to grow adMSC from 26 otherwise healthly patients
undergoing bariatric surgeries and from 23 patients with a variety of diseases
POSTER ABSTRACTS
including ALS, Crohn’s disease, Type 1 diabetes, and ovarian cancer. We saw no
consistent differences in growth kinetics or phenotype. We have successfully
cultured adMSC to more than 25 population doublings with no noticeable
changes. Typically, this protocol will yield 1 x 109 MSC in 3 weeks from a starting
product of 1-2 gms of AT. We have also developed and evaluated ancillary protocols
key to successful cell therapy including freezing, storage, post thaw stability, and
delivery protocols. This effort has led to a robust manufacturing platform useful
for the application of MSC for a variety of diseases. 1Crespo-Diaz, et al., Cell
Transplantation. 2011;20(6):797-811
205
Cell Concentration and Washing using Hollow
Fiber Filtration and ReadyCircuit Assemblies for Cell
Therapy Applications
Catherine Blake,1 Felecia Henderson,1 Kurt Forge,1 Tamara Fedczyna,1 Shannon Eaker,1
Archana Thakur,2 Lawrence Lum,2 1GE Healthcare, Piscataway, NJ, USA, 2Karmanos Cancer
Center, Detroit, MI, USA.
Cell concentration and washing in a closed-system is a critical tool within cell
therapy applications. Although most protocols utilize a centrifugation-based
technology with variable recoveries, we sought to identify technologies that
maintain high recoveries and increased viabilities, while retaining desired cellular
functions (such as cytotoxicity) of the recovered cells. Maintaining this procedure
in a closed system is highly desirable. Hollow fiber filtration is widely used in the
bioprocess field, isolating biologicals such as antibodies, viruses, etc. In these
systems, the culture supernatant is recovered and the cells are considered waste.
We tested the ability of hollow fiber filtration to concentrate and wash human
activated T cells (ATC) grown in a Wave bioreactor (in this case the media is the
waste). A typical concentration/diafiltration circuit was assembled using a single
RTPCFP-6-D-4X2MS filter (0.65 µm porosity, 0.75 mm lumen diameter, 60 cm
path length). Pressure sensors were included to monitor the feed and retentate
pressure throughout the testing. The feed flow rate through the hollow fiber filter
was approximately 1 lpm. The permeate flow was controlled with a peristaltic
pump at approximately 100 mL/minute. Cell recovery was >90% with an excellent
viability (>95%). Recovered ATC showed comparable cytotoxicity against tumor
cells as conventionally harvested ATC. Based on the testing, ReadyToProcess
hollow fiber filters can concentrate and rinse the 5-10 liter WAVE cell culture in
less than an hour. A custom designed circuit can be produced to easily perform
this application and minimize set up time, all performed in a closed system.
206
Conversion of static flask ex vivo expansion methods
to a rotational culture methodology results in
significant cost reduction and improved clinical
feasibility
Howard Voorhies, Ian B. Nicoud, Joseph M. Blake, Daniel Weber, Colleen Delaney, Fred
Hutchinson Cancer Research Center, Seattle, USA, 2University of Washington, Seattle, USA.
1
1
1
1
1,2 1
Background: We developed a novel and clinically feasible method for ex-vivo expansion
of cord blood CD34+ progenitor cells using an engineered immobilized Notch ligand
and retronectin in static culture T-flasks. However, for each 0.5 million CD34+ cells
inoculated, a typical 14 to 16 day culture requires expansion into 18 x 225cm2 flasks.
This current system is resource intensive, requiring multiple individual manipulations
and many hours of labor. To address cost and clinical feasibility, we explored scale-up
using rotational culture.
Methods: CD34+ cells were seeded at 500,000 cells per 75cm2 flask in 20ml serumfree medium plus cytokines and re-fed 10ml complete medium on day 3. On day 6/7,
each flask was passed into a single 850cm2 roller bottle coated with a concentration of
retronectin and ligand equivalent to that used in static flasks based on surface area.
Cultures were maintained in single bottles with supplemental media added every
3-4 days and harvested at day 16. Extensive immunophenotyping was performed
throughout and in vivo repopulating ability was assessed through transplant of NOG
mice.
Results: Total cell and CD34+ cell yields were comparable between single roller bottles
and expanded static flasks when evaluated at harvest. Overall human engraftment in
NOG mice at 2 and 4 weeks post-transplant was similar between roller bottle and
static flask culture methods. Importantly however, roller bottle cultures reduced costs
significantly with less reagents required; retronectin and ligand use was decreased by
85%, media consumption by 72%, and labor by over 70%.
Conclusions: Roller bottle culture maintained a product phenotype similar to cultures
grown in a static system. Materials usage, the number of individual manipulations,
and technician labor were all significantly reduced compared to static culture. This
system reduces the opportunity for product contamination and allows for increased
production capabilities with lower cost, which are vital to clinical application.
207
GENOMIC STABILITY OF MULTIPLE TISSUE DERIVED MSC’S
DURING IN VITRO EXPANSION
Inese Cakstina, PhD1,2 Janis Kungs,1 Vadims Parfejevs,3 Laura Cappiello,1 Janis Ancans, PhD1
Una Riekstina, PhD3 1Faculty of Biology, University of Latvia, Riga, Latvia, 2Cell Transplantation
Center, P.Stradin’s Clinical University Hospital, Riga, Latvia, 3Faculty of Medicine, University of
Latvia, Riga, Latvia.
