2012 Accepted Poster Abstracts

Transcription

2012
Accepted
Poster
Abstracts
(1) Submission ID#13358
Spontaneous in vitro appearance of pericytes in an angiogenesis skin model: implication for their
stem cell lineage.
Author(s)
Francois A. Auger
Director
Centre LOEX de l'Université Laval, Quebec CHU Research Center
Co-Author(s)
Julie Symes - Associate Scientist, Sanofi Pasteur
Nathalie Tremblay - Research Professional, Centre LOEX de l'Université Laval, Quebec CHU Research
Center
Jeffrey A. Medin - Senior Scientist, Ontario Cancer Institute
François Berthod - Senior Scientist, Centre LOEX de l'Université Laval, Quebec CHU Research Center
Topic
Basic Research
Problem
Microvasculature pericytes are nowadays considered as stem cells with a significant plasticity. However,
although their differentiation into various tissues has been well studied there is a paucity of publications
related to their origin. As these cells may be quite useful in Regenerative Medicine any additional information
on their lineage should be quite valuable. We developed an in vitro angiogenesis model in tissue engineered
human skin in which we observed the spontaneous appearance of such microvascular pericytes.
Background
The field of stem cell research concerning mesenchymal stem cells (MSC) has been accelerating greatly
because of the probable relationship with human blood vessel derived stem cells. Human blood vessels are
deemed to have the potential for three stem cell lineage. Thus intima, media and adventitia give rise to the
following stem cells: myogenic endothelial cells, pericytes, and adventitial cells.
However, microvasculature presents only one of these stem cells: the pericyte. These cells in cooperation with
microvascular endothelial cells are essential to the stabilization and maturation of such blood microvessels.
Our group has managed to create a vascularized skin tissue in vitro. Thus, since pericytes have been shown
to appear spontaneously in such a model, we can then also study the origin of such a pivotal cell.
The work presented here has been published as : "F. Berthod, J. Symes, N. Tremblay, J. A. Medin and F. A.
Auger, "Spontaneous fibroblast-derived pericyte recruitment in a human tissue-engineered angiogenesis
model in vitro". J Cell Physiol, 227(5): p. 2130-7. 2012.
Hypothesis
Our in vitro skin model lends itself to a thorough investigation of the origin of the microvascular pericytes that
are observed to appear in it. Indeed, only two cell types are used in the model: fibroblasts and endothelial
cells. We thus set about to unveil which cell type gives rise to in vitro pericytes in our angiogenesis model.
Research
Our group has developed an original in vitro model of endothelialized reconstructed connective tissue (ERCT)
in an appropriate scaffold material. Normal human fibroblasts and endothelial cells were extracted from
donated biopsies and tissues. We thus obtained human dermal fibroblasts, umbilical vein endothelial cells
(HUVEC) and microvascular endothelial cells (HMVEC). Both HMVEC and fibroblasts were treated with
lentiviral vectors to generate cell populations that constitutively expressed enhanced GFP (eGFP). Porous
scaffolds were made with a collagen/glycosaminoglycan/chitosan biomaterial and seeded with human
fibroblasts and either HUVEC or HMVEC. Seeded scaffolds were cultured for 24 days. They were submerged
in the medium for the first 10 days and then elevated at the air-liquid interface for the remaining 14 days.
Biopsies were embedded in OCT and used to make 5 mm thick sections for conventional
immunofluorescence analyses (IF) or 50 mm thick sections for confocal imaging. The antibodies used in the
double staining IF were directed against human Platelet-Endothelial Cellular Adhesion Molecule-I (PECAM-I,
aka CD31), laminin and human alpha-Smooth Muscle Actin (SMA). Nuclei were stained with Hoechst. For
confocal imaging, we used an anti-condroitin sulfate (NG2) antibody or a mouse anti-SMA antibody. Human
PDGF-BB was quantified with an ELISA assay. Cell count and flow cytometry analyses were performed with
cells exposed to antibodies directed against von Willebrand Factor (vWF) or human SMA.
Observations
Pericytes were detected in our Endothelialized Reconstructed Connective Tissue (ERCT) model by both their
alpha-smooth muscle actin (SMA) expression and the colocalisation of SMA-expressing cells with PECAM-1expressing endothelial cells. Moreover, they were shown to also express NG2, a more specific marker of
pericytes. When human endothelial cells were co-cultured with fibroblasts in the sponge, some SMAexpressing cells were detected in the ERCT. Conversely, when fibroblasts were cultured in the sponge
without endothelial cells (RCT), no SMA-expressing cells was detected. The SMA-positive cells in the ERCT
were always observed closely associated with PECAM-1-positive capillary-like tubes (Fig 1A-C), whether
these capillaries were made of Human Umbilical Vein cells (HUVEC) (Fig 1A) or Human Microvascular
endothelial cells (HMVEC) (Fig1B,C). When GFP-expressing HMVEC were used to prepare the ERCT, the
same close association of SMA-expressing cells with GFP-positive capillary-like tubes was shown (Fig1C). A
similar association between SMA-expressing cells and PECAM-1-positive capillaries was observed in human
skin (Fig 1D).
To have a better view of the three-dimensional organization of the capillary-like tubes, a confocal imaging was
performed on thicker 50 µm ERCT sections. SMA-positive cells were detected all along and around the
capillaries (Figure 2B). Moreover, a positive staining using a NG2 antibody showed a similar staining
compared with SMA (Figure 2A). Since NG2 is one of the most convincing markers of pericytes, this is an
additional evidence of the pericyte nature of these cells.
To further confirm that these SMA-expressing cells were pericytes, we tested the effect of increasing
concentrations of PDGF-BB in the medium or blockade of the PDGFR on pericyte distribution. Indeed, PDGF
is well known to play a major role in the recruitment of pericytes during angiogenesis, resulting in proliferation
and migration of mural cells along the new capillaries. PDGF-BB induced a 20% increase in SMA-positive
cells in the ERCT at a concentration of 0.1 ng/ml, whereas this increase was not significantly different
compared to the control. At 1 and 10 ng/ml, no significant effect was observed. In parallel, PDGF-BB had no
effect on the number of vWf-positive cells, or the number of capillary-like structures at 0.1 and 1 ng/ml (Fig 3).
In contrast, 100 ng/ml of PDGF induced a significant 4 times decrease in the number of SMA-expressing cells,
a five-fold decrease in vWf-positive cells and a decrease in the number of capillary-like structures, while the
total number of cells extracted from the ERCT did not change significantly (Data not shown). A concentration
of 10 ng/ml of PDGF also induced a significant decrease of the number of endothelial cells (vWf-positive) and
CLTs.
On the other hand, the blockade of PDGFR using AG1296 induced a 5-fold significant decrease in the
proportion of SMA-expressing cells, a 2.6-fold diminution on the proportion of vWf-expressing endothelial
cells, and a 2.4-fold decrease in the number of CLT in the ERCT treated with AG1296 at 5µM (Fig 4). At 20
µM of AG1296, these decreases were 14.4, 13, and 9.3 times, respectively.
To determine from which cell type SMA-expressing cells differentiated from, GFP-transduced human
fibroblasts and GFP-transduced HMVEC were used, using the same cells as the non-transduced one. Two
types of ERCT were prepared, one made of GFP-fibroblasts and HMVEC, and the other made of fibroblasts
and GFP-HMVEC. A flow cytometry analysis of the cells extracted from the ERCT demonstrated that SMAexpressing cells differentiated from fibroblasts (Fig 5). Indeed, in the ERCT made of fibroblasts and GFP-
HMVEC, SMA-expressing cells were not GFP-positive, thus showing they derived from fibroblasts. In the
ERCT made of GFP-fibroblasts and HMVEC, 78% of SMA-expressing cells were also GFP positive, thus
originating from fibroblasts. The 28% SMA-expressing cells that were GFP negative were probably also from
fibroblast origin, since a sub-population of the GFP-fibroblasts did not express GFP (Fig 5F).
In conclusion, our tissue engineered ERTC indicates that human normal fibroblasts can spontaneously
differentiate in vitro into cells that appear to be pericytes that closely localize with the microtubules of a
microvascular network newly formed from human endothelial cells.
We thus have developed a model, the ERTC, that allows the spontaneous appearance of in vitro pericytes.
This model could be very useful to more clearly define the "stemness" of our pericytes. One example would
be to use a modified ERTC, by adding keratinocytes, in our model of wound healing (Laplante, A., et al.,
FASEB J, 2001. 15: p. 2377-2389) and evaluate their activity in reducing hypertrophic scarring.
A broadly applicable miRNA-based tool for stem cell characterisation and quality control
Author(s)
David J. Mallinson
Dr David J Mallinson, Senior Staff Scientist
Sistemic Ltd
Co-Author(s)
Daria Olijnyk, PhD - Staff Scientist, Sistemic Ltd
Max Bylesjö , PhD - Senior Bioinformatician, Sistemic Ltd
Vincent O’Brien, PhD - CSO, Sistemic Ltd
Topic
Basic Research
Problem
Stem cells derived from both embryonic or adult tissue or from reprogrammed somatic cells have significant
promise for human regenerative medicine. However, despite similarities in developmental potential, several
groups have found 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.
Background
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, especially in
regard to potency of cellular products.
Hypothesis
MicroRNAs are proving highly informative for cell characterisation including stem cells. Cell therapy
developers are now exploring using miRNA profiling for product characterisation. SistemQC™ leverages the
instructive power of miRNA based fingerprinting to provide robust and information-rich characterisation and
QC methods.
Research
Using whole a variety of miRNAome discovery technology platforms combined with subsequent verification
and validation using alternative technologies, Sistemic has developed a novel, reliable, broadly-applicable
monitoring tool that provides both a robust indication of cell identity, homogeneity and potency as well as
providing insights into the underlying changes in gene expression associated with observed biological
phenotypes.
Observations
This patented approach, SistemQC™ utilises a combination of microRNA expression profiling and
customised, multilayered data analysis to provide a simple, robust and cost-effective tool to monitor the
maintenance of pluripotency in stem cell lines across passage, 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. Data will be presented in this poster to support some of these applications
including (1) detection of differences, (2) testing equivalence, and (3) surrogate potency markers.
A safe and efficient method to produce neuronal cells from human bone marrow derived adult
mesenchymal stem cells by modulators of chromatin modifying enzymes and SMAD signaling
proteins
Author(s)
Arshak R. Alexanian
Associate Professor
Medical College of Wisconsin
Co-Author(s)
Topic
Basic Research
Problem
Advances in cell reprogramming technologies to generate patient-specific cells of a desired type will
revolutionize the field of regenerative medicine. The majority of currently used reprogramming technologies
require the exposure of cell nuclei to reprogramming agents via delivery of large reprogramming molecules
into cell cytoplasm or via transfer of cell nuclei into the cells that contain reprogramming molecules. This
raises several technical, safety and ethical issues.
Background
Chemical genetics is an alternative approach for cell reprogramming that uses small, cell membrane
penetrable substances to regulate multiple cellular processes including cell plasticity. Recently, using the
combination of small molecules that involved in the regulation chromatin structure and function and agents
that favor for neural differentiation we have been able to efficiently generate neural-like cells from human
MSCs (hMSCs).
Hypothesis
In the present study, we hypothesized that the neural transformation efficiency could be further improved by
addition to our previous neural induction recipe two specific inhibitors of SMAD signaling pathway.
Research
To this end, MSCs have been manipulated with epigenetic modifiers and inhibitors of SMAD1/3 and
SMAD3/5/8/ signaling pathways in neural growth supporting medium.
Observations
The results demonstrated that this reprogramming approach improved the efficiency of the generation of
neural cells that exhibit several properties of mature neurons.
A versatile, highly efficient, and potentially safer piggyBac transposon system for mammalian genome
manipulations
Author(s)
Angelique Lin
Research Technician
Molecular Medicine Research Center, Chang Gung University
Yaa-Jyuhn James Meir
Assistant Professor
Department of Biomedical Sciences, College of Medicine, Chang Gung University
Role: Author
Co-Author(s)
Meng-Fan Huang - research technician, Molecular Medicine Research Center, Chang Gung University
Jiao-Rung Lin - research technician, Molecular Medicine Research Center, Chang Gung University
Matthew T. Weirauch - Assistant Professor, Center for Autoimmune Genomics and Etiology and Divisions of
Rheumatology and Biomedical Informaticss, Cincinnati Children's Hospital Medical Center
Hsiang-Chen Chou - student, Department of Biomedical Sciences, College of Medicine, Chang Gung
University
Siang-Jin Ashley Lin - student, Department of Biomedical Sciences, College of Medicine, Chang Gung
University
Sareina Chiung-Yuan Wu - Assistant Research Professor, Molecular Medicine Research Center, Chang Gung
University
Topic
Basic Research
Problem
To increase the therapeutic potential of stem cells for clinical applications, an efficient, versatile, and safer
genome manipulation platform is required. Due to the risks associated with viral vectors, developing non-viral
based gene transfer is one of the central focuses in the field of gene and cell therapy.
Background
The piggyBac transposon isolated from cabbage looper moth possesses several desired characteristics, such
as its large cargo capacity and traceless transgene removal, making it one of the most favorable tools for
manipulating mammalian genomes. Nevertheless, to advance piggyBac to clinical applications, many
obstacles remain to be overcome. One of these obstacles is to avoid the potential advert effect of genome
instability caused by the continuing supply of the piggyBac transposase during genetic manipulations.
Hypothesis
Here we address whether a highly efficient recombinant piggyBac transposase can be generated and further
purified for establishing a recombinant transposase-based helper plasmid free piggyBac genetic manipulation
system.
Research
A series of helper plasmids expressing various recombinant piggyBac transposases were generated by
peptide fusion. The transposition activity of these newly constructed helper plasmids was determined in
HEK293, C17.2, CHO, and mouse embryonic stem cells. The piggyBac recombinants of interest were further
evaluated for their ability in promoting iPS formation. A genome wide targeting profiling was conducted to
examine changes, if any, in their targeting preferences.
Observations
Peptide fusion significantly increased the transposition activity of almost all piggyBac recombinant
transposases in a cell type dependent fashion. Among them, TPLGMH and ThyPLGMH, showed the greatest
improvement with a drastic increase in the copy number of integrants. Both TPLGMH and ThyPLGMH display
four-fold increase in generating iPS cells, while only ThyPLGMH showed increased activity in chromosomal
excision, suggesting the potential in generated safer transgene-free iPS by applying TPLGMH for iPS
formation followed by the ThyPLGMH mediated iPS transgenes removal once the transgene is no longer
needed. Genome-wide target profiling revealed that ThyPLGMH exhibits a more random targeting profile with
targets less prone to occur in or near cancer genes, and further away from CpG islands as compared to Myctagged piggyBac and TPLGMH.
Adipose Derived Mesenchymal Stem cells and their efficacy in Skin Injury
Author(s)
Nada M. Alaaeddine
Associate professor, Head of Regenerative and Inflammation lab
Faculty of Medicine, University of St Joseph
Co-Author(s)
Bilal Tarabey - MD fellow, Faculty of Medicine, USJ
Najib Saliba - MD-plastic surgeon, Hotel Dieu de France
Oula El Atat - PhD student, Faculty of Medicine, USJ.
Hussein Hashim - MD-plastic surgeon, Fuad khoury Hospital
Topic
Basic Research
Problem
Recently, plastic surgery has taken a new turn with the use of adipose-derived mesenchymal stem cell (ADMSC). Breast augmentation is one of the major plastic surgery that women compete to perform because they
think it will add to their feministic beauty. Scars in the faces or stretch marks are an additional problem
nowadays where appearances play a major role and have psychological and social impact . No available
permanent and effective treatment are known to drastically reduce or eliminate scars or wounds. Our
objectives were to evaluate the regenerative and repair capacity of adipose-derived stromal vascular fraction
(SVF) and (AD-MSC) on stretch mark treatment, scar, and wound healing.
Background
Several studies have shown that mesenchymal stem cells (MSCs) play an important role in the wound healing
process by secreting cytokines (fibroblast growth factors and vascular endothelial growth factor that promotes
fibroblast proliferation, production and organization of collagen fibers and angiogenesis. In addition, MSCs
behave as pericytes conferring stability to formed vessels, which suggests an involvement of these cells in the
vascularization of wounds, an essential step in the healing process.
Hypothesis
Adipose derived mesenchymal stem cells are known to share with bone marrow mesenchymal stem cells
many characteristics including an immunomodulatory, antiapoptotic, angiogenic, anti-fibrotic, and
chimiotactique effect. Thus they play an important role not only in maintaining tissue homeostasis but also as
potential future therapeutic tools in many tissues injuries including skin repair, wound healing, and scar
reduction.
Research
Autologous AD-MSCs were isolated from thigh or abdomen lipoaspirates, using a well-established protocol.
After patient consent, the synovial vascular fractions (SVF) and/or the cultured ADMSC were injected
subcutaneously on the site of injury.
Observations
20 patients were injected with SVF and/ or AD-MSC for the following indications: 4 patients for scar reduction,
6 for wound healings, 8 for stretch mark and 2 for hair growth. After 40 days of follow up, all the patients were
satisfied with the results and consented to be followed for 12 months. 10 patients out of 12 had their stretch
marks and scars significantly improved in size, color, and texture. The average recovery volume of wounds
and scars was 75% at 12 weeks. The patients with skin scleroderma had hair growth on their eyebrows as
early as 3 weeks. No immediate or delayed adverse effects were seen in any of the patients. Conclusions:
The use of AD-MSCs can be a safe and effective treatment for soft tissue injury and hold a promising future in
regenerative medicine.
Adipose-derived stem cells (ADSC) from aged patients with coronary artery disease keep MSC
properties but exhibit age markers and have an impaired angiogenic potential
Author(s)
Anastasia Yu. Efimenko
MD, PhD, resident
Faculty of Medicine, Lomonosov Moscow State University
Co-Author(s)
Nina Dzhoyashvili - MD, PhD student, Faculty of Medicine, Lomonosov Moscow State University
Natalia Kalinina - PhD, senior researcher, Faculty of Medicine, Lomonosov Moscow State University
Renat Akchurin - Prof., DSc, Russian Cardiology Research and Production Complex of Russian Ministry of
Health
Vsevolod Tkachuk - Prof., DSc, dean, Faculty of Medicine, Lomonosov Moscow State University
Yelena Parfyonova - Prof., DSc, Faculty of Medicine, Lomonosov Moscow State University
Topic
Basic Research
Problem
Mesenchymal stem (stromal) cells (MSC), derived from bone marrow or adipose tissue (ADSC), are
considered to be one of the most promising therapeutic agent for tissue regeneration due to their proliferation
and differentiation potential, ability to stimulate angiogenesis and immunologic privilege. Although ADSC have
already been used in several clinical trials their properties in patients with cardiovascular diseases are poorly
investigated. Most of data regarding ADSC regenerative potential was obtained on cells delivered from
relatively healthy young donors. But it had been known that aging and disease itself may negatively affect
MSC. Impairment of MSC angiogenic properties with age may cause lower effectiveness of augtologous cell
therapy in aged patients with coronary artery disease (CAD) and chronic hind limb ischemia - the most
obvious candidates for therapeutic angiogenesis using stem/progenitor cells.
Background
Cardiovascular diseases including coronary artery disease (CAD) are the most frequent causes of mortality in
most of countries despite of the prominent progress in conservative and surgical approaches for the
stimulation of vascularization. Therapeutic angiogenesis based on the injection of gene constructions with
growth factors or stem/progenitor cells into ischemical tissues provides an attractive novel option to treat such
diseases. ADSC, a promising source for cell therapy, were shown to secrete various angiogenic factors and
improve vascularization of ischemic tissues. It was shown that local and systemic transplantation of ADSC on
the animal models of hind limb ischemia and myocardial infarction (MI) led to the increase in new blood
vessels number and improved blood perfusion within damaged tissue. But age of the patients may be an
important factor affecting ADSC therapeutic potential.
Hypothesis
The aim of the study was to investigate how patient age affects the properties of ADSC with the special
emphasis on their ability to stimulate angiogenesis. We analyzed angiogenic properties of ADSC in the
cohorts of patients both with CAD and without cardiovascular pathology.
Research
ADSC were isolated from surgically obtained subcutaneous fat tissue of patients with (n=64, 43-77 years old)
and without (n=31, 2-82 years old) coronary artery disease (CAD). Age markers such as telomere length,
telomerase activity, expression of p16/INKa and proliferation activity (CFSE assay) were assessed as well as
angiogenic properties of cultured ADSC: ability of ADSC conditioned media to stimulate capillary-like tube
formation by endothelial cells (EA.hy926) on Matrigel, angiogenic growth factors expression (real-time PCR)
and secretion (ELISA) by ADSC.
Observations
ADSC phenotype characterized by flow cytometry was CD90+/CD73+/CD105+/CD45-/CD31- cells for all
samples and cells were capable for adipogenic and osteogenic differentiation. ADSC from aged patients had
shorter telomeres and there was a tendency to attenuated telomerase activity along with decreasing number
of actively proliferating cell and p16/INKa expression in ADSC from patients older than 60 years compared to
younger patients. Conditioned media from ADSC stimulated capillary-like tube formation by endothelial cells
and this effect declined with age both for patients with and without CAD. Level of pro-angiogenic factors
(vascular endothelial growth factor, placental growth factor, hepatocyte growth factor, angiopoetin-1 and
angiogenin) in ADSC conditioned media measured by ELISA significantly decreased with patient age, but
level of anti-angiogenic factors thrombospondin-1 and endostatin did not. There was no age-associated
difference in pro-angiogenic factors gene expression (real-time PCR). At the same time gene expression of
factors related to extracellular proteolysis (urokinase and its receptor (uPAR), plasminogen activator inhibitor1) as well as uPAR surface expression increased in ADSC from aged patients with CAD. in conclusion ADSC
from aged patients express age markers and their angiogenic properties decline due to the decreasing of proangiogenic factors secretion. Our data provide new insights into mechanisms of age-associated impairment of
autologous ADSC therapeutic potential.
Age-dependent Responses to Ischemia-Reperfusion (IR) Injury in Rats After Allogeneic Mesenchymal
Stem Cell (MSC) Transplantation
Author(s)
Rosemeire M. Kanashiro-Takeuchi
Research Assistant Professor
University of Miami
Co-Author(s)
Lauro Takeuchi - Research associate III, University of Miami
Michael Hurtado - Graduate student, University of Miami
Savannah Moon - Volunteer, University of Miami
Qinghua Hu - Assistant Professor, University of Miami
Ian McNiece - Professor, University of Miami
Joshua Hare - Professor, University of Miami
Topic
Basic Research
Problem
Cardiovascular diseases and its complications are major risk factors in the elderly, and myocardial infarction in
that group is associated with increased morbidity and mortality.
Background
It is increasingly appreciated that host factors play crucial roles in the responsiveness to cell therapy for
ischemic heart disease.
Hypothesis
The overall goal of this study is to determine the efficacy of cell based therapies in a rat model of
cardiovascular aging. We tested the hypothesis that advanced age would impair cardiac repair following acute
MI.
Research
Allogeneic bone marrow-derived MSC (5.0x106 cells) from a young (3 month-old) male GFP-transgenic rat or
PBS were injected intramyocardially after ischemia (45 min) and reperfusion (5 min) in young and old (6 and
22 month-old, respectively) female Fisher-344 rats. Cardiac structure and function were assessed before and
after cell transplantation by echocardiography. Hemodynamic measurements by pressure-volume
conductance catheter system were performed at week 8 before sacrificing the animals.
Observations
In young animals, MSC therapy improved EF at week 8 (from 40.5±1.1 to 54.8±1.2%, p<0.01); however, this
effect was markedly attenuated in old rats (39.1±1.1 vs. 43.3±2.9%, placebo vs. treated, same age, p=NS).
Similarly, hemodynamic measurements (table) showed an increase in myocardial contractility - preloadrecruited-stroke-work (PRSW) in young rats (from 28.4±1.4 to 39.3±2.0; placebo vs. treated, p<0.05) but not in
old rats (26.7±3.8 vs. 34.7±4.4; placebo vs. treated, p=NS). Heart rate was similar in all groups.
Table. Hemodynamic measurements
Young
Old
pvalue
Placebo (6)
Treated(5)
Placebo (6)
Treated (9)
223±2.4
219±20.8
217±13.2
222±18
NS
PRSW
28.4±1.4
1
26.7±3.8
34.7±4.4
0.0053
Ees
6.0±1.2
9.2±1.5
6.2±1.4
6.1±0.8
0.0725
Emax
24.9±2.8
32.3±3.7
24.7±5.1
27.3±2.6
0.2877
dP/dt max
6453±475
6911±440
2
2
Heart rate
Contractility
*39.3±2.0
*5217±445
*5518±256
One-Way ANOVA followed by Student-Newman-Keuls pos-hoc analysis. 1* p<0.05 vs. Young placebo and
vs. Young placebo and Young treated.
0.0018
Old placebo; 2* p<0.05
ANALYSIS OF ADMINISTRATION ROUTES OF STEM CELLS FROM HEPATIC BUD OF RATS AS A
MODEL FOR LIVER DISEASE TREATMENT
Author(s)
Maria Angelica Miglino
Professor,PhD, Veterinarian
Universidade de São Paulo, Departamento de Cirurgia, Pós-graduação em Anatomia dos Animais
Domésticos e Silvestres
Co-Author(s)
Amanda Ferreira, FMVZ - MSC veterinary, USP
Lucas Nacif, FMUSP - doctor, USP
Wellington Andraus, FMUSP - PhD, USP
Luiz albuquerque, FMUSP - professor titular, USP
Ana Claudia Carreira, IQUSP - PhD, USP
Mari Cleide Sogayar, IQUSP - professor titular, USP
Durvanei Maria, IB - PhD professor, Instituto Butantan
Topic
Basic Research
Problem
The use of cell therapy with undifferentiated hepatic embryonic mesenchymal stem cells as a model for the
study of degenerative diseases.
Background
Stem cells are virtually capable of differentiating into all cell types and may be used for replacement therapies.
In several diseases. Hepatic cell from embryonic and fetal lineages can be an important source for cell
therapy in patients with liver disease due to its high rate of differentiation into hepatocytes and bile duct
cells, as well as the formation of hematopoietic precursors.
Hypothesis
The biodistribution and colonization of stem cells from rat liver bud is efficient for subsequent treatment
of hepatic insufficiency.
Research
50 inbred rat of Sprague dawley strain underwent a 70% hepatectomy surgery to evaluate the
biodistribution of cell migration, using the image analysis system PET scan. Undifferentiated stem cells
from rat liver bud with 12.5 days of gestation were used, being transduced with lentiviral vector GFP +
and administered to animals at a concentration of 104 cells at the end of every surgery. 10 animals were
used for four routes of administration (oroenteral, intravenous, endotracheal intraperitoneal) and 10
animals for the control group (untreated).
Observations
We obtained the following results: in the oroenteral application there was a significant concentration of stem
cells from bud liver in the lung and liver parenchyma at 7 to 21 days. On intravenous administration, it was
observed that after 15 days the stem cells from liver bud was concentrated in lung and liver parenchyma and
a small portion in areas near the intestines and kidneys. On endotracheal administration there was a high
concentration of stem cells specifically in the lung parenchyma from 7 to 21 days. Moreover, on intraperitoneal
administration there was a large marking of stem cells in kidney and intestine within 7 to 21 days.
Through the chemical analysis performed in this study, cellular migration obtained by PET scan revealed that
in oroenteral via, cells were distributed preferentially in the liver parenchyma and remained significantly viable
throughout the experimental period.
Application of insulin producing cells differentiated from PDX1-transduced human adipose tissue
derived stem cells for diabetes treatment in rats
Author(s)
Zahra Niki
stem cell biology
National Institute of Genetic Engineering and Biotechnology
Ahmad Aleyasin
cellular and molecular biology
National Institute of Genetic Engineering and Biotechnology
Role: Author
Topic
Basic Research
Problem
Diabetes mellitus (DM), which causes about 5% of all deaths globally each year, afflicts 246 million people
worldwide presently and will affect 380 million by 2025.
Background
Diabetes mellitus is caused by insulin deficiency because of auto immune destruction of islet beta cells. The
transplantation of pancreatic cells is a promising approach for cure of diabetes mellitus. however, lack of
suitable donors limits the application.
Hypothesis
Differentiated human adipose tissue derived mesenchymal stem cells (hADSCs) can be a viable therapeutic
source for the treatment of type 1 diabetes.
Research
Human ADSCs were isolated and identified by flow cytometric analysis and were differentiated into adipocyte
and osteocyte by differentiating medium to confirm of their multipotency. PDX1-harboring lentiviruses were
constructed and infecced ADSCs. After infection, ADSCs were cultured in high glucose DMEM medium
supplement by B27, nicotin amid and βFGF for 21 days. Expression of pancreatic markers were confirmed by
immunocytochemistry test and real time PCR.
Observations
The appropriate expression of ectopic PDX1 and insulin were confirmed in the level of protein using
immunocytochemistry analysis. Differentiated cells were secreted insulin (2.32μU/ml) into culture medium in
glucose challenge. Also expression of Ngn3, Glut2, insulin, glucagon, Pax4, PDX1 and somatostatin as
specific marker genes of pancreatic cells was investigated by quantitative RT-PCR. Finally, differentiated cells
were injected into diabetic rats and decreased their blood glucose level.
Artificial epigenetic switches to selectively reprogram the transcriptional machinery conferring to cell
fate
Author(s)
Namasivayam Ganesh Pandian
Research Associate
iCeMS. Kyoto University
Co-Author(s)
Shinsuke Sato, Mr. - Research Assistant, iCeMS, Kyoto University.