With instantaneous development in advanced medical therapy field including
somatic cell therapy medicinal products, the quest for biosafety markers that can
be incorporated in clinical research routine, rises. Mesenchymal stem cells (MSC)
derived from different tissues are in focus for various cell therapy applications.
Current cell therapy trials mostly use untreated and uncultured MSCs. Here we
screen multiple MSC cultures from various multipletissues that are expanded
in vitro. Cells are isolated from three sources: human bone marrow, epidermis
and dermis. The research project has received an approval from local Ethical
committee. Genomic stability monitoring includes tumor related gene expression,
mycoplasma infection testing, karyotyping and polyploidy analysis throughout
prolonged cultivation. In brief, PCR method is used for TERT and Mycoplasma
sp. detection, immunofluorochemistry method for Mycoplasma detection, flow
cytometry for polyploidy screening and Giemsa staining for karyotyping. HeLa
cells were used as a control for TERT expression and karyotype analysis. PA-1 cells
were used as control for polyploidy analysis.
Up to now all MSC cultures (nbm= 11; ndermal=18; nepidermal=5) show normal
karyotype during in vitro propagation. We did not detect Mycoplasma infection
andtumorigenic gene expression in the MSCs. We conclude that the proposed
biomarkers are useful tool to monitor MSC genomic stability during prolonged
cultivation in vitro.
208
Optimization and scale-up of cell therapy clinical
manufacturing processes using continuously
monitored and controlled bioreactors
Jason N. Carstens, Ph.D., Christopher Shellooe, Siddiq Abdul-Alim, Ph.D., Shelly Heimfeld,
Ph.D., Fred Hutchinson Cancer Research Center, Seattle, USA.
There is little information in the research literature on the role of the cell culture
operating conditions (e.g. temperature, dissolved oxygen, pH, media composition)
on manufacturing process performance for cell therapy products. With regards
to dissolved oxygen, there is some evidence suggesting hypoxic conditions may
be beneficial for growth rates, maintain the pluripotency of stem cells, stimulate
cytokine production in T-cells, reduce oxidative damage, and generally influence
therapeutic efficacy. However, in many studies the dissolved oxygen concentrations
are not measured but are instead inferred based upon the gas composition; the
cells are exposed to lower (and unknown) concentrations through oxygen gradients.
63
POSTER ABSTRACTS
The research literature is also sparse on describing the affects of pH, but it is
almost certainly a significant influence on culture performance. Furthermore pH
changes will be driven by cell density and metabolic activity (e.g. lactic acid and
CO2 production) as well as by mass transport (e.g. CO2 accumulation) that in turn
can be a function of the vessel configuration and scale of operation. As many cell
therapy products are in the early stages of research and process development, the
use of on-line instrumentation for continuous monitoring and feed-back control
of cell culture conditions has not yet been widely applied. To this end, an agitated
24-well microbioreactor (Pall Corporation’s Micro-24 MicroReactor) was used to
characterize the operating conditions for T-cells and stem cells in suspension culture.
The microbioreactor was used to continually monitor and control temperature, pH,
and dissolved oxygen in each individual well to study the parameters’ affects on
cell growth rates, peak densities, and viabilities. Off-line spent media analysis was
also performed to characterize the time-course glucose, lactic acid, and amino acid
compositions. This data was used for process optimization and to provide process
performance insight during scale-up into larger vessels and controlled bioreactors.
209
IMPORTANCE OF GATING OUT DEBRIS WHEN PHENOTYPING
COMPLEX STEM CELL PRODUCTS BY FLOW CYTOMETRY
Jerry R. Niedzinski, Elissa J. Flaumenhaft, Chad J. Ronholdt, Russ C. Marians, LABS, Inc.,
Centennial, USA.
BACKGROUND: Flow cytometry is a powerful tool to detect specific cell types
within a complex cellular product. Obtaining true phenotypes from those
products can be hindered by debris, which is found in all samples and can
cause false positive or false negative staining. Here, we showed the importance
of understanding both live cell and dead cell components of a complex mixture
containing abundant debris.
METHODS: An experimental mixture of low passage number mesenchymal
stem cells (MSC), peripheral blood white blood cells (WBC) and demineralized
bone matrix (DBM) was created. DBM is highly processed bone and does not
contain any intact, viable cells. Using both live and dead cell dyes, cell types were
phenotyped individually and in combination for MSC and WBC markers, with and
without eliminating debris. Both cultured MSC and DBM autofluoresced, limiting
the available fluorochromes to high excitation wavelengths (> 600 nm).
RESULTS: This surrogate complex cellularproductcould only be accurately
phenotyped using the combined viable and dead cell dyes because DBM debris
obscured the cells of interest. Before removing debris, individual cell types
represented only 50% of the total number of events. After removing debris, cells
of interest represented ≥ 90% of total events. The same held true in the mixture,
where the cell population doubled after debris removal.
CONCLUSION: Using combined live and dead cell dyes eliminated nucleated
dead cells and non-nucleated debris from the final analysis. More importantly,
the immunophenotype was focused on nucleated live cells, including live
autofluorescent cells, giving more accurate cell counts and a reliable phenotype
of the complex cellular product.
64
210
A MOLECULAR TEST FOR THE MEASUREMENT OF TRILINEAGE
POTENTIALITY OF BONE MARROW-DERIVED HUMAN
MESENCHYMAL STROMAL CELLS
Jessica Carmen, Ph.D., Jordana Levine, Jonathan A. Rowley, Ph.D., Lonza, Walkersville, USA.
Bone marrow-derived human mesenchymal stromal cells (hMSCs) are
multipotent cells. They have the potential to differentiate into either adipocytes
(fat), osteocytes (bone), or chondrocytes (cartilage). The ISCT has recognized
the tri-lineage potential, or tripotentiality, of hMSCs as identifying criteria for
the cell type. Accordingly, many hMSC-based therapeutic products require a
measurement of tripotentiality as a critical quality attribute (CQA) of their product.
Typically, the tripotentiality test is performed by sub-culturing the hMSCs in the
3 differentiating conditions for up to 3 weeks, and then performing histological
staining and assessment of the percentage of the cells that differentiated. This
process is cumbersome and subjective. More importantly, with an average assay
cycle time of one month, therapeutic doses of MSCs cannot be released for
clinical use until results are attained. The traditional trilineage test is dependent
on the expression of lineage-specific proteins, though the expression of lineagespecific genes should be detectable sooner. Furthermore, there is evidence in
the literature to support the feasibility of using molecular testing to demonstrate
hMSC tripotentiality. We have expanded hMSCs and then differentiated them into
multiple lineages for 1 week, extracted total RNA, and performed qPCR in order
to measure the expression of lineage-specific genes expressed by differentiated
cells compared to undifferentiated cells. We have found that hMSCs grown in
osteocyte-inducing medium for 1 week upregulate osteocyte-specific genes 6-8
fold compared to undifferentiated hMSCs. Additionally, we have found that
hMSCs grown in adipocyte-inducing medium for 1 week upregulate adipocytespecific genes 40-40,000 fold compared to undifferentiated hMSCs. Studies
are underway to confirm the detection of chondrocyte-specific genes in hMSCs
grown in chondrocyte-inducing medium. These preliminary results indicate that
a molecular tripotentiality assay for hMSCs may be comparable to the traditional
culture assay, allowing for development of a release test that is quantitative and
fast.
211
CULTURE OF NORMAL HUMAN DERMAL FIBROBLAST CELLS IN A
FUNCTIONALLY CLOSED AUTOMATED CELL EXPANSION SYSTEM
Boah Vang, Michelle Brecheisen, Nathan Frank, Rebecca Peters, Stefano Baila, PhD, Kim
Nguyen, PhD, Terumo BCT, Lakewood, CO, USA.
The large numbers of ex vivo expanded cells that are required in many clinical cell
therapy protocols (>100 million per patient) make standard culture conditions
problematic and expensive, resulting from the need for extensive personnel and
facilities resources and the high potential of contamination. To meet such clinical
demand a robust automated and closed cell expansion method is optimal. The
Quantum Cell Expansion System is a functionally closed, automated hollow fiber
bioreactor system designed to reproducibly grow both adherent and suspension
cells in either GMP or research laboratory environments. The Quantum System
has successfully been used for the ex vivo expansion of clinical-scale quantities
of adult bone marrow-derived mesenchymal stem cells (MSC). It has now been
demonstrated that a second adherent cell type of clinical interest, adult normal
human dermal fibroblasts (NHDF), are ex vivo expandable with the Quantum
System.
This study was an initial proof-of-concept that NHDF may be cultured on the
Quantum System. A total of three expansion runs were performed in duplicate
utilizing a commercially available fibroblast cell line (Lonza Clonetics Adult Normal
Human Dermal Fibroblasts) and the recommended media and supplements for this
cell line (Lonza Fibroblast Growth Media-2 and Supplemental Reagents). Bioreactor
POSTER ABSTRACTS
coating with fibronectin, cell loading, attachment, feeding, and harvest followed the
standard Terumo BCT protocol developed for the culture of MSC. Escalating T25
control flask confluency (70%, 80%, and 90% confluency, respectively) was the
endpoint that triggered harvest time on the associated Quantum experiment. The
initial seeding density for the bioreactor and the control flasks were 1000 cells/cm2,
or approximately 2 x 107 cells per bioreactor and 2.5 x 103 cells per T25 control flask.
Experimental results demonstrate that robust NHDF ex vivo expansion is possible
using an automated hollow fiber-based bioreactor system to reach clinically relevant
cell yields.
212
Comparison Study of Using Culture Bags and G Rex
Flasks to Grow CIK Cells
Madelaine Niam, Senior Lab Officer, Marieta Chan, Lab Director, Cell Therapy Facility, Blood
Services Group, Health Sciences Authority, Singapore, Singapore.
BACKGROUND: We previously established a protocol for large-scale culturing
of CIK (cytokine-induced killer) cells using culture bags for a phase I/II clinical
study for patients with haematological diseases. However, there have been
limitations to this method such as the labour intensive processes due to frequent
media addition and long culture periods (28 days) needed to achieve desired cell
numbers. Therefore, an alternative method was sought to improve our culture
process by evaluating the use of a novel gas-permeable culture device, G-Rex, in
comparison to our current conventional culture method.