Yusuke Nakano, Mr. - Graduate Student, Department of Chemistry, Kyoto University.
Akimichi Ohtsuki, Dr. - PostDoc., Department of Chemistry, Kyoto University.
Abhijit Saha, Mr. - Doctoral Student, Department of Chemistry, Kyoto University.
Toshikazu Bando, Dr. - Associate Professor, Department of Chemistry, Kyoto University
Akira Watanabe, Dr. - Assistant Professor, CiRA, Kyoto University.
Hiroshi Sugiyama, Dr. - Professor, iCeMS and Department of Chemistry, Kyoto University.
Topic
Basic Research
Problem
Transcriptional “ON-OFF” states governed by chromatin modifications aids the storage and retrieval of the
information pertaining to cell fate. Consistent with this notion, artificial induction of pluripotency in somatic cells
through enforced expression of the four factors was achieved to offer new modes of therapy. Notwithstanding
the recent promising breakthroughs, several barriers hamper the clinical translation of induced pluripotent
stem cells (iPSCs). Complete chemical reprogramming is proclaimed as one of the approaches that could
improve the clinical utility of iPSCs. In this context, small molecules that could mimic natural transcriptional
factors and precisely switch “ON” the transcriptional machinery conferring to pluripotency are of increasing
demand.
Background
Taking cues from nature, it is evident that the artificial transcriptional activators should retain both the
sequence-specificity and epigenetic activity of their natural counterparts for effective participation in extremely
complex pluripotency gene networks.
Hypothesis
Our strategy is to construct selective epigenetic switches by conjugating epigenetic modifiers with
programmable DNA binding pyrrole-imidazole polyamides (PIPs) that can permeate the live cells and
selectively recognize each of the four Watson–Crick base pairs. Our notion is that by supplementing
sequence-specificity to the chromatin modifiers, precise up-regulation of gene(s) conferring to pluripotency
could be achieved.
Research
We screened our synthetic small molecules called SAHA-PIP that encompasses the diverse sequencespecific PIPs and chromatin modifying histone deacetylase inhibitor like SAHA for their effect on the iPSC
factors in mouse embryonic fibroblasts (MEF).
Observations
Screening of our first library of SAHA-PIPs revealed that certain SAHA-PIPs could differentially activate the
iPSC factors by triggering epigenetic marks that are associated with transcriptionally permissive chromatin in
MEF (ChemBioChem., 2011, 12, 2822). Synthesis and screening of a series of derivatives of our hit SAHAPIP indicated that our programmable small DNA-binding SAHA-PIPs could be developed to induce the
specific expression of core pluripotency genes (Bioorg. Med. Chem., 2012, 20, 2656). In the second library of
SAHA-PIP with improved sequence recognition ability, a novel SAHA-PIP termed, `δ` dramatically induced
Oct-3/4 and Nanog in MEF (Sci. Rep., 2012, 2, e544). Genome-wide gene analysis suggested that `δ`
induced multiple pluripotency genes by more than ten-fold to initiate cellular reprogramming in just 24 h.
Unlike other small molecules currently employed for stem cells, PIP conjugates can be tailored to bind with
predetermined DNA sequences (Biotechnol. J., 2012, 7, 798). Therefore, strategies to expand our tunable
epigenetic switches could open new vistas of opportunities in cellular reprogramming as they may precisely
send the somatic cells back in time to their embryonic state and/or coax them into totally new types of cells.
Bone marrow endothelial progenitors augment atherosclerotic plaque regression in a mouse model of
plasma lipid lowering
Author(s)
Jana Barlic-Dicen, Ph.D.
Assistant Member
Cardiovascular Biology, OMRF
Co-Author(s)
Longbiao Yao, MD - Staff Scientist, Cardiovascular Biology, OMRF
Janet Heuser-Baker - Senior Research Assistant, Cardiovascular Biology, OMRF
Oana Herlea-Pana, Ph.D. - Assistant Staff Scientist, Cardiovascular Biology, OMRF
Topic
Basic Research
Problem
Atherosclerosis and its complications remain a major cause of mortality worldwide. Deposition of low density
lipoprotein in the vascular subendothelium and the pro-inflammatory reactions of resident cells, which trigger
influx of inflammatory leukocytes into the vascular wall, both lead to development of atherosclerotic plaques
that can rupture, causing myocardial infarction and stroke. Statins (HMG-CoA reductase inhibitors) are the
primary intervention against atherosclerosis. In patients at risk or suffering from cardiovascular disease,
statins effectively lower plasma cholesterol, impede atherosclerosis progression, stabilize plaques and
moderately reduce adverse cardiovascular events. However, recent clinical trials show that long-term
administration of statins causes incomplete plaque regression, leaving patients prone to adverse
cardiovascular events. Thus, understanding of mechanisms that promote complete plaque resolution is
important for the development of novel or improved anti-atherosclerotic therapies.
Background
Endothelial progenitor cells (EPCs) are thought to have an important role in tissue regeneration, especially in
vascular repair. These cells support postnatal neovascularization by homing to and differentiating into mature
CD31-positive endothelial cells in damaged endothelium, promoting vasculogenesis, thereby directly
contributing to endothelial regeneration. EPCs may also produce angiogenic cytokines and growth factors that
promote proliferation of existing resident endothelial cells, activate angiogenesis, and thereby indirectly
contribute to the re-establishment of endothelial homeostasis.
Hypothesis
Given the beneficial effects of EPCs in vascular repair, we hypothesized that these cells play a pivotal role in
atherosclerosis regression.
Research
We tested our hypothesis in the atherosclerosis-prone mouse model in which hypercholesterolemia, one of
the main factors affecting EPC homeostasis, is reversible (Reversa mice). In these mice normalization of
plasma lipids decreases atherosclerotic burden; however, plaque regression is incomplete. To explore
whether endothelial progenitors contribute to atherosclerosis regression, bone marrow EPCs from a
transgenic strain expressing green fluorescent protein under the control of endothelial cell-specific Tie2
promoter (Tie2-GFP) were isolated. These cells were then adoptively transferred into atheroregressing
Reversa recipients where they augmented plaque regression induced by reversal of hypercholesterolemia.
Advanced plaque regression correlated with engraftment of Tie2-GFP EPCs into endothelium and resulted in
an increase in atheroprotective nitric oxide and improved vascular relaxation. Similarly augmented plaque
regression was also detected in regressing Reversa mice treated with the stem cell mobilizer AMD3100 which
in addition to hematopoietic stem cells also mobilizes EPCs to peripheral blood.
Observations
Our investigation based on the use of EPC mobilizer AMD3100 and administration of pure endothelial
progenitors suggests application of EPCs in combination with statins in treatment of atherosclerosis, making
regression of atherosclerotic plaques more efficient than lipid-lowering treatment alone.
C-kit+ cells isolated from developing kidneys are stem/progenitor cells with regenerative potential
Author(s)
Erika Rangel
MD, PhD Postdoctoral fellow
Interdisciplinary Stem Cell Institute
Co-Author(s)
Samirah Gomes, MD, PhD - Postdoctoral fellow, ISCI
Raul Dulce, PhD - Postdoctoral fellow, ISCI
Courtney Premer, BS - research associate, ISCI
Claudia Rodrigues, PhD - Research assistant professor, ISCI
Rosemeire Kanashiro-Takeuchi, PhD - Research Assistant Professor, ISCI
Behzad Oskouei, MD - Assistant Professor of Clinical Medicine, UM
Decio Carvalho, MD - Medical assistant, UM
Phillip Ruiz, MD, PhD - Professor , UM
Jochen Reiser, MD, PhD - Professor, UM
Joshua Hare, MD - Professor, ISCI
Topic
Basic Research
Problem
Chronic kidney disease is increasing at a rate of 6-8% annually in the United States alone, but at present
dialysis and transplant remain the only treatment options. The search for putative stem cells or precursors
within the kidney has been the focus of extensive research. The identification of a kidney stem cell population
would provide important biological insights and could be utilized therapeutically to generate new tubular,
glomerular, and vascular cells for the treatment of both acute and chronic kidney injuries.
Background
The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue
homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell
remains elusive.
One such stem cell marker is the c-kit receptor (CD117), a tyrosine kinase receptor. While c-kit is present on
certain differentiated cells, it has been described as a marker of stem cells in many organs and tissues,
including bone marrow, amniotic fluid, lungs, heart, and liver. Importantly, c-kit+ cells have also been identified
during metanephric mesenchyme (MM) development and its ligand, the stem cell factor (SCF), is abundantly
expressed in the ureteric bud.
Hypothesis
We hypothesized that c-kit+ cells represent a novel population of kidney stem cells isolated from neonatal
kidney and found in a specific niche corresponding to the thick ascending limb of Henle's loop.
Research
We isolated a neonatal kidney-derived c-kit+ cell population that fulfills all of the criteria of a kidney stem cell.
These cells originate in the thick ascending limb of Henle’s loop and exhibit clonogenicity, self-renewal, and
multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Individual clones of these
c-kit+ cells, in nonadherent conditions, formed spheres that expressed markers of stem cells, progenitors, and
differentiated cells. Ex-vivo expanded c-kit+ cells integrated into several compartments of the kidney, including
tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney
following acute ischemia-reperfusion injury in rats.
Observations
Together these findings document a novel neonatal rat kidney c-kit+ cell population that can be isolated,
expanded, cloned, and differentiated. These cells possess the potential to treat renal failure by cellular repair
through multi-compartment engraftment following acute kidney injury and have important biological and
therapeutic implications.
Combination of TGF-β3-releasing pharmacologically active microcarriers and human cartilage
microparticles to enhance hyaline cartilage formation by MIAMI cells for articular cartilage repair.
Author(s)
Gaetan J-R. Delcroix, PhD
Post Doc
University of Miami Tissue Bank
Co-Author(s)
Gianluca D'Ippolito, PhD - Research Associate Professor, University of Miami Tissue Bank, Miami, FL
Lourdes Gomez, MD - Research Associate, GRECC and Research Service, Bruce W. Carter Veterans Affairs
Medical Center, Miami, FL
Teresita Reiner, DVM - Research Health Scientist, Pathology, GRECC and Research Service, Bruce W.
Carter Veterans Affairs Medical Center, Miami, FL
Claudia Montero-Menei, PhD - Associate Professor, INSERM U1066, University of Angers, France
Theodore Malinin, MD - Professor, University of Miami Tissue Bank, Miami, FL
H Temple, MD - Professor, University of Miami Tissue Bank, Miami, FL
Paul Schiller, PhD - Research Associate Professor, GRECC and Research Service, Bruce W. Carter Veterans
Affairs Medical Center, Miami, FL
Topic
Basic Research
Problem
Among the various treatments available for articular cartilage defects, none are completely satisfactory in
terms of biomechanical properties and composition of the newly formed cartilage.
Background
Articular cartilage defects are common, particularly in the young and active population. Microfracture of the
subchondral bone is a widely used technique to treat these, but it frequently produces fibrocartilage.
Autologous chondrocyte implantation (ACI) offers the possibility of hyaline cartilage regeneration, but has
several drawbacks such as graft failure, delamination, tissue hypertrophy as well as harvest site morbidity.
Hypothesis
Our goal is to create an engineered product that can support multipotent mesenchymal stromal cells and
induce cartilage regeneration. We hypothesize that combination of stem cells with a growth factor-delivering
biocompatible and biodegradable biomaterial and/or with human cartilage microparticles will guide and
enhance the formation of hyaline articular cartilage.
Research
A primitive population of mesenchymal stromal cells (MSCs), the Marrow-Isolated Adult Multilineage Inducible
(MIAMI) cells was isolated from swine bone-marrow and used in this study. Use of pharmacologically-activemicrocarriers (PAMs) releasing TGF-β3 has previously been shown to induce the chondrogenic differentiation
of MSCs both in vitro and in vivo, with an increased production of Aggrecan and Collagen II. Use of human
cartilage microparticles (100-200µM in diameter) to fill osteochondral defects created in the femoral condyles
of baboons has also demonstrated a strong potential for cartilage regeneration. The aim of the present in vitro
study was to evaluate the chondrogenic differentiation of swine MIAMI cells in contact with PAMs, human
cartilage microparticles, or a combination thereof. The chondrocytic differentiation of MIAMI cells in contact
with those scaffolds was assessed by RT-qPCR and histological analysis of the neo-cartilage pellets formed in
vitro.
Observations
Attachment of MIAMI cells to either the PAMs releasing TGF-β3 or the human cartilage microparticles
enhanced cell survival in vitro as well as the expression of Aggrecan, a key extracellular matrix (ECM) protein
highly expressed in hyaline cartilage. This supported our previous data obtained with PAMs as well as
recently published studies describing the chondrogenic effect of scaffolds derived from human cartilage. Most
importantly, the strongest effect in terms of cell survival and hyaline-specific cartilage gene expression was
obtained with the combination of PAMs releasing TGF-β3 and human cartilage microparticles. Thus, MIAMI
cells combined with PAMs releasing TGF-β3 hold great promise for hyaline cartilage regeneration, while
combination with human cartilage microparticles could provide additional biomechanical and chondroregenerative support during the healing process of hyaline cartilage. In the future, we expect that combination
of MIAMI cells/TGF-β3 PAMs/cartilage microparticles will help improving the microarchitecture and
mechanical properties of the repaired cartilage tissue in a rapid and minimally invasive fashion.
Culture of human mesenchymal stem cells (hMSC) using a pharmaceutical grade xeno-free cell
culture supplement derived from human plasma
Author(s)
Jose M. Diez
Head of Department
Instituto Grifols S.A.
Co-Author(s)
Juan Ignacio Jorquera, PhD - Vice President R&D , Instituto Grifols, S.A.
Rodrigo Gajardo, PhD - Senior Manager Viral Safety Division, Instituto Grifols, S.A.
Topic
Basic Research
Problem
hMSC are multipotent cells with capacity to differentiate into several lineage-specific cells. hMSC are usually
cultured in media supplemented with foetal bovine serum (FBS).The use of material from animal origin can
cause the risk of introducing transmissible agents.
Background
The use of FBS (or no xeno-free supplements) is appropriate for in vitro research but, due to its animal origin,
it could be a matter of concern if the cells are used for cell therapy. Therefore, it is desirable to perform the
culture in xeno free conditions.
Hypothesis
An industrial, xeno-free, pharmaceutical grade human plasma-derived cell culture supplement (hCCS) is being
developed in Grifols. This material avoids the concern of using FBS or non xeno-free supplements in
advanced therapies, while hMSC are fully functional, undifferentiated and multipotent.
Research
Grifols' hCCS is obtained from industrial cold ethanol human plasma fractionation and it is manufactured
under GMP rules. The cell growth was evaluated subculturing hMSC with different media. Cell viability and
cell yield were calculated with the aid of an automated cell counter (Invitrogen). Cell surface markers were
studied by indirect immunofluorescence assay (IFA) onto cell cultures. Cell differentiation into neuron after
culturing in the desired media was performed using a commercial neurogenic differentiation medium
(PromoCell) .
Observations
Grifols' hCCS is a xeno-free pharmaceutical grade cell culture supplement which supports the growth of
commercial hMSC from bone marrow (viability higher than 90% and cell yield equivalent to other commercial
media) while maintaining the characteristic cell surface marker phenotype of an undifferentiated hMSC as the
IFA showed. The hMSC cultured with hCSS maintains the potential of differentiation into neurons after
differentiation induction. hCCS has high consistency among lots and it is a highly stable gamma irradiated
freeze-dried product. Moreover, hCCS can additionally be used for the culture of several other cell types
(human and animal). Grifols’ hCCS could be an appropriate cell culture supplement for advanced therapies.
Curcumin inhibits and Retinoic acid increases the expression of RANKL in human umbilical cord stem
cells (hUMSCs)
Author(s)
Umadevi Kandalam, Ph.D
Assistant Professor
Nova Southeastern University
Chun YC. Huang
Assistant Professor
University of Miami
Role: Author
Co-Author(s)
Chun Yuh Huang, Ph.D - Assistant Professor, University of Miami
Topic
Basic Research
Problem
This study was intended to investigate their role in regulation of osteogenesis with special reference to the
expression of receptor activator of nuclear factor –κB ligand (RANKL), osteoprotegerin (OPG) in human
umbilical cord mesenchymal stem cells.
Background
Curcumin (CUR), derived from curcuma longa, and Retinoic acid, a derivative of B carotene are known to
influence several cell signaling pathways.
Hypothesis
Our hypothesis is Curcumin and Retinoic acid induce ostegeneic differentiation
Research
The hUMSCs obtained from Sciencell (Carlsbad, CA) were used in this study. The cells were cultured in
complete medium containing low glucose Dulbecco’s modified eagle medium with 10% of fetal bovine serum
and 1% antibiotic and antimycotic solution at 37°C in 5% CO2. 50 to 60 % confluent cells were treated with
curcumin (1µM, 2µM and 5µM) and transferred in to osteogenic medium. Cells grown in similar culture
conditions without curcumin were considered as control. Gene expression of OPG, RANKL was measured by
quantitative PCR. Matrix mineralization was measured by alizarin red staining after 3 weeks of osteogenic
induction.
Observations
Our results demonstrated dose dependent up regulation with maximal at 2 µM was observed in expression of
OPG in curcumin induced cells. Retinoic acid down regulated the expression of OPG and significantly up
regulated RANKL expression than control at all concentrations.
Derivation of functional sensory neurons from human neural progenitor hNP1
Author(s)
Xiufang (Nadine) Guo
Research Scientist
University of Central Florida
Co-Author(s)
Severo Spradling - Undergraduate student, Burnett School of Biomedical Sciences, University of Central
Florida
Maria Stancescu - Research Associate, NanoScience Technology Center, University of Central Florida
Stephen Lambert, Dr. - Associate professor, College of Medicine, University of Central Florida
James Hickman - Professor, NanoScience Technology Center, University of Central Florida
Topic
Basic Research
Problem
Sensory neurons constitute a critical component for the proper function of the nervous system. An in vitro
source of human sensory neurons is highly desirable for developing functional human disease models for
pathological studies and drug screening, as well as providing a renewable cell source for applications in
regenerative medicine.
Background
Stem cells provide a potentially unlimited source for generating specialized neuron subtypes for disease
modeling and cell therapy. In vitro differentiation of functional sensory neurons from human neural stem cells,
in the absence of murine cell feeder layers, has not yet been reported.
Hypothesis
Functional human sensory neurons can be generated from neural progenitors in a defined system.
Research
Human neural progenitor cells (hNP1) were induced to differentiate down the sensory neuron lineage in a
defined, feeder layer-free, in vitro culture system. The identities of derived cells were characterized with
immunocytochemistry and the functional maturation of sensory neurons was evaluated by electrophysiology.
Neural crest (NC) precursors, as one of the cellular derivatives in the differentiated culture, were isolated,
propagated, and tested for their ability to generate sensory neurons.
Observations
Immunocytochemistry characterization indicated that neurons (positive for β III Tubulin and Peripherin) and
Schwann cells (positive for S100) accounted for 51.3 +/- 3.7 % and 48.0 +/- 2.5 %, respectively, of the cells in
culture after differentiation. Patch clamp analysis validated the functional maturation of these sensory
neurons. NC precursors isolated from this culture preserved the potential for generating sensory neurons. In
summary, this study provides a convenient and efficient approach for generating functional human sensory
neurons in vitro from a reliable stem cell source.
DEVELOPMENT AND VALIDATION OF A qPCR ANALYSIS OF MALE PORCINE CELL
BIODISTRIBUTION IN FEMALE PIGS
Author(s)
Maoxiang Li
Senior Scientist, Cellular & Molecular Biology
MPI Research, Inc.
Role: Author
Background
Bio
Haiyan Ma, Ph. D.
Principal Scientist and Manager of Cellular and Molecular Biology
MPI Research
Co-Author(s)
Emily Hill - Research Associate 2, MPI Research
Jeffrey Grover - Research Associate, MPI Research
Valerie Steenwinckel, Ph.D. - Manager, Cardio3 BioSciences S.A.
Mark Wolfe, B.S. - Senior Scientist, MPI Research
Mark Johnson, M.S. - Director, MPI Research
Topic
Basic Research
Problem
Stem cells, whether gene-modified or not, present a number of safety concerns after administration into
human subjects. Major questions have been raised as to where stem cells migrate to after administration,
whether the cells engraft and differentiate, and what their long-term fate is. Therefore, GLP-compliant
preclinical biodistribution and persistence animal studies are recommended by the United States Food and
Drug Administration (FDA) to evaluate the safety of the stem cell therapy products.
Background
The preclinical biodistribution studies are usually performed using immunocompromised rats and mice. While
a large animal assay is required for some of the relevant functional analysis, a sex-mismatched
transplantation may be used to overcome the lack of available immunocompromised large animals. With this
approach, male stem cells are injected into female animals and the Y chromosome specific SPY gene is the
target to be detected by quantitative real time PCR
Hypothesis
For this purpose, a quantitative real time polymerase chain reaction (qPCR) method for biodistribution
analysis of male mesenchymal stem cells (MSC) in female pig tissues has been developed and validated.
qPCR is currently the most sensitive method to analyze the biodistribution of cell and gene therapy products.
Research
In this study, the specificity of the TaqMan probe/primers to the male porcine sex-determining region Y (SRY)
gene sequence was determined by carrying out the real-time PCR of male porcine MSC genomic DNA in the
absence and presence of female pig matrix genomic DNA (gDNA). The condition of the qPCR analysis was
optimized. The intra-assay precision, inter-assay reproducibility, and accuracy were evaluated over five
separate assays. For each assay, one set of standard curve and two sets of quality control (QC) samples
were prepared independently and tested by qPCR.
Observations
The assay specificity, linear range, intra- and inter-assay precision, accuracy and reproducibility, and
acceptance criteria are reported. The low limit of detection (LLOD), low limit of quantification (LLOQ),
and upper limit of quantification (ULOQ) are 5, 50, and 285,000 copies of male porcine MSC gDNA per
micrograms of total DNA, respectively. Assuming male and female porcine cells contain roughly equal amount
of gDNA, the linear detection range of male porcine cells in the background of female porcine cells ranges
from 0.0175% to 100%, and the limit of detection is 0.00175%.
Development of an autologous stem cell therapy for the accelerated healing of the anterior cruciate
ligament following surgical reconstruction
Author(s)
Callie An. Knuth
MRes Stem Cells and Regenerative Medicine
Newcastle University, Newcastle-Upon-Tyne, England
Co-Author(s)
Annette Meeson, Dr. - Professor, Newcastle University
David Deehan, Professor - Orthopedic Surgeon , clinical professor, RVI, Newcastle University
Rachel Oldershaw, Dr. - Post-Doctoral Fellow, Newcastle University
Topic
Basic Research
Problem
During surgical reconstruction of the anterior cruciate ligament (ACL), many grafts fail due to poor
osteointegration at the bone tendon interface. By using autologous stem cells, obtained from traumatic
haemarthrosis fluid, a stem cell treatment can be developed that would improve this osteointegration, thus
improving ACL repair.
Background
Repairing the anterior cruciate ligament (ACL) following injury is slow due to poor osteointegration and
impacts patient recovery. Recently, a mesenchymal stem cell (MSC) population has been identified within
haemarthrosis fluid (HF; bleeding accumulated during ACL injury) which exhibits the capacity for
differentiation and could be used in cellular therapies to facilitate and accelerate the repair of the ACL in injury
situations. By optimizing the condition in which HF-MSC are best derived it will be possible to determine when
these cells are best able to perform and be utilized in a cellular therapy, a therapy which would decrease
patient recovery time and the high economic and sociological costs associated with ACL repair.
Hypothesis
This study was conducted to define the derivation and the effects of in vitro expansion of HF-MSC under
hypoxic oxygen concentrations. In addition normoxic and hypoxic oxygen tension was investigated to
determine if a difference exists in HF-MSC health, proliferation, and differentiation capability. The SP
phenotype within isolated HF-MSC was also investigated to determine if the presence of the phenotype would
decrease interpatient variability observed between cultures.
Research
HF-MSC were incubated in 5% and 20% oxygen and derivation/growth was measured. Phenotypic analysis of
MSC was carried out by immunolabelling and flow cytometry. Cells were cultured in osteogenic media for 28
days to achieve differentiation and morphological features were compared. Metabolic activity and cellular
health was analyzed utilizing an alamar blue assay. Alizarin red (AR) staining was performed to visualize the
presence of calcium deposits in differentiated cell populations, commonly observed in osteocytes. SP
expression was investigated utilizing flow cytometry.
Observations
Under hypoxic conditions significantly more samples derived (Hypoxia: 100%, Normoxia 70%, P
Economical method for the production of cGMP grade Mesenchymal Stem Cells from Umbilical Cord
Tissue, Bone Marrow and Cord Blood
Author(s)
Arati A. Inamdar
Research Associate
Rutgers, The State University of New Jersey/Saiseva Biotech Pvt Ltd
Co-Author(s)
Ajinkya Inamdar - Managing Director, Saiseva Biotech Pvt Ltd
Topic
Basic Research
Problem
The promising role of mesenchymal stem cells (MSCs) in various autoimmune conditions warrants ample
supply of MSCs. MSCs can be obtained from various sources including umbilical cord, placenta, amniotic
fluid, cord blood and bone marrow. The use of fetal bovine or fetal calf serum poses risk to human culture
especially if the grown MSCs are intended for therapeutic usage.
Background
To discuss the economically method for the production of cGMP grade MSCs from different tissue sources
obtained from animal origin free serum supplemented culture media.
Hypothesis
Serum obtained from peripheral blood and cord blood can be used to obtain animal origin free serum.
Research
We have used umbilical cord tissue, bone marrow and cord blood as tissue source for the production of cGMP
grade MSCs. To enrich the culture media with animal origin free serum, we have supplemented the culture
media with the growth factors from pooled platelet lysate obtained from peripheral blood and cord blood to
grow MSCs from these tissues.
Observations
We have assessed the growth potential of these MSCs cultured under our cGMP laboratory conditions using
the serum obtained from these two sources. We report that the rate of growth of MSCs from Umbilical Cord
Tissue, Bone Marrow and Cord Blood in presence of peripheral blood derived serum was better than those
growing in cord blood derived serum. However, there was no significant difference in the morphology of MSCs
obtained in presence of serum from these two sources. We therefore confirm the use of peripheral blood and
cord blood derived serum for the production of cGMP grade cells in economically favorable way.
Effect of Autologous mesenchymal Stem Cells in cerebral metabolism - PET Scan study in Autism – A
case Report Submission Type- Science and Medicine
Author(s)
Anant E. Bagul Sr.
Clinical research officer
Chaitanya hospital,Pune
Co-Author(s)
Sachin Jamadar, CRO - CRO, Chaitanya Hospital
Prakash Jadhavar, CRO - CRO, Chaitanya Hospital
Topic
Basic Research
Problem
Autism is one of those disorders in Autism spectrum disorders (ASD), which characterized by social
interaction abnormalities, impaired verbal and non-verbal communication, and repetitive, obsessive behavior.
Background
Autism is one of those disorders in Autism spectrum disorders (ASD), which characterized by social
interaction abnormalities, impaired verbal and non-verbal communication, and repetitive, obsessive behavior,
while the therapeutic effect of current treatments remains limited progress .Neural hypoperfusion and immune
deregulation are the two key pathologies associated with Autism. Human mesenchymal stem cells have
been shown to have the ability to modulate the immune response and enhance angiogenesis, suggesting the
novel and promising therapeutic strategy. In this study, the safety and efficacy of MSCs transplantation will be
evaluated in patients with Autism.
Hypothesis
Evidence suggests that hypoperfusion and resulting hypoxia is intimately associated with autism, however the
next important question is whether reversion of this hypoxia can positively influence autism.
MSCs show a high expansion potential, genetic stability, stable phenotype, high proliferation rate as adherent
cells, and self-renew capacity
MSCs are known to be potent angiogenic stimulators, having demonstrated positive effects in cerebral
Regeneration and reversal of hypoperfusion
Research
We proposed to study effect of Bone marrow derived mesenchymal cell on hypo metabolic areas of brain .in
22 yrs old autistic female patient We have used PET- CT scan using 4.6 mCi of F18 FDG , following
intravenous and intra-thecal injection of BMMSC in our pilot study.
Observations
As per our study, marked increase in uptake of F18 FDG in fronto temporo parietal region as evident by
picture. Patient also had clinically significant improvement in various clinical paramenter, as
per Childhood Autism Rating Scale and Improvement in the Clinical Global Impression Scale.(Pl check
photos as in the posters)
Effect of Insulin- like growth factor I (IGF-I) on proliferation and viability of bovine type A
spermatogonia
Author(s)
Babak Qasemi-Panahi Sr.
Dr. Qasemi-Panahi
Department of Animal Science, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
Co-Author(s)
Parviz Tajik - professor, Department of Clinical Science, Faculty of Veterinary Medicine, University of Tehran,
Tehran, Iran
Mansoureh Movahedin - professor, Department of Anatomy, Faculty of Medical Science, University of Tarbiat
Modares, Tehran, Iran.
Gholamali Moghaddam - professor, Department of Animal Science, Faculty of Agriculture, University of
Tabriz, Tabriz, Iran.
Topic
Basic Research
Problem
Spermatogonial stem cells (SSCs) are the best candidate for use in genetic improvement of cattle herds. In
fact, SSCs could be transmitting genetic information to future generations. So, in vitro Proliferation and
maintenance of SSCs is important.
Background
a system that supports the proliferation and maintenance of SSCs in vitro could be used to preserve and
expand SSCs numbers as well as increase success in transplantation.