METHODS AND RESULTS: Using cryopreserved peripheral blood mononuclear
cells (PBMNC) from healthy donors as our starting material; the cells were thawed
and washed subsequently before being placed in culture. In our conventional
method, the cells are seeded at a density of 5x106 cells/ml. Additional media as well
as cytokines were added at regular intervals (every 3-4 days) throughout the culture
process. Parallel cultures were grown using both the Permalife culture bags (Origen
Biomedical) and G-Rex flasks (100 cm2, Wilson Wolf). Further optimisation for the
G-Rex cultures were also done to find an optimal seeding density, maximise cell
output and to scale up the process for large-scale manufacturing. Final outcome
parameters used for comparison included determination of cell numbers, fold
expansion, cell viability and % of target CD3+CD56+ cell population. Preliminary
results demonstrated that there was an increased yield of cells with improved cell
viability and decreased cell death but no significant difference was observed for
the % target cell population. There was also decreased risk of contamination as
cultures were subjected to less “manual manipulation” and shortened culture
periods. Overall, there are several advantages to convert to the alternative culture
process but cost (including manpower) would have to be taken into consideration
before the new culturing method could be implemented.
213
DEVELOPMENT OF A CLOSED FILTRATION SYSTEM FOR BATCH
PROCESSING OF POOLED PLATELET LYSATE
Mariluz M. Henshaw, 1 Julie Morris,1 Pavan Gulati,2 Jan Pierce,1 Linda L. Kelley,3 Michael
Boyer,1 1Cell Therapy Facility, University of Utah, Salt Lake City, UT, USA, 2PALL Life Sciences,
Ann Arbor, MI, USA, 3Dana Farber Cancer Institute, Boston, MA, USA.
Human platelet lysate (PL) has been shown to be a comparable if not a superior
substitute to fetal bovine serum (FBS) in human cell cultures. Platelets are a
natural source of growth factors known to enhance human mesenchymal stem
cell (hMSC) expansion. hMSCs are currently being evaluated in several clinical
applications. Availability of a well-characterized PL is critical to manufacture
consistent hMSC products. To date, there is no standard protocol for the
preparation of PL from platelet apheresis units. Current methods employ
repeated freeze/thaw cycles to lyse the platelets and release the growth factors.
This results in a considerable amount of precipitate in the pooled PL product.
In this study, we performed three runs to test different filters and determined
the filter combination that most effectively reduced the amount of precipitate
without compromising growth factor concentration in the final product. Expired
platelet units (within three days post expiration) procured from the American Red
Cross were frozen at -80oC. For each experiment, five PL units were thawed and
pooled. This pooled PL was centrifuged and pumped through the different filters
in series. Samples of filtrates were taken at each step and analyzed for level of
PDGF-AA. PDGF-AA levels post-filtration ranged from 0% - 86% of unfiltered PL.
The best combination of filters in series was selected and a closed PL processing
system was designed consisting of a pooling bag, the filtration train, a collection
bag for the filtered product and a series of 500mL cryobags for final product
redistribution. Prototypes with capacity to prepare 10 and 20 liters of PL were
manufactured; characterization of resulting PL final products for growth factor
levels and ability to promote hMSC expansion are currently underway. This closed
system design for PL batch processing is a first step to having a commercially
available well-characterized PL product.
214
Scaling-up Adherent Stem Cell Cultures: Importance of
Physiochemical Environment Control by Multiplate
Bioreactor
Matthieu Egloff, Product Manager, José Castillo, Global Director, Cell Culture Technologies,
Jean-Christophe Drugmand, Manager, Cell Culture Development, Jonathan Goffinet,
Scientist, Florence Collignon, Scientist, Julien Cardon, Project Engineer, ATMI LifeSciences,
Brussels, Belgium. Scaling-up Adherent Stem Cell Cultures: Importance of Physiochemical
Environment Control by Multiplate Bioreactor 1F. Collignon, 1J. Goffinet, 1J.-C. Drugmand,
1
J. Cardon, 1M. Egloff, 1J.Castillo 1ATMI LifeSciences, rue de Ransbeek 310, B- 1120 Brussels,
Belgium
Delivering patient safety and cost-effective cell production is essential for
sustainable and meaningful commercialization of cell therapies. Single-use
bioreactors represent a solution by minimizing manual handling, realizing
economies of scale and delivering the benefits of a closed system. Subsequently,
process development and industrialization is mandatory to achieve large-scale
GMP manufacturing.
Process development and scale-up is challenging as small variations in
physicochemical parameters, such as surface characteristics, pH and dissolved
oxygen, can heavily impact stem cell growth and behavior. Preserving the cell
culture environment and mitigating risk during process scale-up for large clinical
studies or commercialization is critical to a successful outcomes.
Integrity™ Xpansion™ 2-D multiplate bioreactors have been specially designed and
engineered to enable easy transfer from existing T-flask or multytray stack process
by offering the same cell growth environment and enabling control of the critical
cell culture parameters. Xpansion bioreactors are made up of stacked polystyrene
plates within a closed system. Up to 180 of these plates can be stacked to
produce a unit with a surface area of 11m2, working volume of 19.8L with external
dimensions of 35x60cm. Its compact efficient design enables the elimination of
the gas phase between the plates. This gas phase is replaced by an automatically
controlled aeration system which provides advanced gas diffusion to control the
growth environment to user-defined optimum values of pH and dissolved oxygen.
Presented data will highlight the significant impact of multiplate design and
environmental control systems on process scalability for adherent stem cell
production.
65
POSTER ABSTRACTS
215
217
The Importance of Digital Holographic Microscopy
for Automated, Real-Time Monitoring of Human Adult
Stem Cell Confluence in Large-Scale Cultures.
PEAK SERUM: IMPLICATIONS OF SERUM SUPPLY FOR CELL
THERAPY MANUFACTURING
Matthieu Egloff, Product Manager1 Jean-Christophe Drugmand, Manager, Cell Culture
Technologies1 Jonathan Goffinet, Scientist1 Florence Collignon, Scientist1 Philip Mathuis,
CEO2 1ATMI LifeSciences, Brussels, Belgium, 2Ovizio, Brussels, Belgium. The Importance of
Digital Holographic Microscopy for Automated, Real-Time Monitoring of Human Adult Stem
Cell Confluence in Large-Scale Cultures. 2P. Mathuis, 2S. Jooris, 1F. Collignon, 1J. Goffinet, 1J.-C.
Drugmand, 1M. Egloff 1ATMI LifeSciences, rue de Ransbeek 310, B- 1120 Brussels, Belgium
2
Ovizio Imaging Systems, Engelandstraat 555, B-1180 Brussels, Belgium
To guarantee robust cell expansion on a large scale, an automated control
method becomes essential for obtaining sustainable and useful stem-cell-based
products. Given that both stem cell behavior and the differentiation mechanisms
are sensitive to cell density, monitoring of cell confluence is mandatory. Actual
observation protocols of traditional polystyrene T-flasks, or multitray stacks, are
ineffective for large scale manufacturing.
Integrity™ Xpansion™ multiplate bioreactors have been developed to enable
noninvasive, real-time observation of stem cell growth at large scales. The specific
design of the bioreactor combined with the iLine − a differential digital holographic
microscope (DDHM) of the newest generation − enables automatic multiplate
cell monitoring. The DDHM technology captures 3-D information, enabling labelfree image processing and automatic cell confluence counting.
The data presented will highlight the benefits of differential digital holographic
microscopy as a reproducible and consistent method to track stem cells
confluence during large-scale production.
216
The cell therapy industry (CTI) is emerging as a distinct and competitive
component of global healthcare, creating value for investors and providing lifechanging therapies to patients. Industry growth has necessitated an increased
focus on large-scale manufacturing strategies to meet future demands. One
major challenge is the limited availability of some crucial raw materials used in cell
therapy manufacturing – including bovine serum. Without a sustainable supply
or viable alternatives to these components, the commercial-scale production
of cell therapies will be impossible, halting the momentum of the industry. We
propose that solutions to these challenges are achievable, and can be expedited
by industry-wide collaboration.
Bovine serum is currently used in the majority of cell therapy manufacturing
processes. Current stocks and production rates of serum suitable for GMP
manufacture may only be sufficient to support the production of one blockbuster
cell therapy. Limitations in the availability of bovine serum thus act as a major cost
driver and significant barrier to the commercial success of the industry as a whole.
Thus, without an increase in serum production, or at least a significant increase
in the development and implementation of serum-free production strategies, the
growth and sustainability of the CTI will be severely constrained.
218
BIOASSAYS FOR THE TESTING OF DC MANUFATURED FOR USE
IN HUMAN CLINICAL TRIALS
Quantitative BIOAssays to characterize mesenchymal
stem cells during process development and
manufacture
Maureen Loudovaris, 1,2 Sharron Gargosky,3 Kate Dunster,1,2 Elise Butler,1,2 Gianna
O’Donnell,1,2 Dominic Wall,1,2,4 1Peter MacCallum Cancer Center, Melbourne, Australia, 2Cell
Therapies Pty Ltd, Melbourne, Australia, 3Prima Biomed, Sdyney, Australia, 4Unveristy of
Melbourne, Melbourne, Australia.
Padmavathy Vanguri, Ravi Vyzasatya, Zoe Damian, Linda Yahiaoui, Therese Willstaedt, Lonza
Walkersville, Inc., Walkersville, USA.
Prima Biomed is currently undertaking a global clinical trial called CANVAS.
CANVAS is a multinational, multicenter, randomized, double-blinded, placebocontrolled trial of Cvac as maintenance treatment in patients ovarian cancer in
clinical remission following first-line chemotherapy. Patient who meet all study
criteria will be randomized in a 1:1 double-blinded fashion to either Cvac (active)
group or the placebo group. At least 800 patients will enter the treatment phase of
the study. Patients will be enrolled at approximately 150 centres in Europe, North
America, and Australasia. Currently, there is no internationally recognised “gold
standard” assay for the testing of DC manufacutred for use in clinical trials. Many
parameters exist to quantitate the potency of DC for immunotherapy, including
measuring DC-specific cell surface expression and homing markers, or secretion
of cytokines. However, as DC immunotherapy is typically employed to promote a
cytotoxic T lymphocyte (CTL) response in vivo, it may be better to directly measure
the ability of a DC product to stimulate such a response. This can be measured
through the generation of antigen-specifc CTL cultures using the patient’s cells,
which usually takes 4-6 weeks, and also relies on the ability of the patients immune
cells to respond in vitro. Alternatively, the costimulatory potential of DC can be
measured in an allogeneic setting such as an Mixed Lymophocyte Reaction (MLR).