Hypothesis
IGF-I consists of 70 amino acid residues with three disulfide bridges, is homologous to proinsulin. Synthetic
IGF-I can inhibit of apoptosis and it has mitogenic effect on cells. In this context we examined the effect of
IGF-I on proliferation and viability of bovine type A spermatogonia.
Research
In the present study, bovine SSCs were co-cultured with sertoli cell, with or without Insulin- like growth factor I
(IGF-I). The isolated cells were cryopreserved for one month and then thawed and viability rate of cells were
assayed.
Observations
During 16 days incubation, 100 ng/ml IGF-I showed higher efficacy in increasing of number and diameter of
SSCs colonies than 40 and 150 ng/ml. Furthermore, 100 ng/ml IGF-I increased the frozen/thawed cells
viability rate.
Effect of long term proliferation of adipose derived stem cell (ADSC) on immunomodulation and
carcinogenesis markers
Author(s)
Oula El Atat
Ph.D student
Faculty of Medicine, University of St Joseph
Role: Author
Co-Author(s)
George Hilal - Assistant Professor, Faculty of Medicine, USJ
Diane Antonios - Assistant Professor, Faculty of Pharmacy, USJ
Nabil Hokaim - Professor, Plastic surgeon, Hotel Dieu de France
Hussein Hashim - Plastic Surgeon, Fuad khoury Hospital
Rim Serhal - PhD student, Faculty of Medicine, USJ
Nada alaaeddine - Associate Professor, Faculty of Medicine, USJ
Topic
Basic Research
Problem
Adipose Derived Stem cells (ADSC) are being used therapeutically in many human diseases but also
extensively in plastic surgery. In both cases a large number of cells is essential to generate efficient results,
where the need for culturing and expanding the cells in vitro for several weeks. Our objective is to investigate
the immunomodulatory activity of synovial vascular fraction (SVF) and serial passaged adipose derived stem
cells (ADSC) versus their potential tumorigenic activity depicted by their telomerase and aldehyde
dehydrogenase activity.
Background
Despite all the reports about the therapeutic role of ADSC, many concerns were raised towards their probable
precancerous activity. It has been reported that the ability of stem cell to home to the tumor microenvironment,
promotes breast tumor formation and that adipose-derived human MSCs can be immortalized and
transformed spontaneously after long-term in vitro expansion claiming that MSC may seed cancer. On the
other hand, it has been also reported that MSC inhibit tumor-cell proliferation and tumor growth. Hence,
further studies are required to solve the discrepancy.
Hypothesis
We hypothesize that the secretion profile of ADMSC through passages and their telomerase and aldehyde
dehydrogenase activity may influence their behavior.
Research
ADSC were isolated, purified, and cultured in vitro from lipoaspirates using a well-established protocol. The
immunophenotypic properties of freshly isolated human adipose tissue derived stromal vascular fraction (SVF)
and serial passaged ADSC (P0-P4) were observed by flow cytometry. In parallel, we compared the
telomerase activity and the aldehyde dehydrogenase activity of SVF relative to ADSC (P0-P4) using a
quantitative telomerase detection kit (QTDKit) and Aldehyde Dehydrogenase Based Cell Detection Kit
(ALDEFLUOR™) respectively. The cytokines secretion profile of ADSC during passages was also analyzed
by enzymatic immunoassay kits.
Observations
SVF and ADSC were positive for CD 29, CD 44, CD 73, CD 90, and CD 105, and they were negative for CD
31, CD34, CD45 and CD 106. The telomerase activity was low in SVF and increased during P0 to P4
.However it always decreases at day 21 during each passage. Aldehyde dehydrogenase was detected in SVF
with no changes with serial passages .The level of the cytokine IL-4, IL8, and IL-6 increased significantly
during passages. Though the level of IL-10, IL-17 and GM-CSF was not affected. Thus it seems that the
expansion of ADSC does not confer cells a pretumorigenic activity and that they might be safe to use in
clinical setting.
Effects of Progerin Expression on Adult Stem Cell Functions
Author(s)
Laurin Pacheco
BSc
University of Miami Miller School of Medicine, Miami VA Medical Center, UM Tissue Bank
Co-Author(s)
Lourdes Gomez, MD - Research Scientist, MIAMI VAMC
Claudia Bercerra - Undergraduate Student, Dartmouth College
Paul Schiller, PhD - Associate Professor, University of Miami Tissue Bank, MIAMI VAMC
Topic
Basic Research
Problem
Vascular repair, an essential process for maintaining tissue homeostasis and general health, is critically
reduced during normal aging. Age-induced declines in vascular repair can trigger life threatening vascular
accidents and chronic disease. Efficient vascular repair requires adult stem cell functions that are unimpaired
by disease or aging. Therefore, it is important to define the mechanisms that impair adult stem cell functions
required for vascular repair.
Background
One protein candidate that may diminish vascular repair by modulating adult stem cell functions is progerin,
an alternative splice variant of lamin A. Wild-type Lamin A is a nuclear lamina protein that provides nuclear
structural stability and regulates several important nuclear functions. Progerin is a truncated and permanently
farnesylated mutant that severely disrupts nuclear stability and function. Progerin accumulates in normal
individuals with age due to aberrant splicing, and progerin overexpression diminishes vascular repair in vivo.
High levels of progerin expression caused by a mutation leads to the accelerated aging disorder Hutchinson
Gilford Progeria Syndrome (HGPS). HGPS patients die at approximately 13 years due to vascular accidents
(i.e. stroke, heart attack). Progerin is expressed in atherosclerotic cardiovascular tissues and plaques
collected post-mortem from HGPS patients and aged, non-HGPS individuals.
Hypothesis
We hypothesize that progerin expression promotes vascular dysfunction through a mechanism that disrupts
key adult stem cell functions.
Research
To test our hypothesis, we retrovirally transduced pluripotent Marrow-Isolated Adult Multilineage Inducible
(MIAMI) stem cells, with GFP-progerin, GFP-lamin A, and a vector control. MIAMI cells differentiate into
mature cells found in many tissues affected in HGPS, and participate in vasculogenesis and angiogenesis in
vivo. To assess the effects of progerin expression on markers that promote stem cell self-renewal, we
measured mRNA levels of Notch2, Hes1, Hes5, Oct4, and hTeRT by RT-qPCR. Nuclear morphology and
localization of transcription factors (TFs) Notch2 and Oct4 were evaluated using indirect immunofluorescence.
To determine the effects of progerin on DNA damage, repair, and proliferation, we stained with antibodies
against γH2AX, 53BP1, and Ki67. We also quantified cell growth using a colorimetric assay. To evaluate
differentiation, we grew the cell lines in osteogenic differentiation media for 19 days and quantified ECM
mineralization by alizarin red S staining and mRNA levels of RunX2 and Collagen1α1 by RT-qPCR.
Observations
Progerin expression decreases mRNA levels of Notch2, Hes1, Hes5, Oct4, and hTERT, and leads to
cytoplasmic accumulation of self-renewal TFs Notch2 and Oct4, suggesting deficiencies in nuclear
translocation. Progerin expression increases abnormal nuclear morphology, which is restored by blocking
farnesylation. Progerin expression also increases DNA damage and repair in MIAMI cells. After osteogenic
differentiation, progerin expression decreased the intensity of alizarin red S and mRNA levels of RunX2 and
Collagen1α1, indicating that progerin expression interferes with differentiation. Progerin expression also
decreases proliferation and cell growth. These results further implicate progerin in modulating key adult stem
cell functions that are critical for efficient vascular repair and homeostasis during normal aging.
Endometrial stem cell differentiation into muscular bladder cell: New possibilities to create muscle
tissue for bladder augmentation in women
Author(s)
Javad Verdi
Applied cell Sciences Department, Head of Department
School of Advanced Technologies in Medicine, Tehran University of Medical Sciences
Co-Author(s)
Seyed Abdolreza Mortazavi Tabatabaei - Assistant Professor, Shahid Beheshti University of Medical Sciences
Shiva Sharif - PhD Candidate, Tissue Engineering Department, Tehran University of Medical Sciences
Alireza Shoae-Hassani - PhD candidate, Research center for Science and Technology in Medicine, Tehran
University of Medical Sciences
Topic
Basic Research
Problem
About 400 million people worldwide are suffering from urinary bladder diseases, such as bladder cancer,
neurogenic bladder, exstrophy and interstitial cystitis. The ideal application for tissue regeneration is to
construct a functional urinary bladder for congenital or acquired bladder defects. In many diseases such as
carcinomas the autologous biopsies cannot be used to construct a tissue engineered bladder. Recently
discovery of endometrial stem cells (EnSCs) from the lining of women’s wombs, opens up the possibility of
using these cells for tissue engineering applications such as building up natural tissue to repair prolapsed
pelvic floors as well as building bladder wall. The latter require large amount of smooth muscle cells (SMCs) to
seed the 3D scaffold in fully development of bladder.
Background
Autologous EnSCs could safely be used in women due to the lack of immunogenicity. The differentiation of
human EnSCs into muscular cells of the bladder on the bioabsorbable materials i.e. Polyethylene-Glycolcollagen hydrogels has not been investigated yet. So the present study discusses the human EnSC
differentiation along SMC lineage of bladder.
Hypothesis
Developing a tissue engineered urinary bladder with isogeneic stem cells that obtained easily and in a
massive volume from non-urinary tract is an attractive field that could be critical for the patient’s quality of life
or survival.
Research
The human EnSCs that were positive for CD146, CD105 and CD90 were isolated and cultured in DMEM/F12
supplemented with myogenic growth factors. The Myogenic factors consist of transforming growth factor
(TGF-β1), platelet derived growth factor (PDGF-BB), hepatocyte growth factor (HGF) and vascular endothelial
growth factor (VEGF). Differentiated SMCs on the bioabsorbable polyethylene-glycol and collagen hydrogels
were checked for α-smooth muscle actin, desmin, vinculin and calponin markers by real time RT-PCR,
western blot and immunocytochemistry analyses.
Observations
The histology experiment revealed the growth of SMCs in the hydrogel matrices. Myogenic growth factors
differentiated human EnSCs into SMCs more efficiently on hydrogels matrices and expressed specific SMC
markers in all experiments. Human EnSCs were successfully differentiated into bladder SMCs, using
hydrogels as scaffold. The EnSCs may be used for autologous bladder wall regeneration without any
immunologic complications in women. Currently work in progress using bioabsorbable nanocomposite
materials as scaffold with EnSCs in development of bladder wall.
Establishment and characterization of 3D artificial adipose tissue models using the 3T3-L1 preadipocyte cell line.
Author(s)
Timothy Lyden
Professor of Anatomy and Physiology, Director of Tissue and Cellular Innovation Center
University of Wisconsin-River Falls
Co-Author(s)
Eric Valder - Researcher, UWRF
Brianna Jacques - Researcher, UWRF
Alexander Chibalin - Associate Professor, Karolinska Institute
Juleen Zierath - Professor, Karolinska Institute
Topic
Basic Research
Problem
In the present study, we sought to address the establishment of pre-adipocyte 3D cultures using the mouse
cell line 3T3-L1 and to morphologically characterize the resulting structures. The focus of these studies is to
model adipose-like artificial tissues and examine the cellular behaviors and microenvironments involved.
Background
Our interest in modeling this tissue relates directly to its apparent role in the development and progression of
several major types of cancer, including colon and breast. During earlier work using primary patient tumor
samples in 3D cultures it was noted that adjacent adipose tissues and individual cells were intimately
associated with tumor tissues and were maintained as the tumor spread on the scaffolds as well as over long
culture periods. Therefore, we are exploring the development of artificial adipose 3D constructs with the intent
of using these in future studies on the role of adipose tissues in cancer.
Hypothesis
We hypothesized that in 3D artificial tissue cultures 3T3-L1 cells would undergo differentiation with the
eventual development of mature artificial adipose tissue-like structures that more closely reflect actual tissue
conditions than in standard culture methods. In this study we evaluated that hypothesis relative to structural
and ultra-structural features.
Research
These first experiments with 3T3-L1 cells tested the feasibility of loading pre-adipocyte cultures onto our
standard natural fibrous collagen scaffolding material. These initial constructs were then maintained in culture
for more than 12 months with their development / maturation being monitored and samples taken every week
for SEM analysis during the initial 1 month of culture. Additional samples were also harvested for later
embedding / sectioning and labeling studies to follow. The SEM observations reported here focused on the
development of spontaneous “tissue-like” features and the constituent cellular populations present at each
stage of this adipose AT development.
Observations
In these initial experiments to establish 3D adipose constructs using 3T3-L1 cells, mature adipose-like artificial
tissues were successfully generated and samples harvested for both marker-labeling studies and scanning
electron microscopic evaluation of cell and tissue morphologies. Interesting patterns and distributions of
mature and immature cells were observed in the resulting 3D ATs, with clearly fibroblastic and adipose-like
cells being morphologically in evidence. As the cultures matured, the adipocyte-like cells became prominent
and the artificial tissue surface displayed regional distributions of these and the fibroblastic cells. These
cultures also displayed a tendency to express a thick, viscous material which increased with the age/maturity
of the structures and may represent expression of hyaluronate or other GAGs by the maturing adipocytes
grown under 3D culture conditions. Future studies are planned to examine this question and to explore other
matrix components expressed in these complex 3D artificial tissues.
Establishment and Characterization of 3D Artificial Skeletal Muscle-like Tissues using the Myoblast
Cell Line C2C12.
Author(s)
Co-Author(s)
Brianna Jacques - Researcher, UWRF
Rijada Suljic - Researcher, UWRF
Kathryn Overby - Researcher, UWRF
Alexander Chibalin - Associate Professor, Karolinska Institute
Juleen Zierath - Professor, Karolinska Institute
Topic
Basic Research
Problem
In this report we describe the successful establishment of skeletal muscle 3D “artificial tissues” (ATs)
constructs using the mouse myoblast cell line, C2C12. These structures are essentially self-directed in terms
of development and display a diverse cellular population profile that includes myoblasts, early myotubes and a
small motile population that may reflect the presence of "satellite" cells .
Background
Our laboratory has been engaged for some time in the application of small- scale AT constructs for the in-vitro
3D modeling of cellular interactions and population dynamics. A primary goal of this work is to develop tools
and methods which provide relevant “tissue-like” developmental microenvironments in which to study normal
and pathologic tissue biology. To accomplish this goal we have employed several natural extra-cellular matrix
materials as 3D substrates and a variety of different cell lines as well a primary tissues in studies of skeletal
and cardiac muscle, bone, adipose and brain tissues in addition to various cancerous tumor tissues and cell
lines.
Hypothesis
The working hypothesis of this study was that 3D in-vitro modeling based on natural decellularized extracellular matrix materials would create the needed microenvironments for C2C12 myoblast cells to establish
rudimentary artificial skeletal muscle tissue-like constructs.
Research
These first experiments with C2C12 cells tested the feasibility of loading both immature myoblast cultures and
more mature myotube cultures onto our standard natural fibrous collagen scaffolding material. These initial
constructs were then maintained in culture for 1+ month with their development / maturation being monitored
and samples taken every week for SEM analysis. Additional samples were also harvested for later embedding
/ sectioning and labeling studies to follow. The SEM observations reported here focused on the development
of spontaneous “tissue-like” features and the constituent cellular populations present at each stage of this AT
development.
Observations
This study yielded spontaneous, large-scale organized myotube-containing features in 3D which bear a
striking resemblance to early natural muscle fibers. Additionally, the remaining myoblast populations also
established structural features which appear to provide ancProbhorage points for the maturing myotube
structures. Intertwined with these myotube features were a population of small individual cells that mimic the
relative positions of natural muscle satellite cells. Taken as a whole, this study shows that it is feasible to
develop complex 3D myotube-containing “artificial tissues” using natural extra-cellular matrix materials as a
basic scaffold. Additional analysis of the harvested samples should further elucidate the exact nature of and
relationships between the cells and features observed in this ultrastructural study and help in the comparison
of these constructs with normal in-vivo muscle development.
Expanding the PMC GRN: Genome-Wide Analysis of Ets1 and Alx1 Targets
Author(s)
Kiran Rafiq
Graduate Student
Carnegie Mellon University
Co-Author(s)
Charles Ettensohn - Personal Investigator, Carnegie Mellon University
Topic
Basic Research
Problem
A central challenge of developmental and evolutionary biology is to understand how anatomy is encoded in
the genome, and elucidating the genetic mechanisms that control the development of specific anatomical
features will require the analysis of model morphogenetic processes and an integration of biological
information at genomic, cellular and tissue levels. The formation of the endoskeleton of the sea urchin embryo
is a powerful experimental system for developing such an integrated view of the genomic regulatory control of
morphogenesis. The dynamic cellular behaviors that underlie skeletogenesis are well understood and a
complex transcriptional gene regulatory network (GRN) that underlies the specification of embryonic
skeletogenic cells (primary mesenchyme cells, PMCs) has recently been elucidated.
Background
Establishing linkages between early cell specification networks and specific morphogenetic processes
is crucially important, not just for understanding embryogenesis per se, but also in an evolutionary
context, i.e. for understanding the ways in which evolutionary modifications to genetic networks
have led to changes in morphological features. The PMC GRN is also a powerful model to study
plasticity because this network has the potential to be activated ectopically in other cell types of the
embryo. However, there has been less progress, in using GRNs to explain the complex cell and tissue
behaviors that drive changes in embryonic form.
Hypothesis
We hypothesize that the GRN deployed in skeletogenic primary mesenchyme cells (PMCs) is linked to
genes that control skeletal morphogenesis via transcriptional regulatory inputs from two of the earliest
and well- characterized transcription factors, Ets1 and Alx1.
Research
To provide a comprehensive, genome-wide analysis of the targets of ets1 and alx1,
we performed paired-end, high-throughput cDNA sequencing (RNA-seq). We used Cufflinks, an
open-source software tool for comparing gene and transcript expression under control and
ets1/alx1 knockdown conditions and confirmed a few of the changes by QPCR and the nCounter
Nanostring System. We also perform whole mount in-situ hybridization to identify new mRNAs restricted
as well as enriched in the PMCs to gain insights into new classes of proteins that might play a role in
skeletogenesis.
Observations
The targets of ets1 and alx1 identified previously were almost identical and found to represent only a small
subset of a much larger number of functional targets. Analysis of regulatory genes controlled by ets1 and alx1
identified several common targets, including alx4, foxb, hnf1, and nk7, which could perhaps explain how ets1
and alx1 co-regulate such a large set of common downstream genes. Our transcriptome analysis also allowed
us to identify new classes of proteins that were not previously known to play a role in skeletogenesis. Thus,
our works extends the value of the PMCGRN as a model developmental GRN, expands it to a great extent
and establishes a framework for understanding the genomic circuitry that encodes a major anatomical feature.
Generating Human Retinal Ganglion Cells From Human Induced Pluripotent Stem Cells
Author(s)
Shomoukh Abdulaziz. AlShamekh, MBBS
Research associate
Bascom Palmer Eye institute. Interdisciplinary Stem Cell institute, University of Miami. King Abdulaziz
University Hospital, King Saud University, Riyadh, Saudi Arabia
Co-Author(s)
Jonathan Hertz - Graduate student, Bascom Palmer Eye Institute, University of Miami, Interdisciplinary Stem
Cell Institute
Chen-Ching Yuan - Graduate student, University of Miami
Brooke Derosa - Graduate student, Hussmann Institute for Human Genomics, University of Miami
Shan Uddin - Student, Bascom Palmer Eye Institute, University of Miami, Interdisciplinary Stem Cell Institute
Barbara Corneo - post doc, Nueral Stem Cell Institute
Sally Temple - Primary Investigator, Nueral Stem Cell Institute
Derek Dykxhoorn - Primary Investigator, Husmann Institute of Human Genomics, University of Miami
Jeffrey Goldberg, MD, PhD - Primary Investigator, Bascom Palmer Eye Institute, University of Miami,
Topic - Basic Research
Problem
Glaucoma, and other optic neuropathies, results in the loss of retinal ganglion cells (RGCs) and vision
dysfunction. Therefore, cell replacement therapy may offer promising treatments to protect the degenerating
retina and potentially restore vision function.
Background
Although significant progress has been made to generate photoreceptors from stem cells, there has been little
advancement in efficiently generating RGCs. Therefore, we have been investigating the optimal conditions for
differentiating RGCs from stem cells for cell replacement therapy.
Hypothesis
We hypothesize that cell replacement therapy using human RGCs differentiated from human iPSCs may
repair damaged retina and possibly restore visual function. We also hypothesize that adding SOX4 and Math5
genes to iPSCs will lead to more efficient differentiation to RGCs.
Research
Human induced pluripotent stem cells (iPSCs) were maintained and expanded on feeder layers using
previously published conditions. During embryoid body formation, genes and proteins (SOX4, Math5) were
tested for effects on increasing RGC differentiation efficiency. Differentiated RGCs were identified and
quantified using RGC-specific markers including Brn3. Feeder-free conditions were examined for functional
equivalence.
Observations
iPSCs that express stem cell-specific markers were passaged in proliferative conditions and differentiated into
RGCs in vitro. Feeder-free cell culture conditions allowed iPSC maintenance without the presence of mouse
immunogenicity. iPSCs were found to respond to pro-RGC differentiation signals in similar fashion to rodent
embryonic retinal progenitors. The effeciency of differentiation of iPSCs to RGCs was higher when both SOX4
and Math5 were added, proving that together they play a synergistic role in producing RGCs. These findings
will provide valuable insight in our understanding of RGC differentiation and will pave the way to cure retinal
diseases. The transplantation of these cells in vivo will elucidate the integration potential of these cells in
degenerating retina.
Generation of induced pluripotent stem cells (iPSCs) opens a new avenue in regenerative medicine.
One of the major hurdles for therapeutic applications is to improve the efficiency of generating iPSCs
and also to avoid the tumorigenicity, which requires searching for new reprogramming recipes. We
present a systems biology approach to efficiently evaluate a large number of possible recipes and find
those that are most effective at generating iPSCs. We not only recovered several experimentally
confirmed recipes but we also suggested new ones that may improve reprogramming efficiency and
quality. In addition, our approach allows one to estimate the cell-state landscape, monitor the
progress of reprogramming, identify important regulatory transition states, and ultimately understand
the mechanisms of iPSC generation.
Author(s)
Rui Chang
Assistant Professor
Mount Sinai School of Medicine
Co-Author(s)
Topic
Basic Research
Problem
Converting somatic cells back to the stem cell state, i.e. induced pluripotent stem cells or iPSCs exemplifies
the recent advancement of cellular reprogramming that holds great promise for developing regenerative
medicine.
Background
Generation of iPSCs is often achieved by overexpressing three to four genes in somatic cells that are critical
for regulating pluripotency. Developing optimal reprogramming recipe is a non-trivial task that requires
significant effort.
Hypothesis
We present here a computational method that can facilitate discovery of effective recipes to generate iPSCs
with high efficiency and better quality.
Research
We present here a computational method that can facilitate discovery of effective recipes to generate iPSCs
with high efficiency and better quality. In addition, our approach provides a new way to estimate the landscape
in the cell-state space and monitor the trajectory of cellular reprogramming from a differentiated cell to an iPS
cell.
Observations
This work provides not only practical recipes for iPSC generation but also theoretical understanding of the
reprogramming process.
Genetic Modification of Ckit+ Cardiac Stem Cells to Overexpress Pim1 Enhances their
Cardioreparative Ability After Intramyocardial Delivery
Author(s)
Vasileios Karantalis
Post-Doctoral Research Associate
Interdisciplinary Stem Cell Institute/ University of Miami
Co-Author(s)
Viky Suncion - Post Doctoral Research Associate, Interdisciplinary Stem Cell Institute, University of Miami
Frederic McCall - Research Associate, Interdisciplinary Stem Cell Institute, University of Miami
Jose Rodrigues - MRI Technologist, Interdisciplinary Stem Cell Institute, University of Miami
David Valdes - Senior Veterinary Technician, Interdisciplinary Stem Cell Institute, University of Miami
Wayne Balkan - Research Assistant Professor, Interdisciplinary Stem Cell Institute, University of Miami
Muzammil Mushtaq - Assistant Professor of Clinical Medicine, Interdisciplinary Stem Cell Institute, University
of Miami
Mark Sussman - Professor of Biology, San Diego State University
Joshua Hare - Professor of Cardiology, Interdisciplinary Stem Cell Institute, University of Miami
Problem
Ckit+ cardiac stem cell is a promising new stem cell type that is employed in cardiac regeneration. The poor
engraftment that is observed in preclinical studies may contribute to the subsequent mediocre efficacy of
these cells,
Background
Pim-1 is an oncogene which encodes for the serine/threonine kinase of the same name. Pim-1 is mainly
involved in cell cycle progression, apoptosis and transcriptional activation, as well as more general signal
transduction pathways.
Hypothesis
We tested the hypothesis that overexpression of Pim1 enhances the cardioreparative effect of ckit+ cardiac
stem cells (CSCs)
Research
All experiments on animals were performed in accordance with the protocol approved by the Institutional
Animal Care and Use Committee at the University of Miami. We treated immunosuppressed Yorkshire pigs
(n=29) with human ckit+ CSCs (n=7), Pim1 modified human ckit+ CSCs (n=9) or PBS (n=13) two weeks after
the induction of MI. A transepicardial approach was employed for direct delivery of the product to the
myocardium. Cardiac MRI was obtained before and after cell administration. Four weeks after injection all
animals were humanely euthanized. Data are reported as mean ± SEM. One way ANOVA repeated measures
for within group variations and two-way ANOVA for between groups variations were used for statistical
analyses. All pair wise comparisons were made using Student-Newman-Keuls Method. Significance: P < 0.05.
Observations
The scar tissue in animals that were injected with PIM-1 cells is reduced at 2 (-17.68±3.25%, p<0.001 vs 2
weeks post MI) and continues decreasing at 4 weeks post injection (-25.53±4.3%, p<0.001 vs 2 weeks post
MI. The ckit injected animals also present a scar tissue reduction at 4 weeks (-9.91±3.06%, p=0.012 vs 2
weeks post MI). Peak Eulerian Strain demonstrates the improvement of contractility in the infarct zone of PIM1 injected animals ( 88.35±36.94%, p<0.001 vs 2 weeks post injection) or sustainment of contractility in border
zone after injection. Cardiac Efficiency (CE) is derived from Pressure-Volume measurements and is the ratio
of stroke work to MVO2. At 2 weeks all animals demonstrated a significant deterioration of their CE as a result
of the induced MI. Both stem cell types improve energetics at 4 weeks post injection. The beneficial effect of
intramyocardial delivery of ckit+ CSCs to infarcted porcine hearts was significantly enhanced and persistent
when they overexpress Pim1. This approach provides a rationale for further exploration of the genetic
modification of stem cells and consequent translation to clinical trials.
Growth-Hormone-Releasing-Hormone (GHRH) Receptor Agonist Promotes Cardiac Stem Cell
Proliferation in Rats with Post-Myocardial Infarction (MI)
Author(s)
Victoria Florea
Postdoctoral Research Associate
University of Miami
Co-Author(s)
Rosemeire Kanashiro-Takeuchi, PhD, DVM - Research Assistant Professor, University of Miami
Claudia Rodrigues, PhD - Research Assistant Professor, University of Miami
Lauro Takeuchi - Research Associate, University of Miami
Norman Block, MD - Professor, University of Miami
Andrew Schally, MD, PhD - Professor, University of Miami
Joshua Hare, MD - Professor, University of Miami
Topic
Basic Research
Problem
Mortality from cardiovascular disease has decreased over time as therapeutic advances have become more
elaborate. Despite this progress, no current treatment fully reverses the primary cause of impaired heart
function, the loss of cardiomyocytes. Because the worldwide prevalence of heart failure (HF) continues to
increase, any intervention that improves this condition would promise a beneficial clinical outcome and should
be further explored.
Background
Accumulating data support a cardioprotective role for the growth hormone (GH) axis. The heart harbors
GHRH receptors that can be activated by GHRH and synthetic GHRH agonists, suggesting that the ischemic
heart is also a target of GHRH. Our group has recently shown that a potent GHRH receptor agonist (JI38) is
cardioprotective. Administration of JI38 reduced infarct size and improved cardiac function in the absence of
increasing circulating GH or IGF-1 levels. Cardiac Stem Cells (CSCs) have been shown to contribute to
myocardial repair after infarction and improve cardiac function.
Hypothesis
Based on these findings, we tested the hypothesis that GHRH receptor agonist JI38 promotes activation of
CSCs.
Research
The expression of GHRH receptor was determined in mouse, rat and porcine progenitor cells of cardiac and
mesenchymal (MSCs) origin using fluorescence activated cell sorting. Rat CSCs were treated with the GHRH
agonist JI38 and the antagonist MIA-602 for evaluation of their effect on proliferation, which was assessed by
immunodetection of thymidine analogue incorporation during DNA synthesis. Myocardial infarction (MI) was
induced by coronary artery ligation in 6 month-old Fisher-344 rats. One month post-infarction, animals were
randomly assigned to receive placebo, GHRH receptor agonist JI38, GHRH receptor antagonist MIA-602, or a
combination of JI38 and MIA-602, for a 4-wk period. Cardiac performance and hemodynamics were assessed
by echocardiography and micromanometry-derived pressure-volume loops. The number of ckit+ CSCs was
determined in histological sections.