We have developed a MLR assay for use as a bioassay for the CANVAS study. As
a first step we have generated and qualified 3 HLA-mismatched allogeneic T cell
banks. We have established the MLR assay using 4 normal donor Cvac products
at different Cvac to T cell ratios. Cells are cultured for 7 days prior to analysis using
CSFE dye, CD3 and a live/dead stain by flow cytometry. The MLR assay is currently
under validation and results will be reported.
66
Natasha L. Davie, 1,2,3 Dave A. Brindley,1,2,4 Emily J. Culme-Seymour,5 Mason Chris,1
1
University College London, London, UK, 2The Harvard Stem Cell Institute, Cambridge, USA,
3
Harvard Medical School, Boston, USA, 4Harvard Business School, Cambridge, USA, 5The
London Regenerative Medicine Network, London, UK.
Culture expanded Mesenchymal Stem Cells (MSCs) have emerged as promising
cellular therapeutics in regenerative medicine, and treatment of various diseases.
It is imperative that MSCs developed for clinical use are thoroughly characterized
during process development and manufacture. The phenotype and activity of the
cells often change with time in culture, growth media, culture vessels and expansion
methods. Regulatory requirements dictate that the cells are defined by quantifiable
measures of biological activity and purity to assure consistent safety and potency.
We are developing a comprehensive set of robust and time-efficient tests to
measure these salient properties.
The clinical potential of various MSC-like cells is based upon their 1) tri-lineage
potential or capacity to differentiate along adipogenic, osteogenic, and chondrogenic
lineages; 2) immunosuppressive properties and 3) ability to produce growth factors,
cytokines, and chemokines that can induce cell proliferation, angiogenesis etc.
Trilineage assays are performed in multiwell plates: adipogenesis and osteogenesis
are quantitated with AdipoRed™ and OsteoImage™ assays respectively. A
quantitative measurement of Alcian Blue binding to sulfated glycosaminoglycans
is used to assess chondrogenesis. Immunosuppression by MSCs is quantitated by
determining the reduction in number of T-cell divisions by flow-cytometric analysis
of CFSE-labeled T cells induced to proliferate in vitro. To assess the biologic activity
of factors produced by MSCs we developed immortalized Human Umbilical Vein
Endothelial Cells (HUVECs) that are phenotypically similar to primary HUVECs but
unlike primary cells, maintain functional activities beyond 15 passages. These lines
were expanded to generate a bank of cells to assure long term bioassay consistency.
Proliferation or migration of such indicator cells in response to conditioned
medium from MSCs in culture is evaluated to quantitate the growth promoting
POSTER ABSTRACTS
and angiogenic properties of MSCs compared to known growth factors or VEGF.
Representative data of each of these methods applied to MSC characterization will
be presented.
219
HARVESTING LARGE VOLUMES OF TUMOR INFILTRATING
LYMPHOCYTES (TIL) USING A CLOSED SYSTEM, COMMERCIALLY
AVAILABLE, DEVICE
Renee C Smilee, MT(ASCP), Sabine Ellwanger, MT(ASCP), Albert Ribickas, MT,HP(ASCP),
William E Janssen, PhD, Moffitt Cancer Center, Tampa, Florida, USA.
Tumor Infiltrating Lymphocytes (TIL) have been reported to show significant
responses in the treatment of refractory melanoma. To manufacture TIL in
sufficient numbers for clinical benefit, lymphocytes, extracted from surgically
resected tumors, must be cultured through 12-14 doublings. This, in turn, requires
increasing volumes of culture medium such that, at the conclusion of culturing,
the final TIL product may be suspended in up to 65 liters of medium, contained
within 30 - 40 culture bags, and containing up to 1011 lymphocytes. This volume
must be reduced to less than a liter for infusion, without losing excessive numbers
of the lymphocytes. We have previously reported adaption of the Haemontics™ Cell
Saver 5 instrument for Ficoll density gradient cell separations. We now report a
similar adaption of this intrument to concentrate and wash TIL. We have produced,
harvested, and infused ten TIL products. Two Cell Saver instruments were used in
parallel. Both were set to run the ‘sequestering’ program, and were equipped with
a standard Haemonetics™ disposable set including a 250 mL bowl. With the bowl
spinning, cell suspension was pumped in at a rate of 250 mL/minute. Medium
was pumped through the bowl while centrifugal force held cells against the bowl
wall. Waste bags were changed during the process as each bag become full. At
the conclusion of the cell sequestering, the bowl was stopped, the pump reversed,
and the concentrated cell suspension was pumped out. In use we have processed
from 24.8 to 60.0 mL of cell suspension, recovering from 2.0x1010 to 11.3x1010 cells
in a volume ranging from 474 mL to 775 mL. Total elapsed time ranged from 1hr
39min to 2hr 43min. The Cell Saver instrument has proven to perform capably for
TIL harvesting, and has the benefit of ready availability, ease of operation and low
operating cost.
220
DEVELOPMENT OF MICROFLUIDIC BIOREACTORS FOR CELL
EXPANSION
Huaying Chen, Ryan S Pawell, Jingjing Li, Robert E Nordon, University of New South Wales,
Sydney, Australia.
The role of cell bioreactors is to transform laboratory-based techniques into closed,
scalable cell and tissue production processes for the clinic. Soft lithography and
micro-manufacturing methods offer submicron geometric precision for design
of microfluidics to control cell microenvironment, possibly leading to the next
generation of bioreactor devices.
We describe development of a microwell perfusion bioreactor for expansion of
non-adherent cells such as hematopoietic progenitors or lymphoid subsets.
Microwells protect non-adherent cells from shear stress, and retain cells within
the closed system whilst media is continuously exchanged without the need
for manual cell passage. Prototype devices at laboratory bench scale were
manufactured from polydimethylsiloxane (PDMS) by soft lithography. Wells had a
diameter of 200 microns and depth of 35 microns, and were arranged in a square
lattice with 50 micron intervals between wells. Bioreactor operating parameters
such as perfusion rate and media constituents were optimised at lab-bench scale
by live cell imaging. Cell growth was tracked by time-lapse microscopic imaging;
1500 KG1a cells were simultaneously cultured and scanned every 3 minutes at
100x magnifications for 6 days to determine the efficiency of expansion at the
clonal level. Thus the lab-scale device can also be used for high-throughput, long-
term imaging of haematopoietic and lymphoid clonal growth. PDMS microwell
sheets are not economically manufactured at scale so we have developed hot
embossing methods to manufacture thermoplastic microwell devices. Future
studies will investigate fabrication of multilayer thermoplastic devices for
expansion of therapeutic cell subsets.
221
CLINICAL CORD BLOOD UNITS WASHED WITH AUTOMATED
SEPAX® CELL PROCESSING SYSTEM PROVIDES HIGH QUALITY
POST-THAW RECOVERIES AND VIABILITY FROM CRYOPRESERVED
UNITS
Ronna Dornsife, 1 Ann Kaestner,2 Tiffany Hawkins,2 Melissa Reese,2 Sophia Avrutsky,2 Alexandra
Bolick,1 Joanne Kurtzberg, MD1,2 1Carolinas Cord Blood Bank (CCBB), Duke Translational
Research Institute, Duke University, Durham NC, USA, 2Stem Cell Laboratory, Duke University
Medical Center, Durham NC, USA.
To recover viable and functional cells from thawed cord blood units (CBU) for
transplantation with consistency through automation, clinical cord blood units
were thawed then washed using the Biosafe Sepax® System (final vol ≥ 50mls).
A comparison of cell recoveries and viability from 30 clinical CBU transplant
products (23 CCBB, 7 from other banks) to date versus data from a Sepax®
validation study and from manually washed CBU is presented.
Recovery and Viability: CBU Washed with Sepax® or Manually Washed
Wash
Method,
Setting
CBU
n=
Median
%TNC
Recovery
Median
% CD34+
Recovery
Median
% Total CFU
Recovery
Median
% Post (thaw and)
wash Viability
Sepax®,
Clinical
30
81%
± 13%*
60%
± 14%
29%
± 28%
98%
± 0.8%
Sepax®,
Validation
24
93%
± 8%
76%
± 10%
47%
± 24%
96%
± 1.7%
Manual,
Clinical
195
81%
± 13%
85%
± 68%
22%
± 41%
98%
± 1.7%
(* ± % STDEV)
Quality data on TNC, CD34+ cells, and Total CFU along with post wash viabilities
in these 30 clinical CBU, demonstrated similar but slightly lower recovery values
for TNC, CD34+ and Total CFU with equivalent viability as compared with data
from our in-house Sepax® wash validation data (24 from CCBB). The 30 clinical
CBU washed by Sepax® were found to have equal TNC, slightly better Total CFU,
somewhat lower CD34+ recoveries and identical viability versus data from manually
washed units. None of the CBU (n = 54) washed by Sepax® and tested by BacT/
Alert® had detectable organisms. In conclusion, the combined findings from both
the clinical units to date and validation study CBU washed by Sepax®, support the
ongoing clinical use of the automated, closed kit Sepax® Cell Processing System
to prepare CBU to achieve safe, consistent, and high quality cord blood transplant
products.
222
TOOLS AND METHODS FOR HIGH-THROUGHPUT UNBIASED
CELL SURFACE MARKER SCREENING OF STEM CELLS AND THEIR
PROGENY BY FACS
Rosanto Paramban, Jason G Vidal, Nil Emre, Jody Martin, Christian T Carson, BD
Biosciences, San Diego, USA.
Differentiation of pluripotent stem cells to specific lineages often results in
heterogeneous populations that may require purification for certain applications
such as regenerative medicine and cellular therapy. We have developed a human
cell surface antibody screening panel to enable the identification of unique cell
surface signatures that can be used for enumeration and enrichment of stem cells
and their differentiated progeny. We have further enhanced immunophenotyping
screening by developing various assays to increase throughput and additional
67
POSTER ABSTRACTS
customization. We will describe how we used fluorescence cell barcoding to
increase throughput to enable immumophenotyping of multiple brain cancer
cell samples simultaneously using 242 antibodies to cell surface markers. We
also present a screen in which we combined intracellular marker expression with
cell surface immunophenotyping to identify cell surface signatures of neurons
derived from pluripotent stem cells. Specifically, we performed a surface marker
screen with this panel while analyzing for intracellular expression of neural stem
cell markers Pax6, Sox1 and Sox2 and the neuronal marker doublecortin (DCX).