Observations
Results Cardiac and mesenchymal progenitor cells (CSCs and MSCs) from the three animal species tested
were 96-98% positive for the expression GHRH receptor as compared to positive and negative control cancer
cell lines. We determined the impact of GHRH agonist JI38 activity on rat CSCs division in vitro. Our data
showed an increase in the proliferation rate of CSCs after pretreatment with JI38 (P < 0.05 vs. placebo,
Student’s t test), whereas other treatments did not show a difference. Treatment of rats with JI38 reverses
ventricular remodeling and enhances functional recovery in the setting of chronic MI. Over an 8-week period,
JI38 markedly improved ejection fraction (43±4 vs. 32.7±1.3%, p<0.01). In addition, MI size was substantially
reduced by JI38 (29.3±1.4 vs. 40±1.2%, p<0.05) and capillary density was increased (p<0.05 vs. placebo and
MIA-602). Importantly, all of these effects were completely abrogated by the GHRH receptor antagonist MIA602. Functional recovery was associated with an increase in the number of ckit-positive CSCs with JI38
therapy. The majority of c-kit-positive CSCs was not bone marrow derived; these cells were CD45- and
tryptase-negative, and were clearly distinguishable from resident cardiac mast cells (c-kit-positive/CD45positive).
Conclusions Collectively, these findings confirm for the first time the expression of GHRH receptor in CSCs
and MSCs. Therapy with GHRH receptor agonist after MI substantially improved cardiac performance and
reduced infarct size, suggesting a regenerative process. GHRH agonist treatment stimulates CSCs
proliferation. Accordingly, activation of GHRH receptor signaling pathways represents a novel therapeutic
approach to protect and locally stimulate endogenous CSC population, promoting cardiac repair.
Human bone marrow derived mesenchymal stem cell integration and differentiation depends on
Connexin 43.
Author(s)
Cristina Sanina, MD
Postdoctoral Fellow
Co-Author(s)
Claudia Rodrigues, PhD - Assistant Professor , Interdisciplinary Stem Cell Institute
Michael Bellio - PhD Student, University of Miami
Ivonne Schulman, MD - Assistant Professor , Interdisciplinary Stem Cell Institute
Wayne Balkan, PhD - Assistant Professor , Interdisciplinary Stem Cell Institute
Konstantinos Hatzistergos, PhD - Postdoctoral Fellow, Interdisciplinary Stem Cell Institute
Irene Margitich, MS - Lab Manager , Interdisciplinary Stem Cell Institute
Joshua Hare, MD - Director, Interdisciplinary Stem Cell Institute
Topic
Basic Research
Problem
Stem cell treatment. Mechanism underlying the beneficial effect of stem cell treatment. Role of Connexin 43 in
stem cell integration and differentiation.
Background
Although bone marrow-derived mesenchymal stem cells (MSCs) produce reverse remodeling and improve
regional contractility in the porcine and human heart, the mechanism(s) of actions of MSC integration and
differentiation in the heart remain controversial.
Hypothesis
MSCs express abundant connexin 43 (Cx43) and form functional gap junctions, we hypothesized that cell-cell
connections are crucial for MSC integration into tissues and differentiation into cardiovascular lineages.
Research
Human MSCs were co-cultured with neonatal rat ventricular cardiomyocytes (NRVMs). We assessed the
impact of gap junctional communication by using lentiviral constructs to knockdown (Cx43KD) or overexpress
(Cx43OE) Cx43 in hMSCs. To assess MSC differentiation in co-culture, we evaluated the change/onset of
expression of three endothelial and smooth muscle genes (KDR, PECAM-1, smooth muscle actin, VEcadherin) and seven cardiomyocyte markers (Nkx2.5, Gata4, Troponin I, sodium-ion channel, potassium-ion
channel, calcium-ion channel, sodium-calcium exchanger) using human specific primers.
Observations
Co-culture of Cx43OE or control hMSCs with 7-day-old NRVMs, led to the formation of beating, threedimensional tubes in 10 or 14 days, respectively (n=5, p<0.05), whereas co-culture with Cx43KD hMSCs,
completely abrogated this effect (n=5, p<0.05). Whereas the voltage ions channels were upregulated or
expressed relative to no expression at baseline by co-culture (n=5, p<0.05), and these changes in gene
expression were completely eliminated in Cx43KD hMSCs, expression of cardiac markers, such as Nkx2.5,
Gata4, and Troponin I, was never observed in any co-culture group.
MSC integration into functional cardiac tissue structures and voltage ion channel expression is dependent on
Cx43. Together these findings reveal that cell−cell contact mediated by Cx43 gap junctions is essential for
MSCs to participate in tissue formation and cardiac repair.
Human induced pluripotent (iPS) cell line engineering using Transcription Activator-Like Effector
Nucleases (TALENTM) technology, with further differentiation into mature cell types
Author(s)
Mia Emgard
BD Manager / Assoc. Prof. Karolinska Institutet
Cellectis Stem Cells
Co-Author(s)
Marianne Duhamel - Dr, Cellectis Stem Cells
Sébastien Paris - Dr, Cellectis Bioresearch
Tina Nilsson - B.Sc, Cellectis Stem Cells
Nathalie Ollivier - Eng. Assist., Cellectis Stem Cells
Isabelle Hmitou - Eng., Cellectis Stem Cells
Monika Kaminska - Eng., Cellectis Stem Cells
Jean-Pierre Cabaniols - COO, Cellectis Bioresearch
Johan Hyllner - CSO, Cellectis Stem Cells
Catharina Ellerstrom - Dr, Cellectis Stem Cells
Topic
Basic Research
Problem
Human stem cells, especially patient-derived induced pluripotent stem cells, represent powerful candidates for
improvements to current drug screening approaches, for the development of novel regenerative medicine and
for the generation of better research tools. In some applications however, genome customization is needed for
repair of potential genetic-abnormalities (monogenic diseases).
Background
Generation of mature, differentiated cells, and derivation of endogenous promoter driven reporter cell lines,
can be achieved by genome customization, facilitating mechanistic studies and drug discovery. Efficiency of
targeted engineering is greatly enhanced by the use of selectively cutting endonucleases, especially the
recent TAL effectors nucleases (TALENs™). They can bind to any gene and introduce a specific DNA double
strand break at any selected position, enabling genetic modification in any cell type.
Hypothesis
The use of TALENs™ for target gene integration as an efficient applications for pluripotent stem cells.
Research
Combined with a robust feeder free culture system, DEF –CS™, TALENs™ allow insertion of a “Gene of
Interest” into a defined loci (Rag1, Safe Harbor – SH), as well as fusion of the GFP to iPS and hES
endogenous β-actin gene. This is an efficient, robust and precise approach that opens the door to “on
request” engineering of stem cells.
Observations
Cellectis has now established an iPS Engineering HubTM service that not only take full advantage of the
TALENsTM technology, but also employ the latest technology to reprogram somatic human cells to
undifferentiated pluripotent stem cells. Moreover, using our experience in stem cell differentiation into e.g.
cardiomyocytes and hepatocytes, we have generated differentiated cells exhibiting specific markers and
functional similarities to adult human cells. With high homogeneity, availability in multiwell plate formats, and
with low batch-to-batch variations, the cells are excellent in vitro tools for safety pharmacology and toxicity
assessments.
Indeed, this human pluripotent stem cell technology platform offers a path to discoveries of the future.
Human Neural Stem Cell-Mediated Targeting of Therapeutic Nanoparticles to Solid Tumors
Author(s)
Rachael G. Mooney, Ph.D.
CIRM post-doctoral fellow
Beckman Research Institute of City of Hope
Co-Author(s)
Donghong Zhao, Ph.D. - Staff Scientist, Beckman Research Institute of City of Hope
Elizabeth Garcia, R.V.T - RA II, Beckman Research Institute of City of Hope
Revathiswari Tirughana-Sambandan, B.S. - RA I, Beckman Research Institute of City of Hope
Yiming Weng, Ph.D - Post-doctoral Fellow, Beckman Research Institute of City of Hope
Alexander Annala, Ph.D. - Associate Professor, Beckman Research Institute of City of Hope
Jacob Berlin, Ph.D. - Assistant Professor, Beckman Research Institute of City of Hope
Karen Aboody, M.D. - Associate Professor, Beckman Research Institute of City of Hope
Topic
Basic Research
Problem
Therapeutic nanoparticles (NPs) have potential to improve outcomes for patients with primary or metastatic
breast cancer; however, strategies for better NP retention and tumor-selective distribution are needed.
Background
In pre-clinical models, tumor-tropic neural stem cells (NSCs) can overcome distribution obstacles associated
with even advanced NP-mediated drug delivery: NSCs home to invasive primary and metastatic tumor foci
and avoid off-target spleen and liver deposition. NSCs also penetrate into hypoxic tumor regions, and NSCs
traverse the tumor-blood barrier (TBB) to access intracranial tumor foci. We previously established a clonal,
human NSC line (HB1.F3.CD) that is currently being used to deliver diagnostic iron nanoparticles and a prodrug activating enzyme in Phase I clinical trials for recurrent glioma patients.
Hypothesis
Here we hypothesize that these clinically relevant NSCs can improve the retention, penetration, and
distribution of NPs within solid tumors, and demonstrate the therapeutic potential of 2 different NSC-NP
combinations for treating triple negative breast cancer. The first NSC-NP combination involves endocytosed
gold nanoparticles (AuNPs) that generate tumor-toxic heat upon exposure to otherwise nonharmful nearinfrared stimulation. The second NSC-NP combination involves coupling biotin-conjugated, docetaxel-loaded,
pH-responsive NPs to the surface of avidinylated NSCs using a previously established coupling protocol.
Research
To assess AuNP uptake into NSCs, darkfeild microscopy, transmission electron microscopy and ICP-MS were
employed. NMR was used to confirm polymer synthesis, and dynamic light scattering and zeta-potential
measurements were used to characterize nanoparticles. Flow cytometry, confocal and scanning electron
microscopy were used to confirm surface-localization of docetaxel-loaded NPs. Boyden-chamber migration
and GuavaCyte FACs analysis was used to assess NSC tropism and viability after NP coupling in vitro. In vivo
biodistribution and preliminary efficacy studies have been performed.
Observations
Results confirm efficient NP coupling with 12.5pg AuNPs endocytosed per 1e6 NSCs; and 169 surfaceconjugated polyermic NPs per NSC. Both NSC-NP combinations retained unimpaired viability and migration
to tumor conditioned media in vitro. In vivo biodistribution studies demonstrate that NSC-NP combinations
retain tumor tropism and can cross the TBB to access intracranial tumor foci. Preliminary in vivo therapeutic
efficacy studies with both NSC-NP combinations have been performed and we have confirmed pH-dependent
drug and thermal killing of a triple negative breast cancer line that commonly metastasizes to the brain (MDAMB-231-BR). Together, this research combines human NSCs with therapeutic NPs to achieve significant
improvements in NP-delivery to tumors, which should translate to improved clinical outcomes and minimized
side effects for patients with invasive or drug resistant metastatic breast cancer tumors.
Human Stem Cell Recombineering
Author(s)
Melvys Valledor
Graduate Research Assistant
University of Miami Miller School of Medicine
Paul C. Schiller
Associate Professor of Orthopaedics and Biochemistry & Molecular Biology
University of Miami Miller School of Medicine and the Miami Veterans Affairs Medical Center
Role: Author
Co-Author(s)
Richard S. Myers - Lecturer, University of Miami Miller School of Medicine
Topic
Basic Research
Problem
Highly efficient and specific genome engineering will create opportunities to generate human disease models
and treat human genetic diseases. Current human cell genome engineering technologies rely on toxic induced
DNA damage and/or on human recombination pathways that are highly inefficient for modifying genes.
Background
Homologous recombination is a consequence of DNA damage repair. While induced DNA damage (e.g. by
zinc finger nucleases) stimulates recombination, it is also highly toxic and can lead to unintended genetic
consequences. Human homologous recombination is catalyzed by a consortium of many enzymes in highly
complex pathways. In contrast, viral homologous recombination is accomplished by the coordinated action of
2 enzymes: an exonuclease and a synaptase. These 2 proteins comprise a simple but sophisticated
nanomachine we call a SynExo. The bacteriophage λ SynExo is very efficient for gene editing by
Recombineering in its host, E. coli, with recombination rates on the order of 10% using substrates that are not
damaging to chromosomes. Unfortunately, the λ SynExo is 10,000X less efficient in mammalian cells.
SynExos are common to dsDNA viruses that infect a variety of hosts - including humans - and their
mechanisms are conserved, suggesting that Recombineering can be extended beyond bacteria to humans.
Hypothesis
Recombineering is host-specific because viral SynExo recombinases co-evolve with host proteins. We predict
that a human viral SynExo catalyzes homologous recombination in human cells with efficiencies approaching
that of the λ SynExo in E. coli.
Research
We are building a nanotool set to catalyze human homologous recombination using host-specific viral SynExo
recombinases in human cells. To test the hypothesis, we built a sensitive and quantitative assay for human
gene targeting using oligonucleotides to change YFP-expressing cells to GFP-expressing cells. Human cells
expressing YFP were transiently transfected with a plasmid expressing a human viral SynExo recombinase
subunit and with a DNA oligonucleotide recombination substrate. Recombination was quantified by flow
cytometry.
Observations
A human viral recombinase stimulates gene targeting to levels approaching the efficiency of the λ SynExo
in E. coli. As was previously seen in E. coli, targeting recombination substrates to hybridize to the lagging
strand template in DNA replication forks is more efficient than oligonucleotides directed to bind to the leading
strand template. DNA substrate length was found to be important with 65 nucleotide oligonucleotides being
more efficient than shorter substrates. Optimized SynExo-mediated gene targeting edits genes with ~7%
efficiency with minimal cell toxicity, similar to what is common to bacterial genome engineering by
Recombineering.
Hypoxia cultured human mesenchymal stem cells (hMSCs) exert high in vitro targeted migration
Author(s)
Grigory K. Vertelov
Research Scientist
Stemedica
Co-Author(s)
Givon Sanati - research intern, Stemedica
MG Muralidhar, Dr - Research Scientist, Stemedica
Ludmila Kharazi, Dr - Research Scientist, Stemedica
Timothy Tankovich - research intern, Stemedica
Alex Kharazi, Dr - Chief Technology Officer, Stemedica
Topic
Basic Research
Problem
It is important to preserve major MSC's characteristics during tissue culture expansion, a necessary step
towards the generation of clinically significant cell numbers. One of the major complications at this step is
spontaneous cell differentiation, which can occur particularly in the presence of bovine serum. A possible
approach to alleviate this problem is cultivation of MSC in low oxygen environment. Whilst the effect of acute
hypoxia on MSC is relatively well known, culturing of MSC in permanent hypoxia is underinvestigated.
Background
In vivo MSC reside in an environment with relatively low oxygen (O2) concentration. It has been shown that
hypoxic MSC maintain significantly higher colony-forming unit capabilities and higher levels of stem cellrelated genes. They exhibit higher levels of osteoblastic and adipocytic differentiation markers as well as
increased total protein levels compared to MSC cultured at 20% O2 (normoxic) conditions. Several reports
indicated that MSC cultured in low oxygen, demonstrate increased proliferation activity. A feature which
makes stem cells promising candidates for cell therapy is their ability to effectively migrate into damaged or
diseased tissues. Recent reports demonstrated the increased motility of human mesenchymal stem cells
(hMSC) grown under hypoxic conditions compared to normoxic cells. However, the directional migration of
hMSC cultured in hypoxia has not been investigated.
Hypothesis
Oxygen concentration regulates the intricate balance between cellular proliferation and commitment towards
differentiation, impacts “stemness” of the MSCs. Overall, it appears the hypoxic environment is promoting a
genetic program maintaining the undifferentiated and multipotent status of MSCs. Hypoxic culturing of MSC
may better preserve high motility of MSC.
Research
In this study we examined the in vitro transmembrane migration of hMSC permanently cultured in hypoxia in
response to various cytokines. We compared the directional migration of human hMSCs grown permanently
under normal (21%) and low O2 (5%) conditions until passage 4 using an in vitro transmembrane migration
assay. A series of 17 cytokines was used to induce chemotaxis.
Observations
We found that hMSC cultured in hypoxia demonstrate markedly higher targeted migration activity compared to
normoxic cells, particularly towards wound healing cytokines, including those found in ischemic and
myocardial infarction. hMSC grown permanently in hypoxia have higher clonogenic potential compared to
normoxic cells
Identification of stem cell markers in pancreatic cancer cell lines
Author(s)
Megha Kumar
Undergraduate Research Assistant
California State University, Fresno
Co-Author(s)
Kristin Herring, BS - Graduate Student, California State University, Fresno
Joel Ramirez, BS - Graduate Student, California State University, Fresno
Lulu Wong - Undergraduate, California State University, Fresno
Mina Al-Shahed - Undergraduate, California State University, Fresno
Jason Bush, PhD - Associate Professor, California State University, Fresno
Topic
Basic Research
Problem
Pancreatic cancer is an aggressive disease characterized by rapid progression, invasiveness, and high
mortality rate. Even after surgical resection, multiple rounds of chemotherapy and/or radiation therapy,
patients often have regrowth of the primary tumor. The ability of such tumors to show profound resistance is
believed due to Cancer Stem Cells (CSCs). However, very little is known about the acquisition of CSC
characteristics.
Background
Pancreatic cancer is the fourth leading cause of cancer deaths in the United States with an estimated 37,400
deaths in 2012 and a five-year survival of less than 6% across all stages. Due to lack of effective diagnostic
methods, this disease often eludes detection during its formative stages. Hence, identification of a CSC subpopulation within the bulk tumor population can serve as a mode of earlier detection that is essential for
successful treatment of pancreatic cancer.
Hypothesis
The hypothesis being tested is that cell lines representing the earlier stages of pancreatic cancer have the
highest proportion of CSCs that can be isolated and serve as a model for understanding the intrinsic cellular
pathways responsible for initiating and maintaining the aggressive behavior of tumors even after treatment.
Research
The primary goal was to create an expression profile for stem cell markers against a panel of ten pancreatic
cancer cell lines obtained from different stage of the disease. The most promising cell lines would be used to
further evaluate CSCs by isolating the sub-population using flow cytometry and FACS. In addition, a
proteomic approach was used to analyze secreted material from these cells and identify molecular pathways
responsible for maintaining the stem cell-like state within the cell line.
Observations
High expression of stem cell markers including Nanog, Oct-4, Sox-2, and CD44 were identified in several
human pancreatic cancer cell lines. Focused effort on the BxPc3 cell line have identified a subpopulation of
OCT-4+/SOX-2+ cells (and other subpopulations). In addition, proteomic evaluation of the secretome samples
from BxPc3 showed high levels of Mindbomb-2 (MIB2), an E3 ubiquitin ligase, that positively regulates Deltamediated Notch signaling. Notch signaling is known for its importance in cell/tissue differentiation and taken
together, these results suggest 1) Pancreatic cancer cells are a strong model for identification and isolation of
putative cancer stem cell markers, and 2) BxPC3 cell line has de-differentiated into a stem cell-like state
through modulating expression of the Notch pathway. These findings offer an expanded mechanistic
understanding of the driving force behind CSCs and the potential for a new diagnostic marker(s) to identify
pancreatic cancers at an earlier stage while still treatable.
In Vitro Assessment of Drug Induced Liver Injury Using Human Induced Pluripotent Stem Cell-derived
Hepatocytes
Author(s)
Mark Wolfe
MPI Research, Inc.
Role: Author
Background
Bio
Haiyan Ma, Ph. D.
Principal Scientist and Manager of Cellular and Molecular Biology
MPI Research
Co-Author(s)
Shannon Einhorn, Ms. - Technical Application Scientist, Cellular Dynamics International, Inc
Vanessa Ott - Director of Marketing, Cellular Dynamics International, Inc
Topic
Basic Research
Problem
Drug-induced liver injury is one of the most common reasons that account for the attrition of candidate drugs
during the later stages of drug development. Consequently, early detection of drug-induced hepatotoxicity with
high sensitivity and specificity is essential before compounds are tested in animals and enter clinical trials to
save time and resources.
Background
Primary human hepatocytes represent the gold standard model for drug screening but their use is limited by
the scarce availability and the inter-individual variability. The human hepatocellular carcinoma cell line
HepG2, with phenotypic stability and unlimited availability, is limited in the metabolite toxicity testing because
of its low metabolic activities. The recent discovery and development of induced pluripotent stem cells (iPSC)
present novel opportunities in drug screening.
Hypothesis
The hepatotoxicity risk of a test compound can be determined with high sensitivity and specificity using human
iPSC-derived hepatocytes (iCell® Hepatocytes) in combination with a Thermo Scientific ToxInsight® IVT high
content imaging system and DILI Assay Cartridge. The use of iPSC-derived hepatocytes will overcome the
hurdles from primary hepatocytes and HepG2 cell line.
Research
iCell Hepatocytes were separately treated with a series of known hepatotoxic compounds (Ticlopidine,
Troglitazone, Naladixic acid, Mefenamic acid, Phenylbutazone and Aflatoxin B1) and non-hepatotoxic
compounds (Aspirin, Fluoxetine and Melatonin) and then stained with DILI Assay Cartridge followed by
imaging analysis on ToxInsight. The assay was performed using HepG2 and primary hepatocytes, too, as
comparison. The DILI Assay Cartridge simultaneously detected five multiplexed cellular targets and properties
associated with cell loss, cellular redox stress, and mitochondrial stress in individual compound-treated cells.
The hepatotoxicity of each compound across the three hepatocyte models was evaluated with high sensitivity
and specificity.
Observations
An in vitro high content screening assay to assess DILI of a test compounds using iPSC-derived hepatocytes,
primary hepatocytes and HepG2 cells has been established. The hepatotoxicity prediction of the test
compounds using the multiparametric data generated for the test compound-treated cells demonstrates high
specificity of the assay across the three hepatocyte models but varying sensitivity for each hepatocyte model
system.
IN VITRO EXPANSION AND CHARACTERIZATION OF INSULIN-PRODUCING CELLS DERIVED FROM
HUMAN PLURIPOTENT STEM CELLS CO-CULTURED WITH ENDOTHELIAL CELLS
Author(s)
Dodanim Talavera, MD, PhD
Research Scientist
Cedars-Sinai Medical Center
Co-Author(s)
Soshana Svendsen, PhD - Research Scientist, Cedars-Sinai Medical Center
Clive Svendsen, PhD - Director of the Regenerative Medicine Institute, Cedars-Sinai Medical Center
Donald Dafoe, MD - Director of Pancreas Transplant Program, Cedars-Sinai Medical Center
Topic
Basic Research
Problem
In vitro isolation and expansion of fully differentiated insulin-producing beta cells derived from human
pluripotent stem cells (hPSCs) is still a challenge. Furthermore, mature pancreatic beta cells are difficult to
generate in vitro using conventional differentiation protocols.
Background
Diabetes Mellitus will affect 440 million people worldwide by 2030. Therefore, an adequate source of beta
cells as therapy is crucial. Beta cells can be derived from hPSCs. However, PSC-derived beta-cell lines, that
can be expanded in vitro, have not been established at present. During the normal pancreas development a
“crosstalk” between pre-patterned endoderm and ECs give rise to pancreatic endoderm and fully differentiated
beta cells. These cell signals may also take place in vitro for effective and full differentiation of PSCs into
pancreatic beta cells.
Hypothesis
ECs enhance the differentiation of hPSCs into insulin-producing beta cells and provide suitable conditions for
beta cell proliferation in vitro.
Research
Embryoid bodies (EBs) derived from hPSC lines were cultured alone or together with ECs for twenty-day
periods in presence or absence of growth factors. Following the differentiation protocol, proinsulin expression
was analyzed by FACS. Cultures with enriched proinsulin positive cells were infected with lentivirus that
carried a plasmid with mCherry (red fluorescent) and hrGFP-NLS (green fluorescent) driven by rat insulin and
human ubiquitin C promoters respectively. The mCherry+/GFP+ cells were sorted by FACS and expanded for
further characterization. Sorted cells were analyzed by immunocytochemistry and qRT-PCR for expression of
beta-cell markers. Decrease in quinacrine fluorescence was used as indicator of insulin secretion in vitro.
Observations
Higher expression levels of proinsulin were found in EBs co-cultured with ECs and treated with growth factors
(74%), in comparison to EBs treated only with growth factors (11%). Co-expression of proinsulin C-peptide,
PDX-1, and urocortin 3 was found in mCherry+/GFP+ cells as well as up-regulation of beta cell specific
markers (insulin, GLUT-2, Nkx6.1, Kir6.2, SUR1, GKS, PC1/3, and PC2). Quinacrine fluorescence decreased
along with increasing glucose concentrations. These results suggest that ECs enhance the differentiation of
hPSCs to glucose-responsive insulin-producing cells, and that these cells can be isolated and expanded in
vitro.
In vivo evaluation of the effect of Human Mesenchymal Stem Cells (HMSCs) from the Wharton’s jelly
of the umbilical cord associated with different vehicles in a rat myectomy model
Author(s)
Tiago Pereira
DVM PhD Student
ICBAS - UP
Co-Author(s)
Gärtner Andrea - PhD Student, ICBAS-UP
Irina Amorim, DVM - PhD Student, ICBAS-UP
Jorge Ribeiro, DVM - PhD Student, ICBAS-UP
França Miguel - [email protected], ICBAS-UP
Raquel Gomes - Ph.D student, ICBAS-UP
Paulo Armada-da-Silva, Professor - PhD, FMH-UTL
Ana Lúcia Luís, Professor - PhD, ICBAS-UP
Ana Collete Maurício, Professor - PhD, ICBAS-UP
Topic
Basic Research
Problem
There is evidence both from animal studies and clinical investigations showing that cell therapy involving
different types of stem cells application have promising results considering functional and morphologic
regeneration of the muscle. Mesenchymal stem cells (MSCs) from the Wharton’s jelly of the umbilical cord
possess stem cell properties, are capable of differentiating into neurogenic, osteogenic, chondrogenic,
adipogenic, and myogenic cells in vitro. An alternative approach for the restoration of the damaged skeletal
muscular tissue, considered to be an ultimate treatment of some traumatic or degenerative diseases, is the
transplantation of stem cells that limit the fibrosis and the atrophy of the involved muscle masses, and even
imply the myocytes regeneration and local revascularization. They may therefore prove to be a new source of
cells for cell therapy, including targets such as stromal tissue and muscle, by replacing the degenerated cells,
by producing growth factors or by modulating the inflammatory local response. This will help to avoid several
ethical and technical issues. Taking this into account we thought that the transplantation of MSCs might be
beneficial for skeletal muscle regeneration in severe injuries and for that purpose we initiated this experiment
by the selection of an optimal biocompatible matrix as a vehicle for these cells
Background
Skeletal muscle has good regenerative ability, but the extent of muscle injury might prevent complete
regeneration, especially in terms of functional recovery. Severe lesions, like those originated by trauma
associated with loss of healthy muscular tissue and development of fibrous tissue scar are examples of those
situations where regeneration is limited. Cellular therapy, often envisaged as an ultimate treatment of
degenerative and traumatic diseases, and involving the transplantation of multipotent stem cells, is an
alternative approach for treatment of damaged skeletal muscle. Several studies, both in animal models and
clinical investigations, show that cell therapy involving different types of stem cells, improve the regeneration
of injured skeletal muscle in terms of morphologic and functional recoveries. There are technical or/and ethical
difficulties in obtaining sufficient and appropriate stem cells from the bone marrow or from embryos (obtained
from assisted reproduction techniques or somatic nuclear transfer - cloning) that have limited the application
of this therapy option. When expanded in culture, umbilical cord matrix mesenchymal stem cells (ucmMSCs)
express adhesion molecules (CD44, CD105), integrin markers (CD29, CD51) but not hematopoietic lineage
markers (CD34, CD45). Interestingly, these cells also express significant amounts of mesenchymal stem cell
markers (SH2, SH3). They may therefore prove to be a new source of cells for cell therapy, including targets
such as stromal tissue and muscle, by replacing the degenerated cells, by producing growth factors or by
modulating the inflammatory local response. This will help to avoid several ethical and technical issues
Hypothesis
In this study we tested in vivo the application of Human Mesenchymal Stem Cells (HMSCs) isolated from the
Wharton’s jelly associated to different biocompatible vehicles to induced muscle regeneration in a rat anterior
tibial myectomy model. We questioned the hypothesis that these HMSCs could have a positive role in the
restoration of the muscle architecture after injury. These cells could be important in muscle regeneration as
they can interact with the host cell population in the remodeling of the extracellular matrix
Research
HMSC from Wharton’s jelly umbilical cord matrix were purchased from PromoCell GmbH. The HMSCs were
cultured and maintained in a humidified atmosphere with 5% CO2 at 37ºC. The culture medium was replaced
every 48 hours. The application of human MSCs in rats is possible without inducing any immunossupression
in the experimental animals. Also the karyotype of these cells was evaluated at metaphase before in vivo
application. Sasco Sprague-Dawley male rats with 250-300g were used. After a standardized 5 mm diameter
myectomy lesion of the tibialis anterior muscle, the defect was completely filled with a cellular suspension
containing 1x106 HMSCs in 30 μl of culture medium and 30 μl of fibrin glue containing fibrinogen and
thrombin (MSCFibrin Group). In another group (ConditionedFibrin Group) 30 μl of a conditioned media, a
concentrated media containing trophic factors from HMSCs in culture, was combined with 30 μl of fibrinogen
and thrombin. Control groups were also tested, with the 5 mm diameter myectomy lesion (Control Group) and
with the lesion combined with the addition of 30 μl of fibrin (ControlFibrin Group). Three more groups were
also tested by using an hemostatic matrix (Floseal®) instead of the fibrin glue used previously (ControlFloseal
Group, MSCFloseal Group and ConditionedFloseal Group) and two other groups using a hydrogel composed
of hyaluronic acid, alginate and cerium as a cross-linker (Hydrogel Group and MSCHydrogel Group). After 15
days, the animals were sacrificed and the tibialis anterior muscles of all the groups were collected and fixed in
10% buffered formaldehyde for histological analysis. The International Standard (ISO 10993-6) for biological
evaluation of medical devices was employed for assessment of the local effects after implantation of the
different biomaterials used as vehicles/scaffolds in this study. The local effects are evaluated by a comparison
of the tissue response caused by the tested implant to that caused by the control. In this study the Control
group was obtained by performing the surgical procedure (myectomy) without any biomaterial or cell
implantation. The Control, ControlFibrin and MSCFibrin Groups were also repeated in a different time-point (5
weeks) for evaluation of the muscles after complete regeneration. The surgeries were executed under general
anesthesia with xylazine (1,25 mg/100 g BW im) and ketamine (9 mg/100 g BW im)
Observations
The MSCs exhibited a mesenchymal-like shape with a flat and polygonal morphology. During expansion the
cells became long spindle-shaped and colonized the whole culturing surface. The karyotype analysis to the
HMSCs cell line derived from Human Wharton’s jelly demonstrated that this cell line has not neoplasic
characteristics and is stable during the cell culture procedures in terms of number and structure of the somatic
and sexual chromosomes. The transplanted HMSCs also presented normal morphology and
imunocitochemistry markers for MSCs. Incomplete regeneration after traumatic muscle injury with residual
functional deficiencies is a common problem in orthopedics and traumatology. In our histological qualitative
assessment, the myectomy model proved to be appropriate for a comprehensive and standard evaluation of
the rat skeletal muscular regeneration The histological analysis and ISO 10993-6 scoring proved that fibrin
glue (score of 3,6 points above Control) is less reactive as a vehicle comparing to Floseal® (score of 8,68
points above Control) and hydrogel (score of 10,94 points above Control). Fibrin alone or in combination with
other materials has been used as a biological scaffold for MSCs. Although the inflammatory cell population
was considered to be more abundant in the hydrogel in terms of the ISO scoring, the hemostatic matrix
(Floseal®) produced an exuberant calcification and for that reason was considered inappropriate to be used
as a vehicle for stem cell transplantation in the muscular tissue. In the groups treated with the conditioned
media instead of MSCs a blunted inflammatory response seemed to occur (2,83-4,24 points higher in the
MSCs groups in the ISO scoring). This finding may be justified by the fact that the conditioned media
possesses a variety of growth factors and other molecules that result from the HMSCs in vitro metabolism
during cell culture that will later be important in the host tissue regeneration. In the other and regardless the
fact that HMSCs present low immunogenicity and high immunosuppressive properties due to a decreased or
even absence of Human Leucocyte Antigen (HLA) class II expression it is possible that these cells might
trigger a more pronounced inflammatory response when transplanted in the rat’s skeletal muscle. The HMSCs
associated to fibrin presented promising results in terms of morphologic and functional recovery of the rat
tibialis anterior after myectomy. Further testing will be performed with different time-points of the regenerative
process in order to assess detailed information about distinct stages of muscular regeneration
IN VIVO STUDY OF PRO-ANGIOGENIC CAPACITY OF MESENCHYMAL STEM CELLS AND
ENDOTHELIAL PROGENITOR CELLS IN THE TREATMENT OF ISCHEMIC CARDIOMYOPATHY
Author(s)
Paula Hansen. Suss
Biologist
PUCPR
Role: Author
Background
PhD Student Biologist
Bio
PhD Student Biologist
Co-Author(s)
Luiz Guilherme Achcar Capriglione - PhD Student, PUCPR
Fabiane Barchiki - Master Degree Student, PUCPR
Lye Miyague - Master Degree Student, PUCPR
Andressa Vaz Schittini - PhD Student, Carlos Chagas Institute
Danielle Jackowski - PhD Student, PUCPR
Leticia Fracaro - Master Degree Student, PUCPR
Alexandra Cristina Senegaglia - PhD, PUCPR
Carmen Lucia Kuniyoshi Rebelatto - PhD, PUCPR
Marcia Olandoski - PhD, PUCPR
Alejandro Correa - PhD, Carlos Chagas Institute
Paulo Roberto Slud Brofman - PhD, PUCPR
Topic
Basic Research
Problem
Considering that cardiovascular disease constitute an important cause of morbidity and mortality, it is
necessary to develop new therapeutic strategies as an attempt to reduce the immense social and economic
impact.
Background
Cell therapy which have shown promising results in several preclinical studies and clinical trials. Endothelial
progenitor cells (EPC) and mesenchymal stem cells (MSC) have been shown to be important in regulating
tissue repair process, the formation of new vessels and in the regeneration of ischemic regions.
Hypothesis
The aim of this study was to compare the effect of transplantation of human umbilical cord blood derived-EPC,
human Wharton’s jelly derived-MSC and the combination of these two cell types in the treatment of ischemic
cardiomyopathy (IC) model in rats.
Research
Animal procedures were performed with the approval of the PUCPR Animal Care Committee. Wistar rats
(300-350 g) were used for the infarction and cell transplantation model. Left coronary artery ligation
procedures were performed and baseline echocardiography was performed 7 days after induction of IC. The
animals with left ventricular ejection fraction (LVEF) ≤ 40% were selected for the study. On the ninth day after
induction of IC animals were randomized between experimental groups and were injected with one of the
following in medium: (1) 2000000 MSC; (2) 2000000 EPC; (3) 1000000 MSC plus 1000000 EPC; or (4)
medium only (control). After 30 days post treatment echocardiographic analysis of rat hearts were performed
followed by euthanasia. Histologic assessments were performed by Hematoxylin and Eosin, Masson’s
trichrome and Picrossirius Red staining. Localization of human cells in paraffin-embedded sections of rat heart
tissue was done by fluorescence in situ hibridization (FISH) and quantification of angiogenic growth factors
protein-expression levels in the rat peripheral blood.
Observations
The groups of rats that received cell therapy had an increased level of human angiogenic growth factors in
peripheral blood, there was less collagen deposition in heart tissue and an improved regional myocardial.
When analyzing comparatively the mean values of LVEF in the groups, observed improvement in cardiac
function in terms of increase in LVEF between groups of cell therapy. Low quantities of transplanted cells
were detected in myocardial tissue by FISH. These results suggest that cellular therapy is important in
attenuating the progression of left ventricular dysfunction after IC. Human umbilical cord blood derived-EPC
and human wharton's jelly derived-MSC may represent a promising cell sources for cellular cardiomyoplasty
and efficient therapeutic strategy for improving cardiac function in IC.
Increased Endogenous C-kit+ Cardiac Stem Cells Diminish Myocardial Infarction Damage In Snitrosoglutathione Reductase Mice
Author(s)
Ellena Paulino
Post-Doctoral Fellow
University of Miami Miller School of Medicine/ ISCI
Role: Author
Co-Author(s)
Konstantinos Hatzistergos, PhD - Postdoctoral Fellow, ISCI, University of Miami Miller School of Medicine
Lauro Takeuchi, DDS - Postdoctoral Fellow, ISCI, University of Miami Miller School of Medicine
Rosemeire Kanashiro-Takeuchi, DVM, PhD - Research Assistant Professor, ISCI, University of Miami Miller
School of Medicine
Wayne Balkan, PhD - Research Assistant Professor, ISCI, University of Miami Miller School of Medicine
Joshua Hare, MD - Professor, ISCI, University of Miami Miller School of Medicine
Topic
Basic Research
Problem
Heart disease is the world’s leading cause of death and myocardial infarction (MI)/ischemic heart disease is
the most common type of heart disease. Cardiac tissue has endogenous cardiac stem cells (CSCs), which
proliferate after myocardial infarction (MI) to support cardiac repair. However, the factors that promote CSCmediated cardiac repair are poorly understood.
Background
Mice that are deficient in S-nitrosoglutathione reductase (GSNOR−⁄−), an enzyme regulating S-nitrosothiol
turnover, have elevated levels of S-nitrosothiols. These mice also have preserved cardiac function post-MI.
Hypothesis
We tested the hypothesis that preserved cardiac function and remodeling in post-MI GSNOR−⁄− mice is due to
higher levels of endogenous c-kit+ CSCs.
Research
All experiments on animals were approved by the Institutional Animal Care and Use Committee at the
University of Miami. Myocardial infarction (MI) was performed on 3-month old GSNOR−⁄− mice and their
corresponding wild-type (C57BL/6) controls by permanent occlusion of the left anterior descending coronary
artery (LAD). Cardiac performance was assessed by serial echocardiography. Histological sections were
analyzed for infarct size by using Image J, and endogenous c-kit+/CD45− CSCs at baseline, and 1 and 8weeks post-MI. All images were obtained using fluorescent microscopy. Data are reported as mean ± SEM.
Student t-test, and one or two-way ANOVA. Significance: P < 0.05.
Observations
There was no difference in ejection fraction (EF%) or endocardial volume (EDV, ESV) at baseline or 1 week
post-MI, but at 8 weeks, EF% was improved (30.4±2.2 vs. 21.4±2.8, p<0.05) and EDV and ESV were
preserved in GSNOR−⁄− mice compared with WT (ESV 72.7±6.0 vs. 139.6±19.7 ul; p<0.001, and EDV,
102.1_7.0 vs. 169.7±19.0 ul; p<0.001, respectively). In addition, GSNOR−⁄− mice exhibited smaller scar size
(34.6±2.8 vs. 62.2±7.9%, p<0.05). The number of c-kit+/CD45− CSCs was greater in post-MI GSNOR−⁄− hearts
compared to WT, independent of the zone analyzed (infarct+border zone: 6.3±0.8 vs. 2.3±0.9 cells/mm3,
p<0.05; remote zone: 0.4±0.1 vs. 0.1±0.05 cells/mm3, p<0.05).
Conclusion. Cardioprotection in GSNOR−⁄− mice is associated with Higher c-kit+ levels resulted in decreased
cardiac remodeling which improves cardiac function and mice survival post-MI. Thus, c-kit+ CSCs contribute to
cardioprotection in GSNOR−⁄− mice following MI, and suggest that enhanced S-nitrosylation based signaling
contributes to CSC function following injury.
Induction of Human Endometrial-derived stem cells into enamel-secreting ameloblasts
Author(s)
Jafar Ai
professor of Tissue Engineering and Cell Therapy, Tehran University of Medical Sciences, Tehran, Iran
Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of
Medical Sciences, Tehran, Iran
Role: Author
Azadeh babaee
. in cell molecular biology
Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of
Medical Sciences, Tehran, Iran
Role: Author
Co-Author(s)
Anthem Bah rami - PHD student of tissue engineering, Department of Tissue Engineering, School of
Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran
abdolreza mohamadnia - PHD student of molecular medicine, Hamedan University of Medical Sciences
Topic
Basic Research
Problem
Ameloblasts disappear during tooth eruption, while enamel can be defective due to caries, genetic diseases,
or injury. Thus, other sources of postnatal stem cells are needed as the epithelial component for in vitro
generation of tissue engineered teeth that may be used in replacement therapy. There are a few reports on
the successful transformation of cells into dental epithelial cells. Except for cultured enamel organ cells, a few
cell resources have yet been detected to replace or regenerate ameloblasts.
Background
Previous attempts towards detection of various stem cells in unerupted tooth buds, bone marrow or
embryonic stem cells have provided opportunities for transformation into dental epithelial cells. However,
difficulties in sample preparation, ethical matters and lower potency of stem cells frequently hamper realization
of this purpose.
Human Endometrial-derived stem cells (hEnSCs) are the abundant and easy available source for cell
replacement therapy and can differentiate into numerous types of functional tissue cells.Invitro
transdifferentiation of endometrial stem cell into osteoblasts, adipocytes, chondrocytes and myoblast and
neural cell has recently been confirmed. Also, other studies reported that endometrial stem cells have already
the potential to give rise to odontoblast- like cells. Although hEnSCs have already demonstrated the potential
to give rise to dental mesenchymal-like cells, their ability to give rise to ameloblast-like cells have never been
tested. Here, we investigate the potency of hEnSC in ameloblast differentiation.
Hypothesis
Stem cells from human endometrium are multipotent and accessible source for cell therapy. We
hypothesized here that hEnSCs could be an appropriate cell source for tooth regeneration. The ability of
hEnSC to differentiate into Ameloblasts was examined through co-culturing hEnSCs in the presence of Rat
dental mesenchymal cells and
FGF8.
Research
Single-cell suspensions of EnSCs were obtained from endometrial tissues from 10 women experiencing
normal menstrual cycles, and were cultured. These cells were treated with co-cultured hEnSCs in the
presence of Rat dental mesenchymal cells and FGF8 (100ng/ml) for 14 days. The ameloblastic
differentiation of the hEnSC was assessed by cellular morphology and further
confirmed by immunocytochemistry , Real time PCR and Alizarin red staining.
Observations
We show that morphological appearance in treated cells after 14 days were polarized. Also the key Amelobast
markers including AMTN, AMLEX, AMBN and CK14 were expressed in mRNA level after 14 days post
treatment. The expression AMLEX and AMBN proteins in EnSCs was confirmed by immunocytochemistry too
and the calcification was shown through Alizarin red staining. Our studies identify humanEnSCs as a
potential cell source for in vitro generation of bioengineered teeth that may be used in replacement therapy.
Induction of Stem Cell Differentiation Through Use of Azole Small Molecules
Author(s)
Hector R. Aguilar
Graduate Student
The University of Texas at San Antonio
Co-Author(s)
Ana Cristina Parra-Rivera - Undergraduate Student, The University of Texas at San Antonio
Shane Appel - Undergraduate Student, The University of Texas at San Antonio
Doug E. Frantz, Ph.D. - Assistant Professor, The University of Texas at San Antonio
Topic
Basic Research
Problem
The most significant hurdle in utilizing stem cells effectively and efficiently in regenerative medicine is the lack
of understanding of the biological processes and mechanisms involved in differentiation. Therefore, the use of
molecular probes to delineate these mechanisms are greatly needed.
Background
Stem cell based therapies provide a new tool in regenerative medicine for the treatment of a number of
debilitating diseases. However, many obstacles are present in harnessing the regenerative powers of stem
cells. The most significant hurdle is the lack of understanding of the biological processes and mechanisms
involved in differentiation. Azole small molecules with the ability to induce differentiation towards a cardiac
and neurogenic cell fate have been identified and have great potential in being able to help delineate some of
these processes.
Hypothesis
Our hypothesis is based on the premise that by drastically expanding and optimizing our toolbox of small
molecules synthesized from modern medicinal chemistry, we will identify new molecules that can be used as
probes to help us gain fundamental knowledge about the nature and behavior of stem cell differentiation.
Research
In order to test our hypothesis, the design, synthesize, and evaluation of structure activity relationships (SAR)
of substituted azole analogues has been performed. The synthesis utilizes established cross coupling
chemistry and newly discovered methodology developed in our laboratory. The lead molecules identified can
then be used to synthesize molecular probes to confirm G-protein coupled receptor 68 (GPR68) as the
biological target and help delineate its role in stem cell differentiation. This research outlines the synthesis of
these small molecules and evaluates the structure activity relationships (SAR) in order to identify new lead
compounds with increased potency.
Observations
A high through-put screening of over 150,000 compounds have identified classes of substituted azole small
molecules with the ability to induce differentiation towards a cardiac and neurogenic cell fate. Extensive SAR
studies, have identified lead analogues with the ability to significantly activate GPR68. The lead molecules can
be utilized in synthesizing probes to confirm G-protein coupled receptor 68 as the biological target and will in
turn set the foundation and basic biological understanding to develop molecules with more drug like
characteristics.
Intrathecal Delivery of Autologous Mesenchymal Stem Cells for Amyotrophic Lateral Sclerosis
Author(s)
Nathan P. Staff, M.D., Ph.D.
Assistant Professor of Neurology
Mayo Clinic
Co-Author(s)
Delana Weis - Nurse Research Coordinator, Mayo Clinic
Douglas Padley - Lead Technician, Human Cell Therapy Laboratory, Mayo Clinic
Greg Butler - Technologist, Human Cell Therapy Laboratory, Mayo Clinic
Eric Sorenson, M.D. - Professor of Neurology, Mayo Clinic
Dennis Gastineau, M.D. - Associate Professor of Hematology, Mayo Clinic
Allan Dietz, Ph.D. - Assistant Professor of Laboratory Medicine and Pathology, Mayo Clinic
Anthony Windebank, M.D. - Professor of Neurology, Mayo Clinic
Problem
Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, uniformly fatal disease characterized by
neurodegeneration of the motor neurons in the cerebral cortex (upper motor neuron), as well as the brainstem
and spinal cord (lower motor neuron). Mean age of disease onset is 63 and median survival from diagnosis to
death is 23 months. The only approved agent that alters disease course (riluzole) has a very small beneficial
effect and significant side effects.
Background
Mesenchymal stem cells (MSCs), either naïve or modified, have shown promise as an ALS treatment in
preclinical studies. There is evidence that MSCs (without modification) modulate the immune system, which
has increasingly been demonstrated to be involved in the progression of ALS. MSCs are also excellent
candidates for cell-based delivery, as they can be safely infused into the intrathecal space of animals and
survive for up to 3 months after injection. The cells appear to intercalate between resident neural cells and
maintain their typical morphology and surface marker expression. Furthermore, MSCs may be modified to
secrete biological agents thus serving as a local drug delivery system to the central nervous system. This has
particular relevance in neurological diseases where many therapies do not cross the blood-brain barrier.
Hypothesis
We hypothesize that intrathecal delivery of autologous MSCs is a potential treatment for ALS and have
initiated a dose-escalation safety trial to explore this possibility further.
Research
We have completed pre-clinical safety studies of intrathecal delivery of autologous MSCs in rabbit and have
developed production SOPs in our cGMP cell therapy facility. We have now begun an FDA-approved (IND
#14788) dose-escalation safety study of intrathecal administration of autologous fat-derived MSCs in ALS
patients (Clinical Trials Identifier: NCT01609283). Twenty-five patients with El Escorial definite ALS (less than
two years symptom duration) will be enrolled. Fat-derived autologous MSCs will be delivered intrathecally
during a three-day hospitalization in our Clinical Research Unit. Five groups of five subjects each will receive
treatment using a dose-escalation protocol (group 1: 1 x 10(7), group 2: 5 x 10(7), group 3: 5 x 10(7) (twice
separated by one month), group 4: 1x 10(8), and group 5: 1 x 10(8) (twice separated by one month)).
Observations
Patient reported symptoms, blood, CSF and MRI data will be gathered at specified time points following MSC
treatment. Further details of the trial protocol, patient enrollment statistics, and initial safety data will be
presented.
Isolation of MIAMI cells from cadaveric hands five days post-mortem
Author(s)
Gianluca D'Ippolito
Research Associate Professor
University of Miami Tissue Bank
Role: Author
Co-Author(s)
Gaetan Delcroix, PhD - Research Associate, University of Miami Tissue Bank, Miami, FL
Topic
Basic Research
Problem
Stem cell recovery from cadaveric bone marrow (BM) is of great interest for allogenic stem cell therapy
purposes. However, obtaining a high yield of viable cells remains challenging and BM usually needs to be
harvested (i.e. iliac crest aspirated) from a cadaver within two-three hours post-mortem in order not to
compromise cell viability.
Background
Use of cadaveric organs and tissues holds great promises for transplantation and tissue regeneration. Stem
cells derived from BM are currently being used for the treatment of various disorders. BM is a very wellorganized tissue; its architecture involves stem cells localized in close proximity to the endosteal surfaces, a
niche where oxygen tension is extremely low. More differentiated cells are arranged in a loosely graduated
fashion towards the central longitudinal axis of the bone where oxygen tension is higher.
Hypothesis
We hypothesize that marrow-isolated adult multilineage isolated (MIAMI) cells, a subpopulation of human
mesenchymal stromal cells (MSCs), are localized in the endosteal bone niche and can survive in this location
for long period of time in post mortem donors.
Research
Phalanges and metacarpals were removed surgically respectively from a 12-years-old female, and from 53years-old male donors, twelve-hour post-mortem. Phalanges and metacarpals were left at 4°C for five days in
the presence of RPMI medium. At the fifth day, soft tissue was removed from the fingers/metacarpals and with
an electric saw machine bone was cut into small pieces, approximately 1.5 cm3. Bones were placed into
dishes in the presence of medium, and cells growing out from the bones were analyzed by flow cytometry as
well as for bone differentiation potential.
Observations
Three weeks after placing the bones in dishes, cells started to appear on the plate surface. After two more
weeks, bones were transferred into new plates with medium and cells were counted and processed for
analysis. Flow cytometry analysis showed the expression of MIAMI cell markers such as SSEA-4, CD29,
CD71, CD73, CD81, CD90, CD105, and CD164. These finger-derived MIAMI cells were negative for CD34,
CD45, and HLA-DR while they demonstrated an in vitro capacity to differentiate into bone-forming osteoblasts.
This study identifies for the first time the isolation of MIAMI cells, a highly homogeneous sub-population of
MSCs from cadaveric hand five days post-mortem. These MIAMI cells were able to survive for an extended
period of time in the finger’s bone, and were easily recovered and expanded in high number. Finger-derived
MIAMI cells are highly expressing stem cell markers and are able to differentiate into bone forming cell and
can be a great source of stem cells to be used in regenerative medicine.
Isolation of non-expanded MIAMI cells with GGP-007 Antibody
Author(s)
Co-Author(s)
Gaetan Delcroix, PhD - Research Associate, University of Miami Tissue Bank, Miami, FL
Tracy Anderson - President, Amendia, Inc. and Vivex Inc., Marietta, GA
Topic
Basic Research
Problem
Most cellular therapies require plating and ex-vivo expansion of the cells before transplantation, and therefore
face issues such as a long expansion time before clinical use, increased costs and laborious quality control
procedures. In addition, the regulatory hurdles to bring cell expansion-based therapies to the market are
enormous with investments of several millions of dollars only to demonstrate safety and efficacy.
Background
Adult stem cells are of increasing interest for regenerative medicine purposes in the context of bone and
cartilage repair, skin regeneration or more complex degenerative and neurological disorders. We isolated the
developmentally immature and highly homogeneous mesenchymal stromal cell (MSC) subpopulation MIAMI
cells. MIAMI cells have been isolated from BM of iliac crest, vertebral bodies or long bones from both living
and cadaveric donors. Due to their increased proliferation and differentiation potential, non-expanded MIAMI
(ne-MIAMI) cells are excellent candidates to enhance tissue repair (i.e., bone formation) compared to classical
non-expanded MSCs currently used in clinic (i.e. Osteocell Plus, from NuVasive). In addition to their ability to
differentiate towards cells from the mesodermal lineage, MIAMI cells can also differentiate into cells from the
ectodermal and endodermal lineages, making them attractive for a wide range of clinical applications.
Allogenic MSCs have widely been used clinically without adverse effects and because ne-MIAMI cells are a
well-characterized immuno-compatible developmentally immature subpopulation of stromal cells, we expect
ne-MIAMI cells will be usable in an allogeneic setting. The product potential for ne-MIAMI cells is very broad.
These cells could be isolated directly from living patients or cadavers and used alone or in combination with a
whole host of different matrices and scaffolds. Being able to directly isolate MIAMI cells, i.e. without
expansion, will reduce the time needed between BM harvesting and treatment when used in an autologous
setting, while it will greatly reduce the cost and regulatory hurdles faced by such therapies. The ne-MIAMI
cells alone, or in combination with matrices (i.e., decellularized dermal matrix, micronized dermal matrix, bone
microparticulate, demineralized bone matrix) can be implanted into patients with non-healing wounds or
ulcers, nerve or spinal cord injuries, non-healing bone defects or bone non-unions, repair of osteoarticular
defects and other conditions that are difficult to treat.
Hypothesis
Our goal is to achieve a direct isolation of MIAMI cells from BM without any expansion. We hypothesize that
GGP-007 antibody will recognized the developmentally immature population of MIAMI cells within BM white
blood cells, thereby avoiding the expansion step usually required to isolate those cells.
Research
Isolation of ne-MIAMI cells was performed on fresh human BM. After ficoll gradient separation mononuclear
cells (MNCs) were incubated for 30 minutes at 4°C in the presence of anti-GGP-007 phycoerythrin
conjugated. FACS analysis demonstrated that 3.5% of MNCs are GGP-007+ cells (the MIAMI cells) which are
about 3,500 more than the number of MSCs isolated from BM using the empirical isolation procedure.
Observations
Our procedure allows the direct isolation of a larger amount of cells, without the need for expansion.
Subsequently, GGP-007+ cells were separated using a cell sorter. The final product contains 99.3% of GGP007+ cells, containing the ne-MIAMI cells. After plating, the ne-MIAMI cells acquire the typical morphology of
MIAMI cells within 24-hours. To verify the differentiation capacity of ne-MIAMI cells, cells were plated in the
presence of osteogenic differentiation medium for 21 days. After adherence to plastic surface ne-MIAMI cells
start to grow rapidly. At the end of the incubation time cells were stained for osteoblastic markers, as well as
alkaline phosphatase and calcium deposition. Osteoblastic-differentiated ne-MIAMI cells express high levels
of osteoblastic markers and alkaline phosphatase and phenotypically direct calcium deposition. This
preliminary data shows that ne-MIAMI cells are able to differentiate, at least, into bone forming cells.
Additionally, we determined the expression of MIAMI cell markers after plastic adherence and expansion of
the ne-MIAMI cells isolated and compared them to MIAMI cells isolated with the original method. Ne-MIAMI
cells appear to be a more immature population. The number of cells expressing immature markers was almost
double in ne-MIAMI cells compared to adherence-isolated (ad)-MIAMI cells. Other markers, such as CD29,
CD105, CD63, CD71, and CD164 were lower in ne-MIAMI cells, implying that ne-MIAMI cells appear to
express lower levels of markers of more committed cells.
Additional experiments are underway to further characterize ne-MIAMI cells which hold great promise to bring
to the market a stem cell therapy product with minimal cell-handling time, costs and regulatory hurdles.
Mass Spectroscopic Detection of Genetic Material in Salivary Microvesicles in Healthy and Diabetic
Patients in Support of CHEN’s Hypothesis of Genetic Exchange.
Author(s)
Wilfred Gregory. Chen, M.B., B.Ch., B.A.O., M.D. (N.U.I.)
Medical Doctor / Director
Urology Research Unit
Co-Author(s)
Topic
Basic Research
Problem
Our lab has been engaged in the fundamental problem of intercellular and intertissue communication between
stem cells, adult cells, cancer and metastatic cancer cells, healthy and diseased cells. Recently with the
heightened interest in microvesicles and their cargo of proteins and genetic material, as intercellular carriers of
information, the focus has been in easily accessible human salivary microvesicles, in healthy and diabetic
patients, observing several factors, especially miRNA and miDNA molecules, using mass spectroscopy
Background
In both embryological, and adult tissues, it is well established that stromal epithelial interactions are important
for development and differentiation. It is not clear as to the nature of these intercellular factors.
Hypothesis
A central biological dogma has been that DNA is stable and fixed, since Avery 1944 established that DNA is
the essential genetic material, Crick and Watson 1953 determined the double helix structure of DNA and
Nirenberg and Koranna 1961-1966 etc elucidated the genetic code. Contrary to this law the author CHEN,
W.G. 1968, 2011, 2012 proposed the Hypothesis of Genetic Exchange which postulates that “there exists a
system of exchange of intercellular and intertissue factors including genetic material DNA/RNA between cells
and tissue, stem cells and adult cells. This is important for the homeostatic control of growth and
differentiation. As a corollary to this hypothesis, disturbances of the exchange system, or the messages
themselves, result in abnormal growth even cancer". An updated version of this hypothesis was recently
published 2011.
Research
On the basis of this hypothesis, mass spectroscopic analysis was performed on salivary microvesicles of
healthy and diabetic patients. Microvesicles are effective intercellular carriers of information. Saliva is an
accessible secretion, and may be useful in disease detection and monitoring. Mass spectroscopy is an
accurate method to determine the structure and biochemical composition of macromolecules both within cells
and outside. It involves direct analysis of the specimen, in the standard state, without the disturbances by ultra
centrifugation, anticoagulants, the presence of lipoprotein particles and small platelets.
Observations
Results revealed microvesicles enclosing DNA molecules, some approximately 25 nucleotides, RNA
molecules, some approximately 25 nucleotides, and proteins, in the saliva of both healthy and diabetic
patients. This is the first demonstration of DNA molecules in salivary microvesicles in healthy and diabetic
patients.
Further studies are continuing. The presence of proteins and genetic material in microvesicles make them
possible carriers of intercellular information and lend support to the Hypothesis of Genetic Exchange.
Key Words: Intercellular Genetic Exchange, DNA, RNA, Stem Cells, Adult Cells, Differentiation, Cancer,
Saliva
Mesenchymal Stem Cells Facilitate Neuron Recovery after Oxygen-Glucose Deprivation Injury
Author(s)
Peng Huang
Research Fellow
Mayo Clinic Florida
Co-Author(s)
James Meschia - Professor of Neurology, Mayo Clinic Florida
Thomas Brott - Professor of Neurology, Mayo Clinic Florida
Abba Zubair - Associate Professor of Laboratory Med/Pathology, Mayo Clinic Florida
Topic
Basic Research
Problem
Following stroke, neurons in the area surrounding the infarct core (penumbra) are vulnerable to delayed but
progressive damage as a result of ischemia, reduced ATP levels, lack of protein synthesis and inflammation.