Finally, we will illustrate how mulitpotent stromal cells (MSC) can be characterized
by immunophenotpying and propose that this surface screening technique can be
used for comparative analysis of MSC lines. Overall, these tools and methods
enable the characterization of cell population heterogeneity, establish quantitative
benchmarks between different batches of cells, and allow for FACS isolation of
homogenous cell populations from a heterogeneous pool of cells.
223
MESENCHYMAL STEM CELLS TRACKING USING QUANTUM DOTS
IN AN ANIMAL MODEL OF SPINAL CORD INJURY
Sara Ranjbarvaziri, Nasser Aghdami, Royan Institute, Tehran, Iran.
In the field of stem cell therapy, non invasive cell tracking could provide
valuable information regarding both engraftment efficiencies and the underlying
mechanisms by which transplanted cells can possibly contribute to functional
improvements. The use of quantum dots (QDs) as new and promising fluorescent
probes with superior optical properties, hold great potential in long term in vivo
imaging, however there are many uncovered issues regarding their competency.
In the present study, human bone marrow mesenchymal stem cells (BMSCs) were
labeled with QD585 and QD800 by using positive charged peptides. To investigate
QD stability in vivo, labeled cells were locally injected to the sites of thoracic (T10)
spinal cord midline lateral hemisection injury in rat.
Results demonstrated that one week following transplantation, some cells with
human origin were resided at the implantation site, although no QDs were
observed in their cytoplasm. Clear QD fluorescence signals could be only detected
in cells which were negative for human nuclei staining, which indicated the
deposition of QDs into the host cells possibly due to their active excretion from
labeled cells or release after cell death.
This study showed that QDs leaked out from MSCs in vivo and the released
particles were able to re-enter adjacent cells over time. Excretion of QDs from
labeled cells could remarkably affect their potential for long-term stem cell
tracking in vivo.
224
A BROADLY-APPLICABLE MICRORNA-BASED MONITORING TOOL
FOR STEM CELL QUALITY CONTROL AND DIFFERENTIATION
MONITORING
Daria Olijnyk, David Mallinson, Max Bylesjö, Vincent O’Brien, Sistemic Ltd, Glasgow,
Scotland.
Stem cells derived from both embryonic and adult tissue or from reprogrammed
somatic cells have significant promise for human regenerative medicine. However,
despite similarities in developmental potential, several groups have found
68
fundamental differences between stem cell lines that could impact on the potency
and/or safety of the resultant cell populations but which were not predicted using
current monitoring procedures based on flow cytometry and analysis of panels
of mRNAs. There is a requirement for reliable tools to monitor cell populations
during the processes of stem cell line development, directed differentiation and
scale-up to safe, therapeutically-useful cell populations. Sistemic have developed
a novel, sensitive, reliable, broadly-applicable monitoring tool that provides both
a good indication of cell homogeneity and insights into underlying biological
effects associated with any observed alterations in microRNA expression profiles.
We demonstrate here that our approach also provides an assessment of the
likely impact of the observed miRNAs changes on cell phenotype. We will present
case studies to illustrate that SistemQCTM, represents a simple, robust and costeffective tool to monitor the maintenance of pluripotentcy in stem cell lines
across passages, the staging of directed differentiation from embryonic, iPS or
direct reprogramming strategies and, post scale-up, an assessment of functional
attributes and safety profile of the cells.
225
Will not be presented
226
Functional stability of hematopoietic stem and
progenitor cells in ex vivo expansion product of cord
blood CD34+ cells at +4°C.
Zoran Ivanovic, Pascale Duchez, Bernard Dazey, Jean Chevaleyre, Xavier Lafarge, Philippe
Brunet de la Grange, Jean-Michel Boiron, Marija Vlaski, EFS, Bordeaux, France.
We developed an ex vivo expansion procedure starting from cord blood CD34+
cells, enabling several hundred expansion fold of total cells, more than one
hundred fold of CD34+ cells and committed progenitors, and without a negative
impact on stem cells exhibiting both short- and long-term repopulating capacity
(Ivanovic et al, Cell Transplant 2011). On the basis of these data, a clinical
scale procedure was set up (Macopharma HP01®medium in presence of SCF,
FLT3-L (100 ng/ml each), G-SCF (10 ng/ml) and TPO (20 ng/ml) (Duchez et al,
Cell Transplant 2012 in press) and is in use for an ongoing clinical trial (adult
allogeneic context) yielding excellent preliminary results (Milpied et al, ASH
2011). In order to test the possibility to use the expanded cells in transplantation
centers worldwide, we studied the functional stability at 4°C (usual temperature
of transportation) of hematopoietic progenitors and stem cells 48 hours after
expansion. If the cells were washed and resuspended in 4% albumin solution
(actual procedure for immediate injection), only one half of total nucleated and
34+ cells and 30% of committed progenitors survived. This condition has also
an evident negative impact on stem cells in expansion product as demonstrated
on the basis of reconstitution of NOG/Scid mice bone marrow by human CD44,
CD33, CD19+ cells as well as by human committed progenitors (CFC). Surprisingly,
if the cells were stored 48 hours at +4°C in the culture medium, very good survival
of total and CD34+ cells (90 to 100 %) and CFC (around 70%) was obtained,
as well as a full maintenance of stem cells (the same in vivo essay with NOG/
Scid mice). These data point to a possibility of maintenance of full functional
capacity of expanded grafts for two days, time necessary for its transportation in
any transplantation center worldwide.