Currently there is no effective treatment to rescue such dying neurons.
Background
Intracerebral hemorrhage (ICH) is the least treatable form of stroke and is associated with high morbidity and
mortality. There is currently no effective treatment for ICH. Current management focuses on treatment of
precipitating factors like hypertension and coagulopathy. We are proposing to use mesenchymal stem cells
(MSC) to alleviate the hallmarks of ICH (hematoma-induced neurotoxicity and immune activation) and to
promote neuro-regeneration. MSC are multi-potent cells with the capacity to induce tissue repair and
modulate immune response.
Hypothesis
We hypothesized that mesenchymal stem cells (MSC) can rescue damaged neurons following exposure to
Oxygen-Glucose Deprivation (OGD) stress.
Research
To test our hypothesis, we first derived MSC from the bone marrow collected from a healthy volunteer. The
MSC were cultured and expanded to passage 5 (P5). Following our established MSC culture expansion
method, we demonstrated using flow cytomeric analysis that the MSC positively expressed CD73, CD90 and
CD105 while they lacked expression of CD34 and CD45. Furthermore, we demonstrated that the MSC can
differentiate into bone, cartilage and fat tissues in the appropriate differentiation medium. Also they can form
colony forming units (CFU) when seeded at very low confluence. All these indicate our cell preparation meets
criteria for MSC characteristics as defined by International Society for Cellular Therapy (ISCT). We then
evaluated the capacity of the MSC to facilitate neuron recovery and regeneration following OGD injury. M17
cell line, which was derived from neuroblastoma, was used in the OGD stress model.
Observations
Using a proliferation assay, we showed that M17 proliferation was significantly decreased after 48 hours of
OGD. In addition, we used annexin V and propidium iodide stains to demonstrate increased rate of apoptosis
after 48 hours of OGD. These effects were alleviated via co-culture with MSC. Neuron recovery was observed
within 24 hours after exposure to MSC. The effect was maximal by 48 hours and disappeared by 72 hours. In
our model, MSC were co-cultured in an insert above but in the same medium with the OGD stressed M17
cells and without direct cell-cell contact. These experiments suggest that MSC can facilitate neuro-recovery
and neuro-regeneration following OGD injury by secretion of trophic factors.
Mesenchymal Stromal Cells (MSCs) from embryonic & adult sources differ in their gene expression,
which mirrors their biological properties.
Author(s)
Vivek M. Tanavde, Ph.D
Principal Investigator
Bioinformatics Institute
Co-Author(s)
Candida Vaz, Ph.D. - Post Doctoral Fellow, Bioinformatics Institute
Betty Tan, B.Sc - Senior Research Officer, Bioinformatics Institute
Qianyi Lee, B.S - Doctoral Student, Stanford University
Sai Kiang Lim, Ph.D. - Principal Investigator, Institute of Medical Biology
Topic
Basic Research
Problem
MSCs from different tissue sources are increasingly being used in the clinic for treating a variety of disorders.
The International Society for Cellular Therapy (ISCT) has established guidelines for defining MSCs being used
in the clinic. Although MSCs from different tissue sources confirm to the ISCT definition, they often differ in
their biological properties especially in the transplanted tissue. These differences in biological properties are
not explained by gene expression studies that compare transcriptomes of MSCs from different sources.
Background
Differences in gene expression between MSCs from bone marrow & adipose tissue have been reported, but
do not explain the cellular mechanisms that explain biological differences seen in these cells.
Hypothesis
We compared gene expression patterns of MSC's derived from Embryonic stem cells, Fetal limb and Bone
Marrow with a view to correlate this with their biological properties in vitro.
Research
Each microarray dataset comprised of an undifferentiated control and three differentiated tissue categories :
Adipocytes, Osteocytes and Chondrocytes taken at an early stage (Day7) and a terminal stage (Day14). The
differentially expressed genes obtained between the differentiated tissue and the undifferentiated control were
then compared across the three sources.
Observations
A meagre of 2-4% of the differentially expressed genes were found to be common among all the three
sources. The functions of the common genes were cell death, cellular movement, cellular growth and
proliferation and cell cycle. The topmost functions for each of the sources were very different. For the human
embryonic stem cell derived MSC's, the key functions were cellular assembly and organization and DNA
replication and repair. For the fetal limb derived MSC's, the top functions were cardiovascular system
development and function and angiogenesis. For the bone marrow derived MSC's the top functions were
tissue and connective tissue development. Thus, although the MSC's derived from each of these sources
have the same surface phenotype they use different mechanisms for differentiation. These differences in gene
expression could give clues to explain biological differences.
Neural Stem Cells and A-beta-driven Hippocampal Neurogenesis: An early biomarker of Alzheimer's
disease?
Author(s)
Miguel A. Lopez-Toledano
Research Assistant Professor
Florida Atlantic University. Center for Molecular Biology and Biotechnology
Co-Author(s)
Farzaneh Modarresi - Assistant Professor, University of Miami
Mohammad Faghihi - Assistant Professor , University of Miami
Diane Baronas-Lowell - Research Associate Professor, Florida Atlantic University
Xiaoping Wu - Technician, Florida Atlantic University
Herbert Weissbach - Director and Distinguished Research Professor, Florida Atlantic University
Claes Wahlestedt - Professor of Psychiatry and Behavioral Science, University of Miami
Topic
Basic Research
Problem
Alzheimer’s Disease (AD) is an age-related progressive neurodegenerative disorder characterized by
impairment of cognitive function and progressive memory loss. It is the most common form of dementia
affecting 5% of adults over 65 years of age and there is no cure. Following diagnosis, the brain damage is
very severe and the life expectancy is five to seven years. A specific test for early diagnosis that can: 1) detect
the disease when damage could be potentially reverted and 2) understand disease development before the
appearance of cognitive impairment and brain damage is urgently needed. A key characteristic of AD is the
increased production and self-aggregation of A-beta. The hippocampus is one of the brain regions more
affected by A-beta deposition. In the hippocampus, neural stem cells (NSC) differentiate to neurons and some
studies show their contribution to the formation and consolidation of new memories. These factors make the
study of hippocampal neurogenesis in AD very appealing. Our data indicate that A-beta induces increased
neurogenesis of NSC obtained from the hippocampus in vitro. In J20, a transgenic mouse, widely used as an
AD animal model, that overexpresses APP (amyloid precursor protein) our results show an increase in
hippocampal neurogenesis vs. same-age wild type mice. This increase happens only in young animals, before
memory impairment or A-beta deposition. In older mice, when signs of memory impairment or A-beta deposits
are already present, there is a decrease in neurogenesis as compared to wild type. The neurogenic effect of
A-beta in vitro also produces changes in the profile of several microRNAs (miRNA), non-coding RNAs
(ncRNA) that act as post-transcriptional regulators of gene expression. Studying the specific changes in
ncRNA might be essential for AD early diagnosis, before other symptoms like plaque formation and memory
loss occur. Background
The aggregation state of A-beta plays a role in both its killing and neurogenic potentials in AD. A-beta
aggregation causes H2O2 accumulation and the generation of free radicals in neurons. Several antioxidants,
such as vitamin E, can protect the cells against A-beta neurotoxicity. In fact, methionine sulfoxide reductase A
(MsrA) has been shown to play a role in cell death protection against oxidative damage, acting both as a
protein repair enzyme and a scavenger of ROS. The levels of MsrA decrease in AD patients and the oxidation
level of the amino acid methionine located in the position 35 in A-beta (Met35) seems to increase A-beta
aggregation into oligomers. MsrA could play a role in modifying both A-beta aggregation and the neuronal
differentiation on NSC. As the neurogenic effect of A-beta seems to appear prior plaque deposition and
memory impairment in the mice, we hypothesize that the up regulation of MsrA activity could reduce Met35
oxidation and help to control the aggregation state of A-beta.
Recent large-scale studies of human and mouse transcriptomes have found numerous transcripts that do not
encode proteins. Tens of thousands of non-coding RNA (ncRNA) transcripts are expressed from mammalian
genomes, in various cells and tissues, and demonstrate a complex interlaced and overlapping network from
both DNA strands. CNS development, synaptic plasticity and the stress response are all tightly linked to
epigenetic mechanisms that drive gene expression. Many ncRNAs are involved in adult neurogenesis,
neuronal differentiation and the onset of neurological diseases. For this reason, it is likely that A-beta
treatment could modify the ncRNA profile and modulate neurogenesis.
Hypothesis
Based on our investigations, we hypothesize that: 1) specific A-beta aggregates induce increased
neurogenesis 2) A-beta-mediated neurogenesis is one of the first symptoms of AD, 3) the oxidative stage of
A-beta determines its aggregation potential and neurogenic ability, 4) MsrA modulates A-beta oxidation and
aggregation, 5) specific aggregates of A-beta induce increased neurogenesis by changing the ncRNA profile
and 6) changes in ncRNA levels could potentially be used as a biomarker for AD early detection.
Research
1.- Study of the aggregation capacity of different oxidative states of A-beta: Following the same aggregation
protocol, we studied the different aggregates formed with wild type A-beta (WT-A-beta) and two A-beta
analogues: one with methionine sulfoxide in position 35 (Met35(O)-A-beta) and another with norleucine, an
aminoacid non susceptible to oxidation, in position 35 (Nle-A-beta). We examined the different aggregates
formed by the three A-beta analogs by western blot analysis using a specific antibody that recognizes A-beta
(6e10).
2.- Study of the neurogenic potential of the different aggregates of A-beta: We used the aggregates obtained
in experiment 1 to study neuronal differentiation on WT-NSC. We measured the total number of neurons by
immunocytochemistry, using specific antibodies for neuronal differentiation (Beta-Tubulin III, NeuN).
3.- Measurement of MsrA levels and activity: To study the effects of internal cellular production of A-beta in
vitro from the human mutated form of APP, we obtained NSCs from the hippocampus of J20 APP-Tg mice
(J20-NSC). We also compare the MsrA levels of the hippocampi of J20 mice at different times. MsrA levels
was determined by western blot using a specific anti MrsA antibody (ab16803, Abcam) and the activity was
measured by using a colorimetric assay based on the reduction of the substrate 4-dimethylaminoazobenzene4'-sulfonyl-L-methionine-S-sulfoxide and the reduced product separated from the substrate by high-pressure
liquid chromatography (HPLC).
4.- Study of the neurogenic potential of the internal production of A-beta in vitro using J20-NSC: We studied
the effect of WT-A-beta on WT-NSC and J20-NSC by immunocytochemistry.
5.- Study of the number of NSC, cell proliferation and neurogenesis in the dentate gyrus and subgranular cell
layer in WT, J20 and Tg19959 mice at different ages: Experiments in vivo on mice were developed following
National Institute of Health guidelines and approved by The Animal Care and Use Committee at Florida
Atlantic University. In the present experiments, we used wild type (C57BL/6J), J20 and Tg19959. All mice will
be purchased from Jackson Laboratory (Main, USA), with the exception of Tg19959 (kindly provided by Dr. M.
Leissring, Mayo Clinic). All animals were bred born in house and we performed the protocols previously
described (17, 89). We sacrificed the animals at different ages between two months old up to eleven months
old. One hemibrain per animal was fixed in phosphate-buffered 4% paraformaldehyde, pH 7.4, at 4◦C for 24
hr, washed in phosphate buffer saline (PBS) three times and embedded in 3% agarose for vibratome
sectioning (Micron HM 650V, Thermo Scientific). 50 μm sections of hemibrain were made in the sagittal axis.
Sections were permeabilized in PBS with 1% TX-100 and maintained in the same plus 10% FCS and primary
antibodies two days with agitation. As a negative control, adjacent brain slices were similarly processed,
except that the primary antibody was omitted. Rabbit anti Ki67 (Vector) immunofluorescence secondary goat
anti mouse or goat anti rabbit AlexaFluor 568 and 488 antibodies were used for detection.
6.- Study of ncRNA profile changes after treatment with neurogenic A-beta in vitro: In order to study the
mechanisms underlying A-beta driven neurogenesis, WT-NSCs were treated with A-beta for 24 hours, after
which the miRNA profile was elucidated.
Observations
To see if A-beta oxidation affects self-aggregation and neurogenic potential, we studied WT-A-beta and two Abeta analogues: Met35(O)-A-beta and Nle-A-beta. Using the same protocol for oligomeric aggregation (74),
Met35(O)-A-beta showed increased fibrillar aggregation. On the contrary, Nle-A-beta resulted in stronger
bands corresponding to monomers. These data suggest that A-beta oxidation in Met35 increases its ability to
aggregate. In order to test their neurogenic ability, WT-NSCs were exposed to different analogues of A-beta
for 24 hours on the seventh day post plating. Under these conditions, Met35(O)-A-beta was able to increase
neurogenesis at the same level as WT-A-beta. However, Nle-A-beta did not increase the number of neurons
over that observed in the control. These observations suggest oxidation of A-beta (either WT-A-beta or
Met35(O)-A-beta) is needed for the formation of the neurogenic form of A-beta.
To study the effects of internal cellular production of A-beta in vitro from the human mutated form of APP, we
obtained NSCs from the hippocampus of J20 APP-Tg mice (J20-NSC). We found a significant increase in
MsrA activity in floating neurospheres (completely undifferentiated cells) of J20-NSC compared with WT-NSC,
perhaps as a cellular response to increased oxidative stress. In addition, J20-NSCs had an increase in
neuronal differentiation as compared to WT-NSCs, suggesting that A-beta produced by cleavage of APP is
able to increase neurogenesis.
To study the neurogenic effect of A-beta in vivo, we used two animal models of AD that overexpress the
mutant form of human APP: J20 and Tg19959. Our in vivo results showed increased proliferation and
neuronal differentiation in J20 three-month-old animals that disappeared with age. Tg19959 showed increased
proliferation in the dentate gyrus vs. wild type controls at all ages. Tg19959 also exhibited increased levels of
NSC (as measured by nestin production) and neuronal progenitor cells (as measured by DCX expression);
nestin and DCX levels were measured by both protein and RNA analyses at all ages. These data suggest a
dysregulation in hippocampal neurogenesis that manifests very early in the development of the AD models.
In order to study the mechanisms underlying A-beta driven neurogenesis, WT-NSCs were treated with 1 uM of
A-beta for 24 hours, after which we studied the miRNA profile. Our results identified thirteen miRNAs
statistically different in the A-beta-treated group as compared to the control group. The most striking result
was miR-376b, which showed a 236% increase in expression. In addition, treating with anti-miR-376b prior to
A-beta addition abolished the neurogenic effect of A-beta in WT-NSC, suggesting that A-beta-driven
neurogenesis is mediated by miR-376b. In summary, our preliminary results suggest that the A-beta peptide is
able to induce neuronal differentiation of NSC in vitro and in vivo. This neurogenic effect depends on the
aggregation state of A-beta, which depends, in part on the oxidation level of Met35. A-beta-mediated
neurogenesis corresponds with ncRNA dysregulation, and increased neurogenesis is observed in young APPoverexpressing mice, seemingly one of AD’s first symptoms. Therefore, we believe that miR-376b could be a
good candidate for early diagnosis of AD.
New methods for autologous Limbal Stem Cells expansion
Author(s)
Maxim Y. Gerasimov
ophthalmology, cornea tissue engineering
Ural State Medical Academy
Co-Author(s)
Ramil Rachmatullin - postgraduate, Orenburg State University
Anastasy Zvereva - postgraduate, Ural State Medical Academy
Topic
Basic Research
Problem
In this study we present promising methods for Limbal Stem Cell expansion. They include a new method of
corneal biopsy, new conception of co-cultivation autologous corneal epithelial progenitors and keratocytes,
and new biodegradable unmodified poly-hyaluronan matrix for successful ex-vivo Limbal Stem Cell expansion.
Background
Autologous Limbal Epithelial Stem Cells (LESc) transplantation is a well-known regenerative technology for
cornea surface reconstruction and limbal stem cells deficiency (LSCD) treatment. Most authors use small
corneal biopsy and lethally irradiated mouse 3T3-fibroblast feeder layer for cultivation. They used different
matrixes such as allogenic human amniotic membrane, autologous fibrin gel, contact lens and platelet poor
plasma for cell attachments and transplantation.
Hypothesis
It is known that the corneal stromal cells (keratocytes) do not only produce the main components of the
stroma, but also can be cultured in vitro. So they may be used like a feeder layer for LESc expansion,
therefore we developed a new protocol for corneal biopsy. Due to the fact that hyaluronic acid has a positive
action for corneal epithelization we have tested a new biodegradable nanostructured poly-hyaluronan matrix
for LESc expansion.
Research
Small slices of cornea (1-2 mm2) from limbal zone were taken from 3 patients before cataract surgery
(approved by the Ural State Medical Academy Local Ethics Committees). Taking into account that the maxim
quantity of keratocytes is in 10% of stroma surface, we used deep cuts biopsy (120 micron). Limbal explants
were cultured in DMEM (high glucose) with 10% fetal bovine serum. Corneal epithelial progenitors and
keratocytes mix cell cultures were seeded on biodegradable nanostructured poly-hyaluronan matrix and
cultured being submerged in media or using an «air-liquid» method. Samples were harvesting after 14 days in
formalin fixed and embedded in paraffin. Sections were stained with hematoxylin & eosin (H&E) for pilot
morphological results.
Observations
Phase contrast microscopy showed that the primary cells from limbal explants formed a feeder-like corneal
stromal cells monolayer with epithelial colony areas. Using submerged cultured in media 2-4 epithelial cell
sheet on poly-hyaluronan matrix was yielded. Using an «air-liquid» method 3-5 epithelial cell sheet on polyhyaluronan matrix, including the basement membrane was yielded.
Novel angiogenic therapies: gene-modified adipose-derived stromal cells and combined gene delivery
Author(s)
Pavel I. Makarevich, MD
Research Fellow
Russian Cardiology Research and Production Complex, Laboratory of Angiogenesis
Co-Author(s)
Evgeny Shevchenko, PhD - Research fellow, Russian Cardiology Research and Production Complex
Zoya Tsokolaeva, PhD - Research fellow, Russian Cardiology Research and Production Complex
Veronika Sysoeva, PhD - Assistant professor, Lomonosov Moscow State University
Alexander Shevelev, PhD - Research fellow, Russian Cardiology Research and Production Complex
Alina Gavrilov, BS - Student, Universitat Tubingen
Evgeny Gluhanyuk - Student, Lomonosov Moscow State University
Griogorieva Olga - PhD student, Lomonosov Moscow State University
Vlasik Tatyana, PhD - Head of laboratory, Russian Cardiology Research and Production Complex
Yelena Parfyonova, MD, Dsc - Prof., Head of Laboratory, Russian Cardiology Research and Production
Complex
Topic
Basic Research
Problem
Most studies on gene and cell therapies show high efficacy in animal studies yet lacking long-term tissue
protection in clinical trials thus indicating the need for development of new approaches in therapeutic
angiogenesis.
Background
Induction of angiogenesis in ischemic tissue is an important therapeutic goal. It can be achieved by gene
transfer of angiogenic growth factor genes or by cell therapy using wide variety of cell types. In most cases
such therapies show high efficacy in animal studies yet lacking long-term tissue protection in clinical trials thus
indicating the need for development of new approaches in therapeutic angiogenesis.
Hypothesis
Hypothesis behind our study is that more effecient treatment of ischemia can be developed using VEGFoverexpressing adipose derived stromal cells or combined delivery of growth factors in plasmid vectors
injected directly to ischemic tissue.
Research
The first emerging solution is application of new vectors. In our study we have tested a panel of constructs
based on a novel pC4W plasmid that is characterized by higher in vitro protein output compared to pcDNA3
used as reference. Next step we tried to use gene transfer of several therapeutic genes to enhance
angiogenic response in tissue. We cloned cDNAs of human VEGF165, HGF, angiopoietin-1 and mouse
urokinase to pC4W and tested efficacy of sole genes or their pair combinations in murine hind limb ischemia
model. We found that gene transfer of VEGF+HGF, VEGF+ANG1 or ANG1+HGF amplified limb perfusion
compared to sole vectors yet uPA combinations failed to show synergistic interaction. Our findings were
confirmed by histological studies and showed reduced necrosis, higher CD31+ and SMA+ density in
combined gene transfer groups. We also managed to identify possible changes in cytokine expression in
endothelial cells stimulated by VEGF+HGF. In particular, MCP-1 and IL-8 might be involved in enahnced
angiogenic response after administration of a combination of plasmids. Next widely used approach is cell
therapy using adipose-derived stromal cells due to their feasibility and well-characterized tissue-protecting
properties. In our study we tried to use gene-modified ADSC overexpressing human VEGF165.
Overexpression was induced by rAAV2-mediated transduction of human ADSC taken from different donors
during surgery. In vitro tests confirmed stable and potent secretion of VEGF165 for at least 30 days after
transduction. Further in vivo studies revealed that VEGF-expressing ADSC induce higher vascularization of
Matrigel plug in nude mice compared to unmanipulated ADSC or GFP-transduced cells. Vessel density
(identified by CD31 and SMA staining) was higher in Matrigel plugs from VEGF-ADSC animals indicating
VEGF-driven formation of mature arterioles. Hind limb ischemia model test showed increased limb perfusion
after injection of VEGF-ADSC compared to controls and furthermore we found that VEGF-ADSC-treated mice
had higher perfusion than animals that received VEGF-carrying plasmid.
Observations
Thus we can conclude that combined gene transfer and gene-modified cell therapy can be used to enhance
angiogenic response in ischemic tissue and can present an attractive target for further translation after
appropriate safety tests.
Novel neuroendocrine progenitor cells of the olfactory epithelium function in neural stem cell
maintenance and regeneration
Author(s)
Mridula (Meera) Vinjamuri
Graduate Student
University of Rochester
Co-Author(s)
Catherine Ovitt, PhD - Assistant Professor, University of Rochester
Topic
Basic Research
Problem
Challenges facing the development of neuroregenerative cell-based therapeutics include the identification of
potential donor cells, and characterization of their behavior in a transplant environment. The identification of
neuroendocrine cell progenitors, which possess the ability to stimulate tissue regeneration through the
induction of endogenous stem cells, would be a critical advancement for engineering neuroregeneration.
Background
The olfactory epithelium (OE) is uniquely characterized by constant neurogenesis throughout the life of the
organism. The stem cell niche responsible for the continuous replacement of olfactory neurons is located in
the basal layer of the epithelium. It includes the quiescent horizontal basal cells and the progenitor globose
basal cells, which are marked by the expression of transcription factor Ascl1 (Achaete-scute-like 1).
Supporting microvillar cells secrete neuropeptide Y (NPY), a neurotransmitter that induces proliferation of
neuronal progenitors by signaling through the NPY1 receptor expressed on the basal progenitors in the OE.
Previous studies in salivary glands identified bipotential progenitors marked by expression of transcription
factor Ascl3. In this study, we report that Ascl3 expression marks a novel neuroendocrine progenitor cell
population in the murine OE. These neuroendocrine cells function in the normal maintenance of OE, as well
as during tissue repair, through crosstalk with the basal stem and progenitor cell pool.
Hypothesis
We hypothesize that the Ascl3+ neuroendocrine progenitors play a critical role in maintenance and
neuroregeneration of the OE through the production of signaling molecules that directly regulate proliferation
and differentiation of neural stem cells.
Research
Neuroendocrine progenitors were identified by lineage tracing, using a mouse model in which Cre
recombinase is driven by the Ascl3 promoter. Descendants of Ascl3-expressing cells were traced by mating
the Ascl3-Cre mouse with a LacZ reporter strain. Ascl3+ neuroendocrine progenitor cells were specifically
ablated by mating the Ascl3-Cre mouse with a strain carrying a conditional DTA (diphtheria toxin A) gene.
NPY knockout mice were used to compare olfactory stem cell maintenance and regeneration in the absence
of NPY alone. Cell specific marker analysis was performed using either immunohistochemistry or in situ
hybridization. EdU (5-ethynyl-2´-deoxyuridine) incorporation and the Click-iT EdU cell proliferation assay kit
were used to measure cell proliferation. Olfactory epithelial lesions were induced following intra-peritoneal
injections of methimazole into adult mice.
Observations
We demonstrate by genetic fate mapping that Ascl3-expressing cells are precursors of the neuroendocrine
microvillar cells of the OE. These are the primary source of neuroproliferative factor NPY. Cell-specific
ablation directly demonstrated the precursor-progeny link between Ascl3+ cells and microvillar cells, although
we find no evidence of direct regulation of NPY by the Ascl3transcription factor. In comparison to the
phenotype of NPY knockout mice, the specific ablation of Ascl3-expressing progenitor cells has significant
effects on the neuronal stem and progenitor cells in the OE. This demonstrates the critical role that microvillar
cells play in regulating stem and progenitor cell maintenance and proliferation. To confirm these findings, we
analyzed the ability of the OE to regenerate after major injury. We find that in wild type mice, an early step in
the OE regeneration process is the reappearance of the Ascl3+ neuroendocrine progenitor cells. However, in
the cell-specific ablation model these progenitor cells are blocked from reappearing, leading to limited and
disorganized regeneration of the OE.
Paracrine Activity and Angiogenic Properties of Adipose Tissue–derived Mesenchymal Stem Cells
from Patients with Ischemic Heart Disease complicated by Diabetes Mellitus Type 2 and Heart Failure.
Author(s)
Nina Dzhoyashvili
M.D., PhD student
Medical Faculty of Lomonosov Moscow State University
Co-Author(s)
Anastasia Efimenko, M.D., PhD - Resident Doctor, Medical Faculty of Lomonosov Moscow State University
Natalia Kalinina, PhD - Senior Researcher, Medical Faculty of Lomonosov Moscow State University
Yelena Parfyonova, M.D., PhD,D.Sci - Professor, Medical Faculty of Lomonosov Moscow State University;
Head of the Angiogenesis Laboratory at Russian Cardiology Research and Production Center
Topic
Basic Research
Problem
Therapeutic effects of mesenchymal stem cells (MSCs) in the treatment of many diseases may be due to their
paracrine actions. The regenerative potential of MSCs can be deteriorated in age-related diseases such as
coronary artery disease, diabetes mellitus, arterial hypertension. This knowledge is necessary to provide new
insights into molecular and cellular mechanisms underlying many pathological conditions. In addition, these
findings will allow us to increase therapeutic potentials of stem/progenitor cells including autologous stem cells
and to develop novel approaches for stimulation of endogenous regenerative processes.
Background
Cardiovascular diseases (CVD) is today the largest reason of global mortality and will continue to dominate in
the future. There is substantial evidence that among many factors diabetes mellitus is a significant contributor
to CVD risk. The magnitude of the morbidity and mortality is more than two-fold greater in people with both
diabetes mellitus and CVD compared to non-diabetics. Most of cardiologic conditions result in permanent
cardiac tissue damage followed by heart failure. Current therapies primarily aim to treat the pathological
remodeling that occurs after cardiac injury. Animal studies over the last decade indicate that the systematic
administration of mesenchymal stem cells possesses the regenerative potential and can improve cardiac
function. MSCs are considered to be one of the most promising therapeutic agents for cell therapy.
Hypothesis
Adipose tissue-derived mesenchymal stem cells (ADSCs) have already been used in several clinical trials.
However, their properties in patients with cardiovascular diseases and diabetes mellitus type 2 are poorly
investigated. We presumed that age-related diseases such as atherosclerosis and diabetes mellitus type 2
may deteriorate MSCs' ability to secrete multiple angiogenic factors for tissue regeneration and newly vessels
formation.
Research
We investigate paracrine effects and angiogenic properties as well as proliferative activity of AD-MSCs from
patients with ischemic heart disease (IHD) , diabetes mellitus type 2 (DM2) and heart failure (HF). AD-MSCs
were isolated from subcutaneous adipose tissue of patients with IHD (n=23); IHD and DM2 (n=20); IHD and
HF (n=12); IHD, DM2 and HF (n=6) and control donors (n=15) with age ranging from 44 to 80 and cultured for
2-3 passages. Proliferation activity (CFSE assay), angiogenic factors gene expression (real-time PCR) and
secretion by ADSC (ELISA) were measured. Conditioned media harvested from ADSC cultures were
assessed for their ability to stimulate tube formation by human EA.hy926 endothelial cells on Matrigel.
Observations
ADSC phenotype characterized by flow cytometry was CD90+/CD73+/CD105+/CD45-/CD31- for all groups, and
cells were capable for adipogenic and osteogenic differentiation. No differences were found in cell proliferation
among all groups. Total tube length formed by EA.Hy926 cells was lower in both groups IHD +DM2 (p=0, 01)
and IHD+HF (p=0,029) compared to healthy donors. There was a decreasing trend in the group of IHD
(p=0,101). We showed that despite the increased mRNA expression levels of VEGF and PlGF there wasn’t
increase in VEGF and PlGF secretion in both groups IHD +DM2 and IHD+HF. ENDS and TbS-1 mRNA levels
were significantly increased in all groups of patients compared to the control group. We could not show an
increase in the secretion of these factors. It may be due to a low stability of these proteins. We found the
increase in mRNA and protein expression of PAI-1 in all groups compared to the control group. We showed
negative correlations between the total length tube of endothelial cell EA.hy 926 and gene expression of
ENDS and TbS-1 in ADSCs from patients in IHD+HF and IHD groups. Hence, we presume that decrease in
angiogenic potentials of ADSCs from patients with IHD complicated by diabetes mellitus type 2 and heart
failure is explained by disturbance in coordinated network of pro- and anti-angiogenic growth factors: proangiogenic action is insufficient in the presence of more potent activity of anti-angiogenic growth factors.
Pharmacologically active microcarriers carrying stem cells for tissue engineering in an organotypic
model of neurodegenerative disorders
Author(s)
Nicolas Daviaud
PHD student
UMR S_1066 - LUNAM university, Angers, France
Co-Author(s)
Elisa Garbayo, PhD - Pharmacy and Pharmacetical Technology Department, University of Navarra,
Pamplona, Spain
Laurence Sindji, Assistant engineer - INSERM 1066, Angers University, Angers, France
Miguel Perez-Pinzon, Professor - Miller School of Medicine, Department of neurology, University of Miami,
Florida, USA
Paul C Schiller, Associate Professor - Bruce W. Carter Veterans Affairs Medical Center, and GRECC,
University of Miami, Florida, USA
Claudia N Montero-Menei, Associate Professor - Angers University, INSERM U1066, Angers, France
Topic
Basic Research
Problem
Neurological disorders as Parkinson’s disease, Alzheimer’s disease, Huntington’s disease or ischemia are
characterized by the loss of neurons or glial cells in the brain. Cell therapy for central nervous system
disorders is a very promising strategy, but, grafted cells survival and integration within the host tissue is poor .
Only around 10% of the grafted cells survive and differentiate into neuronal cells.
Background
Pharmacologically active microcarriers (PAMs), are biodegradable and biocompatible poly(lactic-co-glycolic
acid) microspheres, presenting a 3-dimensional biomimetic surface and releasing a therapeutic protein. These
combined parameters stimulate survival and differentiation of the cells conveyed on their surface. Moreover
the cell/PAM complexes may also affect their microenvironment. We have recently shown in hemiparkinsonian rats that PAMs with a laminin biomimetic surface, releasing neurotrophin-3 (NT-3) enhance the
survival and differentiation of marrow-isolated adult multilineage-inducible (MIAMI) cells leading to the
neuroprotection and repair of the nigrostriatal pathway. Neural Stem Cells (NSC) aer specific stem cells of the
central nervous system and are able to differentiate into neurons. MIAMI cells are adult stem cells that are
able to differentiate into neuron-like cells and secrete tissue-repair factors. Both cells can protect or repair the
nigrostriatal pathway.
Hypothesis
We hypothesized that these cells will protect or repair the nigrostriatal pathway by two different means. NSC
will mainly replace the lost dopaminergic cells while MIAMI cells will protect this pathway mainly by secreting
tissue-repair factors.
Research
In this study we first developed an organotypic model of PD to enable an easy, rapid and reproducible
investigation model of tissue engineering approaches for this pathology. Using this PD organotypic model, we
next studied the potential of MIAMI cells compared to NSC to protect or repair the nigrostriatal pathway. We
then examined the beneficial effect of delivering these cells forming complexes with laminin-coated PAMs
releasing NT3. Finally, we focused on the possible mechanism underlying their tissue-regenerative effect.
Observations
Organotypic model of PD was made of 400µm sagittal slices which developed the disease after a mechanical
lesion within two days. Stem cells alone or in combination with laminin-coated PAMs releasing NT3 were
injected into the striatum of the PD organotypic model and their behavior were analyzed at 1 day and 2 weeks
after implantation.We showed that the implanted stem cells differentiated into neuron-like cells expressing β3tubulin and tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis. Dopamine levels are
currently being evaluated. Moreover, survival and neuronal differentiation was enhanced when combining the
stem cells with the PAMs. In addition, repair of the nigrostriatal pathway was observed 2 weeks after injection
of the stem cells/PAM complexes, particularly when using PAMs releasing NT3. Gene expression analysis
revealed that NSCs express stanniocalcin-1 and MIAMI cells are able to express and secrete particularly
vascular epithelial growth factor among other neuroprotective factors. This factor might be responsible of their
neurorepair effect through the protection of the microvasculature around the graft, which was enhanced after
implantation of MIAMI cells combined to PAMs. In conclusion, these results show the potential interest of this
approach for tissue engineering strategies. In this regard, we are currently developing a similar therapeutic
procedure for Huntington’s disease.
Pluripotency Prediction Using A Key Set Of Six Epigenetic Biomarkers
Author(s)
Jill E. Petrisko
Scientist
Zymo Research Corporation
Co-Author(s)
Lam Nguyen - Senior Research Associate, Zymo Research Corporation
Manuel Krispin - Director of Applied Epigenetics, Zymo Research Corporation
Larry Jia, M.D. - President, Zymo Research Corporation
Topic
Basic Research
Problem
The ability to accurately characterize the pluripotent state of human embryonic stem (ES) cells or induced
pluripotent stem (iPS) cells is paramount to the field of human stem cell research. Pluripotent stem cells
display cell morphology, cell surface markers, a gene expression profile, and an epigenetic signature distinct
from that of partially or fully differentiated cells. Routine measures employed to characterize the pluripotent
state of an ES or iPS cell line include embryoid body formation, karyotyping, expression of transcription
factors OCT4, SOX2, and NANOG, expression of specific cell surface antigen markers such as SSEA and
TRA, and more recently, characterization of the cells’ epigenetic profile.
Background
Genome-wide DNA methylation signatures for human ES and iPS cell lines have been studied using both
reduced-representation bisulfite sequencing (RRBS) (Bock et al. 2011) and bead array platforms (Bibikova et
al. 2006). While these genome-wide methods are the most accurate means for characterizing the epigenetic
variability among ES and IPS cells lines, they are expensive and cumbersome for high-throughput research
laboratories wanting to routinely monitor the quality of their pluripotent cell lines.
Hypothesis
The OneStep qMethyl Human Pluripotent Stem Cell Panel I uses methylation-sensitive restriction enzyme
(MSRE) digestion and real-time PCR to predict the pluripotent state of an ES cell or an iPS cell based upon a
specific epigenetic profile of six key genes: RAB25, NANOG, PTPN6, MGMT, GBP3, and LYST. These gene
regions have been established in the literature (Nishino et al. 2011, Calvanese et al. 2008, and Deb-Rinker et
al. 2005) to show differential DNA methylation in their promoter regions between human ES and iPS cells, and
differentiated cells based upon bisulfite sequencing.
Research
In order to test the effectiveness of promoter regions of RAB25, NANOG, PTPN6, MGMT, GBP3, and LYST
as epigenetic biomarkers for pluripotency prediction in ES cells, genomic DNA from five human ES cell lines
ESI-017, ESI-035, ESI-049, ESI-051, ESI-053, (BioTime), one mesenchymal stem (MS) cell line (PromoCell),
one neural progenitor stem (NS) cell line (Millipore), fully differentiated liver cells, and non-methylated and
methylated human DNA standards (Zymo) were run in triplicates for each of the six promoter regions using
the OneStep qMethyl Human Pluripotent Stem Cell Panel I protocol. All samples were run in a 96 well plate on
the Applied BioSystem 7500 Real-time thermocycler and Ct values were converted to methylation
percentages based upon the 2-∆CT method (Livak and Schmittgen, 2001). In a separate experiment
conducted at Rutgers University, the OneStep qMethyl Human Pluripotent Stem Cell Panel I protocol was
tested on iPS cell lines reprogrammed from fibroblasts and cryopreserved lymphocytes.
Observations
The methylation patterns of the five ES cell lines ESI-017, ESI-035, ESI-049, ESI-051, ESI-053, (BioTime) and
the iPS cell lines were consistent with the patterns of DNA methylation in the promoter regions of RAB25,
NANOG, PTPN6, MGMT, GBP3, and LYST shown in the reported literature, while differentiated liver cells had
a distinctly opposite pattern of DNA methylation. The MS cell line and the NS cell line showed patterns unlike
that of either the ES cell lines or the fully differentiated cells. Multiple passaging or sub-culturing of an iPS cell
line for 5 to 22 passages showed that the DNA methylation pattern was stable in each of the six gene regions.
The benefit of this epigenetic profiling panel is that it can be used for pluripotency prediction and quality
control of human ES and iPS cell lines quickly and easily in the laboratory. This method does not require
bisulfite conversion of DNA or complex bioinformatic analysis. The OneStep qMethyl Human Pluripotent Stem
Cell Panel I can be run as single-plex or multiplexed real-time PCR reactions on a variety of real-time
thermocycler platforms.
Preclinical experience and perspectives of a clinical trial using CD133 stem cells
Author(s)
Yvan Torrente
Assistant Neurologist
Università degli Studi di Milano
Co-Author(s)
Mirella Meregalli - biologist, Univesità degli Studi di Milano
Andrea Farini - biologist, Università degli Studi di Milano
Marzia Belicchi - biologist, Università degli Studi di Milano
Daniele Parolini - biologist, Università degli Studi di Milano
Clementina Sitzia - biologist, Università degli Studi di Milano
Paola Razini - biologist, Università degli Studi di Milano
Letizia Cassinelli - biologist, Università degli Studi di Milano
Luis Garcia - Professor, Institut de Myologie-UPMC-Paris 6
Marcelo Baruffi - Professor, AADM/UNAERP Ribeirao Preto-SP-Brazil
Luciana Jardim - Veterinary, AADM, University of Sao Paolo.
Topic
Basic Research
Problem
Muscular dystrophies are a heterogeneous group of inherited disorders presenting a large clinical variability
regarding age of onset, patterns of skeletal muscle involvement, heart damage, rate of progression and mode
of inheritance. Attempts to repair muscle damage in Duchenne muscular dystrophy (DMD), the most severe
case, are facing several problems and no therapy is available for this disease as for all muscular dystrophies.
Background
Cell therapy is one promising approach to correct genetic diseases by contributing to tissue regeneration;
stem cells can be isolated from a healthy donor or, when possible from the same patient. In the first case cells
will be transplanted under a regime of immune suppression while in the second case, cells will have to be
genetically corrected before transplantation in the same patient from which they were derived. The recent
identification of different types of multi-potent stem cells, some of which are suitable for protocols of cell
therapy, has disclosed new perspectives in the treatment of genetic diseases.
Hypothesis
Our previous work indicated that CD133+ stem cells, a recently identified population of progenitor cells,
produce functional improvement upon intra-arterial injection in a mouse model of muscular dystrophy. Thus it
could be possible to focus upon this type of stem cell for autologous transplantation in DMD animal models.
Research
Recently, transplantation of engineered dystrophic canine muscle-derived CD133+ cells has given promising
results in Golden Retriever Muscular Dystrophy (GRMD) dogs, the most reliable animal model that shows a
form of dystrophy very similar to DMD (and even more severe in most cases). We isolated CD133+ stem cells
from muscle biopsies of GRMD dogs, we then expanded them characterized the CD133+ cells by FACS
analysis. The issue was then to use a specific-designed lentiviral vector capable of eliminating the mRNA
segment from exon 6 to 8 in canine dystrophin gene. We tested different concentration of lentiviral vectors and
verified the restoration of dystrophin transcript in culture by RT-PCR. Two dystrophic dogs were treated with
serial intrarterial injections of autologous transduced cells. The transplanted animals were analysed at
different times; most of the biopsies in all muscles were morphologically less affected than those of untreated
dogs. Functional improvement of treated dogs was also assessed by a 15 min timed running test and by a
combined clinical grading score. The untreated littermates became slower over the treatment time, whereas
two treated dogs ran faster after treatment.
Observations
Because of these results, we plan a pilot clinical trial, based on intra-muscular and intra-arterial transplantation
of autologous engineered muscle derived CD133+ cells. Efficacy and possible adverse effects will be
evaluated to test whether this approach may represent a first step towards an efficacious therapy for muscular
dystrophy.
RECOMBINANT HUMAN LAMININ E8 FRAGMENTS (LM-E8s) SUPPORT THE EFFICIENT ADHESION
AND EXPANSION OF DISSOCIATED HUMAN PLURIPOTENT STEM CELLS UNDER DEFINED AND
XENO-FREE CONDITION
Author(s)
Eihachiro Kawase
Lecture
Facility of Cell Processing, Department of Embryonic Stem Cell Research, Institute for Frontier Medical
Sciences, Kyoto University
Co-Author(s)
Takamichi Miyazaki - Postdoc fellow, Institute for Frontier Medical Sciences, Kyoto University
Sugiko Futaki - Assistant professor, Institute for Protein Research, Osaka University
Hirofumi Suemori - Associate professor, Institute for Frontier Medical Sciences, Kyoto University
Yukimasa Taniguchi - Postdoc fellow, Institute for Protein Research, Osaka University
Masashi Yamada - Assistant professor, Institute for Protein Research, Osaka University
Miwa Kawasaki - Research fellow, Institute for Protein Research, Osaka University
Maria Hayashi - Research fellow, Institute for Protein Research, Osaka University
Hideaki Kumagai - Graduate Student, Institute for Frontier Medical Sciences, Kyoto University
Norio Nakatsuji - Professor, Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University
Kiyotoshi Sekiguchi - Professor, Institute for Protein Research, Osaka University
Topic
Basic Research
Problem
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent cells,
have the potential to provide infinite sources of tissues for regenerative medicine. Although defined and xenofree media have been developed, culture conditions for reliable propagation of hPSCs still require
considerable improvement.
Background
Human pluripotent stem cells (hPSCs), including human embryonic stem cells and induced pluripotent cells,
have the potential to provide infinite sources of tissues for regenerative medicine.
Hypothesis
Although defined and xeno-free media have been developed, culture conditions for reliable propagation of
hPSCs still require considerable improvement.
Research
Here, we show the long-term undifferentiated culture of dissociated hPSCs using recombinant E8 fragments
of laminin isoforms (LM-E8s). The LM-E8s are the functional minimal forms that retain full integrin binding
activity, and they are easier to be produced as the recombinant proteins than intact laminins.
Observations
Small volume of LM-E8s was sufficient to coat the culture vessels, but the LM-E8s promoted greater adhesion
of hPSCs than intact laminin isoforms and Matrigel. We successfully maintained four hPSC lines on the LME8s in three defined media for 10 passages with single-cell passaging. Furthermore, we could culture the
dissociated hPSC on the LM-E8s in defined and xeno-free medium for over 30 passages, sustaining a high
level of expression of pluripotent markers, a normal karyotype, and the potential of differentiation into all three
germ layers. This culture system using the LM-E8s with single-cell passaging allows the robust expansion of
hPSCs for therapeutic applications.
Regenerative Medicine is 'Going to the Dogs': Treating Canine Dilated Cardiomyopathy with MSCs
Author(s)
Richard Vulliet, PhD, DVM
Professor and Founder, ReGena-Vet Laboratories, LLC
U C Davis and ReGena-Vet Laboratories
Co-Author(s)
Pamela Rosman - Co-ordinator`, ReGena-Vet Laboratories, LLC
Topic
Basic Research
Problem
Non-ischemic dilated cardiomyopathy (DCM) results from the premature death of cardiomyocytes (CM). As
the CMs are lost, they are replaced with scar tissue and in rare cases, fatty infiltration. The heart enlarges to
compensate for this loss of the contractile cardiomyocytes. Eventually, the heart cannot compensate for the
lost CMs, and cardiac output is reduced. Congestive heart failure and pulmonary edema occur following this
loss. A heritable form of DCM occurs in Doberman Pinschers at approximately 6 to 8 years of age. Affected
dogs are usually euthanized for humanitarian reasons when pulmonary congestion and edema cannot be
controlled by conventional medical treatments. Fifty percent of affected dogs are euthanized within three
months and ninety-five percent are euthanized within six months following onset of clinical signs.
Background
Several studies have examined the effects of MSCs in dilated cardiomyopathy. Nagaya et al
demonstrated that intra-myocardial injection of MSCs significantly increased capillary density, decreased
amount of collagen and improved cardiac contractile function in a myocarditis-induced rat model of dilated
cardiomyopathy. Notably, some engrafted MSCs were positive for cardiac myocyte markers (troponin T,
connexin-43), while others formed vascular structures, indicating that these cells could differentiate into
different cell types in this environment. Shabbir et al., using the Syrian hamster model of dilated
cardiomyopathy, demonstrated that intramuscular injection of MSCs would improve cardiac function. They
also found signs of myocardial regeneration and a decrease in fibrosis in the dilated myocardium. The
therapeutic potential of MSCs in canine dilated cardiomyopathy has not been investigated.
Hypothesis
Administered canine MSCs will reverse the progressive decrease in cardiac contractility associated with nonischemic dilated cardiomyopathy in Doberman Pinchers. The MSCs will increase cardiac function, improve
cardiac parameters and increase survival of critically ill dogs.
Research
Following systemic administration of canine MSCs, histology revealed the presence of fluorescently tagged
MSCs in close proximity to damaged cardiomyocytes thirty-six hours after infusion. The administration of stem
cells reversed the precipitous decline in fractional shortening (FS%) , a parameter commonly used by
veterinary cardiologists to determine the pumping action of the heart. Treated dogs had an improvement in
exercise tolerance and/or cardiac parameters. We have improved the quality and quantity of life with bone
marrow stem cells using our treatment protocol.
Observations
Using dog patients with terminal diseases allows the development of the optimal protocol. During the course
of treatments, our protocols have evolved for safety, efficacy and convenience. We have extended the life of
canine DCM patients for over a year and improved their quality of life. Further, we have also identified several
adverse or potential reactions following administration of MSCs. We conclude that dog patients with naturally
occurring terminal diseases can be used to develop treatment protocols for human diseases. Dog patients are
excellent predictors of response to stem cell therapy. In comparison with laboratory animals, they can be used
to optimize routes of administration, dose, and other parameters necessary prior to treating human patients.
They may also identify adverse reactions due to the longer duration of treatment and life span.
Regulation of Human Embryonic Stem Cell Pluripotency and Self-renewal by a Novel RNA Binding
Protein L1TD1.
Author(s)
Maheswara Reddy Emani
Post Doctoral Fellow
Turku Centre for Biotechnology, University of Turku.
Co-Author(s)
Elisa Närvä - Ph.D student, Turku centre for Biotechnology
Nelly Rahkonen - Ph.D student, Turku centre for Biotechnology
Juha-Pekka Pursiheimo - Senior scientist , Turku centre for Biotechnology
Kari Nousiainen - Ph.D student, Aalto University School of Science
Miro Viitala - Masters student, University of Turku
Harri Lähdesmäki - Professor and Academy Research Fellow, Aalto University School of Science
Riikka Lund - Senior scientist, Turku centre for Biotechnology
Riitta lahesmaa - Director, Professor, Turku centre for Biotechnology, University of Turku
Topic
Basic Research
Problem
Human pluripotent stem cells (hPCSs), including embryonic and induced pluripotent stem cells (hESCs and
hiPSCs), offer a wide range of opportunities for cell therapy and disease modeling. The detailed
understanding of the molecular mechanisms regulating the stem cell status is still elusive.
Background
We have identified a novel RNA binding protein, L1TD1, with previously unknown function that is highly
expressed in hESCs and iPSC, but not in normal somatic cells.
Hypothesis
We hypothesize that L1TD1, in complex with RHA-LIN28, regulates OCT4 translation in hESCs and plays an
important role in maintaining hESCs pluripotency and self-renewal.
Research
By the experiments of transcriptional profiling a panel of novel genes with unknown function characteristic for
hESCs was identified (Lund et al. manuscript in preparation). Moreover, while being highly expressed in
hESCs, these genes were rapidly down regulated during differentiation of the cells to embryonic bodies. In
addition, these genes were also highly expressed in iPS cells. One of the top genes selectively expressed by
hESCs is L1TD1, also known as ECAT11 or FLJ10884. L1TD1. Further using siRNA approach we have
studied L1TD1 function in hESCs.
Observations
Here we demonstrate that L1TD1 regulates self-renewal and is a novel marker for hESCs. L1TD1 is highly
expressed in hESCs and rapidly down-regulated during differentiation. Moreover, our results show that
silencing of L1TD1 induces downregulation of known regulators of pluripotency OCT4, NANOG and SOX2
leading to differentiation of the cells. L1TD1 is also highly expressed in testicular and colorectal cancers. We
have further demonstrated that L1TD1 interacts indirectly (via RNA) with LIN28 and directly with RNA helicase
A (RHA), and is required for hESC self-renewal and cancer cell proliferation.
Reprogramming to Pluripotency Can Conceal Somatic Cell Chromosomal Instability
Author(s)
Liviu Malureanu
M.D.
Mayo Clinic
Co-Author(s)
Masakazu Hamada - Ph.D., Mayo Clinic
Wei Zhou - , Mayo Clinic
Tobias Wijshake - , Mayo Clinic
Jan van Deursen - Ph.D., Mayo Clinic
Topic
Basic Research
Problem
One of the primary safety concerns of iPSCs is genetic instability, which is associated with cancer and various
other diseases, and includes abnormalities in both chromosomal structure and number. Whereas certain
structural chromosome changes have been shown to preclude somatic cell reprogramming, reprogrammed
cells have successfully been generated from somatic cells that undergo stable inheritance of an abnormal
number of chromosomes, such as Down syndrome. This implies that aneuploidy (an abnormal number of
chromosomes) is not a barrier to reprogramming. However, the extent to which defects that promote the
continuous reshuffling of whole chromosomes during mitosis, a condition referred to as Whole Chromosome
Instability (W-CIN), interfere with efficient reprogramming of somatic cells is unknown.
Background
The potential to restore pluripotency to mature somatic cells has generated new prospects in the
establishment of patient-specific regenerative therapies and has also offered new options for more advanced
and specific modeling of human disease. However, several obstacles remain prior to the therapeutic
application of iPSCs, including the risk of introducing loss of genomic integrity. Recent studies revealed that
somatic cell reprogramming introduces changes at the nucleotide level. Both cell culture length and conditions
were identified as key determinants of this type of genetic variation. In contrast to changes at the nucleotide
level, reprogramming seems to be less permissive to certain types of structural chromosome damage, such as
short telomeres and double strand DNA breaks. Cells with these kinds of aberrations are thought to be
eliminated during the early stages of reprogramming by induction of p53-dependent apoptosis. Here we have
examined the relationship between somatic cell reprogramming and W-CIN, a type of genomic instability
associated with cancer and other human disorders. High rates of chromosomal instability pose a potential
safety risk in regenerative therapies based on iPSCs, since aneuploidization is potentially tumor promoting.
Furthermore, because aneuploidy can alter the metabolic and proliferative properties of cells, aneuploidization
of cultured iPSCs may impact the analysis of studies using disease-specific iPSCs derived from patients. The
molecular mechanisms that underlie chromosome segregation and that safeguard the process are highly
complex and remain incompletely understood. More than one hundred genes are expected to contribute to
chromosomal instability in mammals, including genes implicated in chromosome condensation, sister
chromatid cohesion and decatenation, kinetochore assembly and function, spindle formation, mitotic
checkpoint control and attachment error correction.
Hypothesis
Structural chromosome defects are known to limit reprogramming of somatic cells into iPSCs, but whether
defects that cause whole chromosome instability (W-CIN) preclude reprogramming is unknown. We
hypothesize W-CIN is not a barrier to reprogramming and that aneuploid cells can be selected for or against
during reprogramming depending on the W-CIN gene defect.
Research
To begin to address the impact of numerical chromosome instability on stem cells we examined the impact of
two distinct W-CIN gene defects on somatic cell reprogramming. The first defect involves the BubR1 gene,
which encodes a core component of the mitotic checkpoint. While complete loss of BubR1 results in cell death
by mitotic catastrophe, cells with low amounts of BubR1 are viable despite frequent chromosome
missegregation and development of near-diploid aneuploidies. The second defect involves the RanBP2 gene,
which encodes a giant nuclear pore complex (NPC) protein with SUMO E3 ligase activity. Complete
inactivation of RanBP2 gene expression results in cell death, but cells with low levels of RanBP2 survive and
proliferate normally despite incomplete DNA decatenation, frequent chromosome missegregation and
aneuploidization. We investigated the impact of W-CIN on cell reprogramming using MEFs derived from
BubR1 (BubR1H/H) and RanBP2 hypomorphic (RanBP2–/H) mutant mice. Earlier work demonstrated that
BubR1H/H MEFs generate ~10% of normal BubR1 protein levels and RanBP2–/H mice ~26% of normal
RanBP2 protein levels. We selected BubR1H/H and RanBP2–/H MEFs for our studies because their
aneuploidy rates are quite similar, with BubR1H/H cultures having 36% aneuploid cells at passage 5 (P5) and
RanBP2–/H cultures 33%. Moreover, entirely different mechanisms drive aneuploidization in BubR1H/H and
RanBP2–/H MEFs. To induce reprogramming to pluripotency, Oct-3/4, Sox2, and Klf4 expression constructs
were introduced in passage 5 wildtype, BubR1H/H, and RanBP2–/H MEFs by retroviral transduction. c-Myc
was omitted because its overexpression has been associated with aneuploidization. We have quantified the
number of iPSC colonies emerging from BubR1H/H, RanBP2–/H and from wildtype MEFs. We used
immunofluorescence to screen the iPSC colonies for the presence of ES cell-associated markers. We tested
by several methods the developmental potential of BubR1H/H, RanBP2–/H and wildtype iPS cell colonies: 1)
RT-PCR analysis of retroviral transgene silencing and endogenous pluripotency-associated gene induction. 2)
qRT-PCR analysis of EBs derived from mESCs and iPSC clones for expression of embryonic germ layer
markers. 3) Analysis of teratomas derived from iPSC clones. 4) Viable chimera mouse formation from
aneuploid iPSCs injected into BALB/c host blastocysts. Decatenation of centromeric DNA by Topoisomerase
IIA (Top2a) is essential for proper chromosome separation of sister chromatids. Previously, we showed that
targeting of Top2a to inner centromeres of mitotic chromosomes is regulated by RanBP2-mediated
sumoylation. We have examined iPSC clones derived from wildtype and RanBP2–/H MEFs for Top2a
centromeres localization. We performed chromosome counts on metaphase spreads of independent
RanBP2–/H and BubR1H/H iPSC clones to determine whether there might be a bias against reprogramming
of aneuploid MEF cells and to compare W-CIN rates before and after reprogramming. Then, we selected
independent BubR1H/H iPSC clones, prepared subclones from single cells and analyzed again their
karyotypes.
Observations
Our studies provide several important new insights that should improve the efficacy of iPSC use in future
clinical applications. First, we demonstrate that W-CIN does not pose a barrier to reprogramming. Second, we
show that W-CIN iPSCs are capable of differentiating into all three distinct germ layer cell types, contribute to
chimera formation and to germ-line transmission. Third, we show that although MEFs with two distinct W-CIN
defects fully reprogram to iPSCs, with similar efficiency as wild-type MEFs, they do so with highly contrasting
outcomes on chromosome number integrity and stability: BubR1 hypomorphic iPSC clones preferentially
originate from aneuploid MEFs, while RanBP2 hypomorphic iPSC clones preferentially stem from MEFs with
normal diploid chromosome numbers, indicating that aneuploid cells can be selected for or against during
reprogramming depending on the genetic defect driving the chromosome number instability. The observation
that BubR1H/H aneuploid MEFs preferentially dedifferentiate raises the possibility that BubR1 might be a key
component of a surveillance pathway that prevents aneuploid cells from reprogramming. Knowing that BubR1
levels decrease with aging, we hypothesize that reprogramming of somatic cells from elderly individuals into
karyotypically normal and stable iPSCs may be particularly challenging. Consistent with correction of W-CIN
upon reprogramming, RanBP2–/H iPSCs localized Top2a to the inner centromeres with similar efficiency as
iPSC derived from wildtype MEFs. Fourth, our data uncovered the fascinating concept that a W-CIN gene
defect (RanBP2 hypomorphism) responsible for severe aneuploidization in somatic cells can become dormant
upon reprogramming, indicating that testing of both iPSCs and the iPSC-founding cells for chromosome
number instability will be necessary for the safe application of iPSC technology in regenerative medicine. Prior
Publication Disclosure: Part of this work was recently published in: Hamada M, Malureanu LA, Wijshake T,
Zhou W, van Deursen JM (2012) Reprogramming to Pluripotency Can Conceal Somatic Cell Chromosomal
Instability. PLoS Genet 8(8): e1002913. doi:10.1371/journal.pgen.1002913 Part of this work was recently
presented at the 18th Annual Mayo Clinic/Karolinska Institutet Research Meeting, September 20-22, 2012.
Retinoblastoma Regulates Cardiac and Mesenchymal Stem Cell Niches during Adult Heart
Regeneration
Author(s)
Konstantinos Hatzistergos, PhD
Postdoctoral research associate
University of Miami, ISCI
Co-Author(s)
Adam R. Williams, MD - Postdoctoral Research Associate, University of Miami, ISCI
Wayne Balkan, PhD - Research Assistant Professor, University of Miami, ISCI
Irene S. Margitich, MSc - Sr. Manager, Research Support , University of Miami, ISCI
Joshua M. Hare, MD - Professor, University of Miami, ISCI
Topic
Basic Research
Problem
Cardiac (CSCs) and mesenchymal stem cells (MSCs) stimulate heart repair, both primarily and secondarily by
stimulating endogenous cardiac regeneration; however, the underlying mechanisms remain elusive and
controversial.
Background We recently showed that transplantation of a combination of MSCs and c-kit+ CSCs improves
engraftment and doubles the regenerative response compared to the individual cell types in a porcine model
of cell therapy. In other regenerative tissues, similar interactions between c-kit+ stem cells and MSCs control
the regenerative response by regulating retinoblastoma (pRb) activity. To understand this interaction in the
heart, we quantified the levels of Ser-608 hyperphosphorylated pRb (Rbser608+), a marker previously
documented only in actively regenerating cardiomyocytes (CM) in salamanders, and Ink4a alternate reading
frame product (ARF), an inhibitor of muscle regeneration in mammals.
Hypothesis
Interactions between CSCs and MSCs regulate Rbser608 and ARF expression in endogenous progenitors and
CMs to enhance cardiac regeneration.
Research
Twenty adult swine underwent myocardial infarction (MI), followed 2 weeks later by intramyocardial injection
of 200M human (h)MSCs (n=3), 1M hCSCs (n=3), 200M hMSC plus 1M hCSC (MCC, n=3) or placebo (n=3).
Tissue from immunosuppressed animals was analyzed 4 weeks later for exogenous cell engraftment (human
Alu). Expression of ARF and Rbser608 were assessed in GATA4+ progenitors and mitotic (phosphoH3+) CMs.
Observations
In situ hybridization demonstrated Alu+ cells in all groups. Engraftment was ~7-fold greater in the MCC group
compared to hCSCs or hMSCs (7.8±4.6, 0.2±0.2, 1.4±0.7 Alu+/cm3 respectively, p=0.0003) and infarct size
was reduced 2-fold in MCC vs either cell type alone. Interestingly, Rbser608+ was expressed both in porcine
GATA4+ progenitors as well as in mature CM. There were 3 times as many Rbser608+/GATA4+ progenitors
(150M/cm3) in the MCC hearts compared to the other 3 groups (p<0.0001). In addition, ~20% of the CM were
Rbser608+/Ink4a(−), suggesting that the MCC hearts contained CM capable of completing the cell-cycle
(P<0.0001). There was also a 46-fold increase in mitotic CMs in MCC hearts vs. either cell alone (P<0.0001).
Importantly, MCC recipients contained a subpopulation of Ink4aARF(−)/phosphoH3+ CM, supporting the
existence of transient amplifying CM with the capacity to undergo multiple cell divisions.
Conclusions: Together these findings demonstrate a novel mechanism of heart regeneration, where hMSC
and hCSC interactions propagate Rbser608 in CM and CSCs to regulate cell-cycle and stem cell fate decisions.
Semaxinib treatment increased expression of stem cell markers in vascular endothelial cells:
Implications for resistance to cancer therapy.
Author(s)
Jayanta Das
Post Doctoral Research Associate
Florida International University
Role: Author
Co-Author(s)
Quentin Felty, PhD - Associate Professor, Florida International University
Problem
Tumor drug resistance may be a result of increased expression of stem cell markers that allow for the
reprogramming of vascular endothelial cells to a stem-like state that enhances tumor angiogenesis.
Background
The theory that malignancies depend on a small population of stem-like cells for proliferation has received
much attention. Solid tumors have been postulated to be composed of two populations of stem cells: CSC that
may ultimately drive tumor growth and progression and non-tumor forming cells. Since the spread and growth
of cancer tumors depends on infiltrating blood vessels, a better understanding of how non-tumor forming
vascular cells may become resistant to anticancer drugs may lead to novel therapies.
Hypothesis
It has been previously reported by others that the overexpression of Oct4 and Sox2 in HUVEC generated
induced pluripotent stem cells (iPSCs). Here, we determined whether exposure to Semaxanib (SU5416), a
selective synthetic inhibitor of VEGF receptor tyrosine kinase could induce the expression of stemness genes
(Oct4, Sox2, and CD31) in human microvascular endothelial cells.
Research
Brain microvascular endothelial cells (hCMEC/D3) were used to study the effect of SU5416. Both wildtype and
Magnetic-activated cell sorting (MACS) separated CD31 endothelial cells were treated with SU5416 [5µM] for
three hours and grown for four days in both adherent and non-adherent liquid cell culture. Confocal
microscopy studies analyzed the expression of stem cell markers Oct4 and Sox2 in both monolayer and cell
spheroids.
Observations
Confocal microscopy studies showed that exposure to SU5416 significantly increased the expression of Oct4
and Sox2 compared to vehicle control. This pattern of stem cell marker expression was also observed in
endothelial cell spheroids grown in B27 media liquid culture. Remarkably, SU5416 treatment increased the
diameter of endothelial spheroids compared to vehicle control. SU5416 treatment has been reported to induce
the formation of reactive oxygen species (ROS). Therefore, we determined whether pre-treatment with the
ROS scavenger N-acetylcysteine (NAC) would prevent the expression of stem cell markers. Pretreatment with
NAC showed a significant reduction in the expression of Oct4 and Sox2 compared to vehicle control.
Furthermore, NAC inhibited the SU5416 induced increase in the diameter of endothelial spheroids.
Interestingly, conditions that allow for the formation of ROS such as hypoxia have been reported to induce
stemness. Our findings suggest that drug induced stemness in vascular endothelial cells may occur through a
redox dependent signaling. Hence, tumor drug resistance may be a result of increased expression of stem cell
markers that allow for the reprogramming of vascular endothelial cells to a stem-like state that enhances
tumor angiogenesis.
SIMULATED MICROGRAVITY INDUCES INDIRECRLY CYTOSKELETON CHANGES TO PROLIFERATE
HUMAN MESENCHYMAL STEM CELLS.
Author(s)
Yumi Kawahara
President
Space Bio-Laboratories Co.,Ltd.
Co-Author(s)
Masaya Matsumoto - graduate student, Graduate School of Biomedical and Health Sciences, Hiroshima
University
Takahiro Fukazawa - graduate student, Graduate School of Biomedical and Health Sciences, Hiroshima
University
Takeshi Imura - graduate student, Graduate School of Biomedical and Health Sciences, Hiroshima University
Elham Khalesi - graduate student, Graduate School of Biomedical and Health Sciences, Hiroshima University
Sun Ya-Nan - graduate student, Graduate School of Biomedical and Health Sciences, Hiroshima University
Hiroyuki Uwatoko - graduate student, Graduate School of Biomedical and Health Sciences, Hiroshima
University
Kyousuke Nakata - graduate student, Graduate School of Biomedical and Health Sciences, Hiroshima
University
Louis Yuge - Professor, Graduate School of Biomedical and Health Sciences, Hiroshima University
Topic
Basic Research
Problem
Our studies have shown that microgravity is suitable for maintaining stem cell culture in an undifferentiated
state. However, it is unclear why stem cells can proliferate and maintain undifferentiating state easier under
microgravity environment than under normal 1G environment.
Background
Previously, we cultured mouse bone marrow stromal cells, human mesenchymal stem cells (hMSCs), and
mouse embryonic stem cells. In the research using hMSCs, we demonstrated the number of hMSCs
increased under simulated microgravity environment compared with under normal 1G environment. Moreover,
FACS analysis showed that the cells under simulated microgravity maintained undifferentiating state, but a
part of the cells under 1G went to differentiating.
Hypothesis
In space biology, immunostaining methods have shown that microgravity causes changes in cellular structure.
We consider that cells cultured in simulated microgravity are also changed cellular structure not only on the
level of protein expression but also on that of gene expression.
Research
The 3D-clinostat, simulated microgravity machine is a multidirectional G-force generator. By controlled
simultaneous rotation of two axes, the 3D-clinostat cancels the cumulative gravitational vector at the center of
the device, producing an environment with an average of 10−3G over time. This is accomplished by the
rotation of a chamber at the center of the device to disperse the gravitational vector uniformly within a
spherical volume at a constant angular velocity. We examined hMSCs cultured under 1G environment and
simulated microgravity environment to analyze immunostaining and DNA microarray. hMSCs in microgravity
stained with vinculin and stress fiber showed decreasing number of focal contact and weakening cytoskeleton.
Cell proliferation and morphological changes attributed to some of the more than 500 genes examined.
However, the expression of genes related to cytoskeleton and focal contacts (ACTG1, VCL, and others) was
unchanged compared with cells cultured under normal 1G condition.
Observations
Focal contact is considered as gravity sensor in space biology. We showed hMSCs cultured under simulated
microgravity had poorer cytoskeletons and fewer focal contacts, which agree with research on space biology.
However, DNA microarray data was unexpected and revealed changes in some interesting factors such as
actin polymerization-related molecules. Our results suggested that the cytoskeleton changing in hMSCs is
associated with stem cell proliferation and differentiation in simulated microgravity. Microgravity can provide
us new insight into stem cell biology.
Single and Double Encapsulation of Cell and Cell Clusters for Regenerative Medicine and Cell
Transplantation
Author(s)
Vita Manzoli
Senior Research Associate
Diabetes Research Institute - University of Miami
Chiara Villa
PhD Student
Diabetes Reasearch Institute - University of Miami
Role: Author
Co-Author(s)
Simona Maciotta Rolandin, Ph.D. - Postdoctoral Fellow, Diabetes Research Institute - University of Miami Miller School of Medicine
Christopher Fraker, Ph.D. - Assistant Research Professor , Diabetes Research Institute - University of Miami Miller School of Medicine
Antonello Pileggi, M.D. Ph.D. - Associate Professor of Surgery, Microbiology & Immunology, and Biomedical
Engineering, Diabetes Research Institute - University of Miami - Miller School of Medicine
Cherie L. Stabler, Ph.D. - Associate Professor, Diabetes Research Institute - University of Miami - Miller
School of Medicine
Yvan Torrente, M.D. Ph.D. - Director of Stem Cell Laboratory, Department of pathophisiology and
transplantation - University of Milan - IT
Juan Dominguez Bendala, Ph.D. - Research Associate Professor of Surgery, Diabetes Research Institute University of Miami - Miller School of Medicine
Luca Inverardi, M.D. - Professor of Medicine, Microbiology and Immunology, Diabetes Research Institute University of Miami - Miller School of Medicine
Camillo Ricordi, M.D. - Distinguished Professor of Medicine, Biomedical Engineering, Microbiology and
Immunology, Diabetes Research Institute - University of Miami - Miller School of Medicine
Jeffrey A. Hubbell, Ph.D. - Professor, Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne
(EPFL), CH
Alice A. Tomei, Ph.D. - Assistant Research Professor, Diabetes Research Institute - University of Miami Miller School of Medicine
Topic
Basic Research
Problem
Conventional alginate microencapsulation has been tested in pre-clinical animal models, including non-human
primates (NHPs) and in humans with unsatisfactory results. This is likely because of the large (600-1000µm)
diameter of standard microcapsules that impairs oxygen and nutrients exchange and causes delays in
secretion of trophic factors following cell stimulation by the host; and limited choice for transplantation sites
due to the high total volume of the encapsulation product; finally long-term applications might fail due to the
poor mechanical properties and poor in vivo stability of conventional encapsulation materials.
Background
Successful encapsulation of autologous, allogeneic or xenogeneic cells with a semipermeable barrier can be
beneficial for many therapeutic applications, including improving preservation and shipment of coated cells
and guaranteeing immunoprotection after transplantation for regenerative medicine. Especially in cell
transplantation for treatment of autoimmune diseases, including type-1 diabetes, immunoisolating properties
of capsules might allow transplantation in absence of or with reduced systemic immunosuppression.
Hypothesis
We hypothesize that by developing novel encapsulation technologies that address most concerns associated
with conventional cell microencapsulation we can turn cell encapsulation into an effective and safe therapy
and that by designing the procedure to be versatile, reproducible, and scalable we can facilitate its
translatability to clinical applications.
Research
As an alternative to conventional alginate microencapsulation, we developed two novel encapsulation
technologies: a “conformal coating” microencapsulation and a “double conformal coating” encapsulation and
compared to alginate micrencapsulation. Conformal coating is achieved by exploiting a fluid dynamic principle
that allows to wrap cell clusters with a 10-20µm layer of polymer, minimizing diffusion barriers and size of
encapsulated cell product and allowing a wider choice of transplantation sites. The double coating technology
confers a conformal coating as an extra layer to improve mechanical, permselectivity and immunoisolating
properties of enclosed alginate microcapsules (400-600µm in diameter) that are obtained with a conventional
micro droplet generator. Our combined encapsulation methods are versatile because they can be applied to
any coating materials and to any cell type and are reproducible and scalable for clinical translation.
Observations
We have shown that both the conformal coating and alginate microencapsulation technologies do not to affect
cell viability and function (i.e. secretion of trophic factor upon stimulation) of cell clusters of different origins
and we are currently optimizing the double coating technology by combining the two encapsulation methods.
We strongly believe that our platform for cell encapsulation will allow us to compare different encapsulation
technology and tailor them for different therapeutic applications as a valid and may be superior alternative to
standard alginate microencapsulation.
S-nitrosoglutathione Reductase (GSNOR) Enhances Vasculogenesis by Mesenchymal Stem Cells
Author(s)
Samirah A. Gomes
MD, PhD , Postdoctoral Associate
Interdisciplinary Stem Cell Institute University of Miami
Co-Author(s)
Erika Rangel, MD.PhD - Postdoctoral Associate, ISCI/University of Miami
Courtney Premer , BS - Research Associate, ISCI/University of Miami
Raul Dulce, PhD - Postdoctoral Associate, ISCI/University of Miami
Yenong Cao, GS - PhD student, ISCI/University of Miami
Victoria Florea , MD - Postdoctoral Associate, ISCI/University of Miami
Wayne Balkan, PhD - Research Assistance Professor, ISCI/University of Miami
Claudia Rodrigues, PhD - Research Asst. Professor , ISCI/University of Miami
Andrew Schally, PhD - Full Professor, University of Miami
Joshua Hare, MD - Full Professor, ISCI/University of Miami
Topic
Basic Research
Problem
Mesenchymal stem cells (MSCs) can differentiate into endothelial cells; however, the mechanism(s)
underlying this process remain elusive. We assessed the role of nitric oxide (NO) by comparing MSCmediated vasculogenesis in mice lacking S-nitrosoglutathione reductase (GSNOR−⁄−), a denitrosylase that
metabolizes intracellular GSNO.
Background
Nitric oxide (NO) and vascular endothelial growth factor (VEGF) signaling promotes vasculogenesis by
endothelial progenitor cells (EPCs).Although nitric oxide (NO) signaling promotes differentiation and
maturation of endothelial progenitor cells, its role in the differentiation of mesenchymal stem cells (MSCs) into
endothelial cells remains controversial. The impact of NO signaling in the differentiation of MSCs into
endothelial cells has not been previously tested. Given the similar signaling involved in endothelial
differentiation of EPCs and MSC, we reasoned that NO plays an equivalent role in this process.
Hypothesis
We tested the hypothesis that NO signaling, mediated by small molecular thiols, promotes MSC differentiation
into endothelial cells
Research
Bone marrow-derived MSCs isolated from WT and GSNOR−⁄− mice or humans (hMSCs) were grown in
endothelial growth media followed by 24h in Matrigel (in vitro), in the presence of either vehicle, L-NAME (a
nitric oxide synthase (NOS) inhibitor) or GSNO and SNAP (nitric oxide donors) and with JI-38 a growth
hormone-releasing hormone (GHRH) agonis . Vasculogenic markers such as VEGF, PDGFR and VEGFR2,
NO production and NOS expression by MSCs was assessed. Additionally, used an allograft assay to study
the in vivo angiogenic potential of the murine MSCs.
Observations
GSNOR–/– MSCs exhibited markedly diminished capacity for vasculogenesis in in vitro and in vivo relative to
WT MSCs. This decrease was associated with downregulation of the platelet derived growth factor α (PDGFα)
receptor (PDGFR) in GSNOR–/– MSCs, a receptor essential for vascular endothelial growth factor (VEGF)-A
action in MSCs. Pharmacologic inhibition of NO synthase and stimulation of growth hormone-releasing
hormone receptor (GHRHR) with GHRH agonists augmented VEGF-A production and normalized tube
formation in GSNOR–/– MSCs, whereas NO donors or PDGFR antagonist reduced tube formation ~50% by
murine and human MSCs. The antagonist also blocked the rescue of tube formation in GSNOR–/– MSCs by LNAME or GHRH agonists, JI-38, MR-409 and MR-356. Therefore, GSNOR–/– MSCs have deficient capacity
for endothelial differentiation due to depressed VEGF-A production related to a NO/GSNOR imbalance.These
findings unravel important aspects of modulation of MSC activation of PDGFR by VEGF-A, and illustrate a
paradoxical inhibitory role of S-nitrosylation signaling in MSC-vasculogenesis. Accordingly, disease states
characterized by NO-deficiency may trigger MSC-mediated vasculogenesis. These findings have important
implications for therapeutic application of GHRH agonists to ischemic disorders.
S-nitrosylation Regulates the Balance of Adipogenesis and Osteogenesis in Mesenchymal Stem Cells
Author(s)
Yenong Cao
Graduate Student
ISCI, University of Miami Miller School of Medicine
Co-Author(s)
Samirah Gomes - Post Doctoral Associate, ISCI
Erika Rangel - Post Doctoral Associate, ISCI
Wayne Balkan - Associate Professor, ISCI
Joshua Hare - Professor, ISCI
Topic
Basic Research
Problem
Previous studies have shown that Nitric Oxide (NO) is crucial for the regulation of adipogenic and osteogenic
differentiation. Nevertheless, the mechanism of bone marrow-derived mesenchymal stem cells (BMMSCs)
lineage bifurcation into adipocytes and osteoblasts remains to be elucidated.
Background
Circulating bone marrow-derived mesenchymal stem cells (BMMSCs) are attracted to the arterial wall where
they can undergo adipogenic or osteogenic differentiation to form ectopic aggregates of fat and bone. We
assessed the role of nitric oxide (NO), an inhibitor of lipid formation and vascular calcification, in BMMSCs
differentiation.
Hypothesis
We hypothesized that S-nitrosylation mediated-NO signaling controls the balance between adipogenic and
osteogenic differentiation of BMMSCs.
Research
-/-
BMMSCs were isolated from wild type mice (WT) and mice deficient in S-nitrosoglutathione reductase (GSNOR ),
a denitrosylase that governs levels of protein S-nitrosylation. Cells were cultured in either adipogenic or osteogenic
differentiation media followed by functional and gene expression assays.S-nitrosylation was tested by SNO-RAC
assay.
Observations
-/-
BMMSCs derived from GSNOR mice had reduced fat droplet formation and expression of the adipogenic
-/markers PPARγ (1329±415.3-fold increase in WT vs. 158±65.61-fold in GSNOR , P<0.05)
-/-/and FABP4 (11.06±3.29-fold in WT vs. 4.06±0.62-fold in GSNOR , P<0.05). Conversely, GSNOR MSCs exhibited
enhanced osteogenic differentiation as indicated by greater calcium deposition and higher expression of the
-/osteogenic marker Osteopontin (1.48±0.17-fold in WT vs. 16.18±5.26-fold in GSNOR , P<0.05). Interestingly,
-/-/GSNOR cells had higher baseline expression of Osteopontin, Osteocalcin and Runx2. Treatment of GSNOR
MSCs with L-NAME, a nitric oxide synthase (NOS) inhibitor, decreased osteogenic differentiation without affecting
adipogenic differentiation. Baseline level of S-nitrosylated PPARγ, a master regulator of adipogenesis and
-/osteogenesis, was increased in GSNOR MSCs.
SoxC Family Members Regulate Retinal Ganglion Cell Differentiation
Author(s)
Jonathan Hertz
Graduate Student
Co-Author(s)
Xiao-Lu Jin - Research Associate, ISCI
Brooke Derosa - graduate student, UM Medical School
Janet Li - Research Associate, UM Medical School
Praseeda Venugopalan - graduate student , UM Medical School
Daniel Valenzuela - Undergraduate, UM
Roshni Patel - undergraduate, UM
Kristina Russano - Research Associate, UM Medical School
Kenneth Muller - Professor, UM Medical School
Veronique Lefebvre - Professor, Cleveland Clinic
Derek Dykxhoorn - assistant professor, UM Medical School
Jeffrey Goldberg - Professor, UM Medical School
Topic
Basic Research
Problem
The cell-autonomous regulators or extrinsic signals sufficient to specify retinal ganglion cell (RGC) fate in the
developing retina or from adult stem cells remain largely obscure. What are the molecular signals that regulate
neural cell fate?
Background
For example, retinal ganglion cells (RGCs) are born from multipotent retinal progenitor cells (RPCs) during
embryonic development, but little is known about the cell autonomous mechanisms and environmental signals
that specify RGC fate.
Hypothesis
Unknown regulatory pathways function in parallel to Math5 and upstream of Brn3b.
Research
Through gain and loss of function experiments, both in vitro and in vivo, we have found a new molecular
pathway involving Sox4/Sox11 in parallel to GDF-11/Math5 signaling that is required for RGC differentiation.
Observations
ganglion cell (RGC) fate in the developing retina or from adult stem cells remain largely obscure. Here we
report a new molecular pathway involving Sox4/Sox11 in parallel to GDF-11/Math5 signaling that is required
for RGC differentiation from retinal progenitor cells in vitro and in vivo and sufficient to potentiate the
differentiation of electrophysiologically active human RGCs from induced pluripotent stem (iPS) cells. We
further describe regulation of Sox4 by REST and the TGFβ family member GDF-15, and SUMOylationdependent regulation of compensatory Sox11 activity by Sox4. Through this work we also uncovered a
mechanism for SUMOylation regulation of Sox11 localization and function. These data define a novel
molecular network necessary and sufficient for RGC fate specification and suggest a pro-RGC molecular
manipulation which may provide potential promise for cell replacement-based therapies.
Stem Cell Chemotaxis and Antineoplastic Effects in Ovarian Cancer Cells
Author(s)
Mary E. McAsey, PhD, ELD/HCLD, CC (ABB)
Associate Professor, SIU Fertility and IVF Center Laboratory Director, Director of Research
Southern Illinois University School of Medicine, Fertility and IVF Center, Department of Obstetrics and
Gynecology
Craig Cady, PhD
Associate Professor
Biology Deparment, Bradley University
Role: Author
Co-Author(s)
Jennifer Li, BS - Researcher III, Department of Obstetrics and Gynecology, Southern Illinois University School
of Medicine
Isaac Schafer - Student, Biology Department, Bradley University
Kerri Houser, BS - Medical Student, Southern Illinois University School of Medicine
Megan Logsdon, BS - Medical Student, Southern Illinois University School of Medicine
Topic
Basic Research
Problem
Epithelial ovarian cancer is the most lethal of the gynecologic cancers and the fourth most common cancer in
women. Although significant advances in chemotherapeutics have improved the 5-year survival rates for all
stages of ovarian cancer, the survival rate beyond this has not changed significantly over the last 20 to 30
years for metastatic ovarian cancer. Clearly new and novel therapies must be developed to treat this
devastating disease. Stem cells may offer a new cell-based therapy for ovarian cancer. Bone marrow
mesenchymal stem cells (BMSC) are excellent candidates for cell-based therapies because they effectively
target and persist in hard-to-reach tumor regions.
Background
BMSC are effectively recruited to tissues following hypoxic stress or injury. BMSC may specifically target the
most refractory of ovarian cancer cells, those residing within hypoxic tumor regions. Use of engineered,
autologous stem cells has the potential to significantly alter the approach to treatment of ovarian cancer. This
approach to stem cell gene therapy does not require bone marrow ablation, may reduce the required dose of
cytotoxic therapies and may improve the sensitivity to radiotherapy for effective tumor ablation.
Hypothesis
Our hypotheses are: 1.) BMSC preferentially migrate to ovarian cancer cells 2.) Mechanisms of migration
involve secretion of specific growth factors and cytokines from ovarian cancer cells.
Research
Tumor spheroids were generated from renilla luciferase-transduced HEY and SKOV3 ovarian cancer cells.
GFP-transduced human BMSC were incubated for 24h with the tumor spheroids and the spheroids examined
using fluorescence microscopy for incorporation of BMSC. For co-culture migration assays, BMSC (1.5 x 104
cells/ml) were labeled with 5-(6)-carboxyfluorescine diacetate, succinimidyl ester (CFDA-SE, Invitrogen) and
50 μl were added to the center of cloning rings mounted on glass coverslips. HEY, SKOV3, 2774 C10 and
OCCI, the fibroblast cell line CLL171 and the immortalized, non-tumorigenic human ovarian surface epithelium
cell line, HOSE cells (0.5 ml, 1.5 x 103 cells/ml) were added to each slip, outside of the cylinder. After 3h, the
cylinders were removed and the cells incubated for 48h. The cells were then labeled with 100 ng/ml 4′-6′Diamidino-2-phenylinole dihydrochloride (DAPI, Sigma) and observed under fluorescence microscopy. Cells
that migrated beyond the outer boarder of the cloning cylinder were counted. Controls included coverslips
onto which BMSC were plated in cylinders without ovarian cancer cells plated outside of the cylinders. Other
controls included coverslips onto which BMSC were plated in cylinders and additional, unlabeled stem cell
plated outside of the cylinders.
For invasion assays, BMSC (1x104 cells) were plated in the upper chamber of 8μm transwell inserts, (Becton
Dickenson, BD) coated with matrigel and placed in wells containing 0, 20, 40, 80 or 100% serum-free
conditioned media from either HEY or SKOV3 cells and incubated for either 24h or 48h. Positive controls
included media containing 16% serum. After 24h or 48h the BMSC were removed from the upper surface of
the insert membrane and the BMSC that migrated to the underside of the membrane were stained with DifQuik, photographed and counted.
To assess if lipid-soluble factors were involved in BMSC chemotaxis, BMSC were plated on 8μm transwell
membranes (BD), placed in wells containing heat-inactivated (56°C for 30 min, 75°C for 15 min, or 95°C for 2
min) conditioned media from SKOV3-ip1 ovarian cancer cells and incubated for 24 h. Positive controls
included media containing 16% serum. The BMSC were removed from the upper surface of the insert
membrane and the BMSC that migrated to the underside of the membrane were stained, photographed and
counted.
For evaluation of potential cytokines that may induce BMSC migration, BMSC were plated in transwell inserts
with various cytokines added to serum-free media within the wells. BMSC were incubated for 24h and the
migrated BMSC stained and counted as above. To determine if ovarian cancer cells expressed VEGF, DNA
was extracted from HEY, SKOV3, 2774 C10, OCCI and SKOV3-ip1 ovarian cancer cells and rtPCR performed
with primers specific for VEGF-A, -B, -C, and -D. VEGF-A protein was assessed with an ELISA using
conditioned media from HEY, 2774 C10, BMSC, CLL171 and HOSE cells. Migration assays as described
above were used to test if increasing levels of VEGF-A (50, 100, 200 or 400 ng/mL) induced BMSC migration.
BMSC co-culture migration assays were performed as above using CLL171 fibroblast cells transduced to
overexpress VEGF and non-transduced CLL171 cells as a negative control.
Data were analyzed using Prism for Macintosh (GraphPad Software, San Diego, CA). Statistical differences
among treatment groups for invasion assays were assessed using a one-way analysis of variance (ANOVA)
followed by Dunnett’s post-hoc testing when indicated by statistical significance during ANOVA. Unpaired ttests were performed to assess differences in stem cell migration between groups with and without ovarian
cancer cells at 24 and 48 hours. Probability values <0.05 were considered statistically significant.
Observations
For tumor spheroid migration assays, BMSC were observed surrounding and within tumor spheroids following
24h. In co-culture assays, there was significant migration of BMSC toward all ovarian cancer cell lines at 24
and 48 h (p<0.0001). Co-culture with CLL171 or HOSE cells did not induce BMSC migration. For the invasion
assays, 40, 80 and 100% conditioned media from the ovarian cancer cell lines HEY and SKOV3 significantly
induced BMSC invasion compared to serum-free media (p<0.001). For heat-inactivation experiments to test
for lipid-soluble factors that may stimulate BMSC migration, there was a significant reduction in migration for
heat-inactivated conditioned media relative to non-heat-inactivated conditioned media (56°C, p< 0.05, 75°C
and 95°C, p<0.001). Multiple cytokines stimulated BMSC migration. Relative to serum-free control, VEGF-165
exhibited a 29% increase, VEGF-C, 22%, VEGF-121, 26%, mVEGF 15%, FGF, 21%, Wnt-1, 32%, SDF-1A
27%, HGF-1 16% and TGF-beta1, 19%. IGF-1, ANG-1, ANG-1, GM-CFF, IL-3 and ET-1 all exhibited less than
1% increase in migration relative to the serum-free control. In rtPCR experiments, VEGF gene expression was
demonstrated in all ovarian cancer cell lines. VEGF was detected with ELISA in conditioned media from 2774
C10 (3249 ± 242 pg/mL), SKOV3-iP1 (612.8 ± 102.3), HEY (1171.7 ± 2.5). The control cell line CLL1781
exhibited a much lower concentration of VEGF, (36.7 + 1.4), whereas HOSE cells had non-detectable levels
of VEGF. For dose-response migration assays, all concentrations of VEGF tested resulted in significant
migration of BMSC compared to migration observed with serum-free media (p<0.001). In co-culture migration
assays with CLL171 cells overexpressing VEGF, BMSC migration was significantly greater at both 24 and 48h
compared to migration of BMSC with non-transduced CLL171 cells (for 24h, p<0.01, for 48 h, p<0.05).
Our results suggest that BMSCs migrate toward ovarian cancer cells and that soluble factors, including VEGF
may mediate this migration. Additional research is vital for understanding the underlying mechanisms involved
in stem cell targeting and for the development of new therapies. Engineered BMSCs may specifically target
the most refractory of ovarian cancer cells, those residing within hypoxic tumor regions. Use of engineered,
autologous stem cells has the potential to significantly alter the approach to treatment of ovarian cancer. This
type of stem cell therapy does not require bone marrow ablation, may reduce the required dose of cytotoxic
therapies and may improve the sensitivity to radiotherapy for effective tumor ablation. At the very least it may
become another addition to the growing armamentarium for the treatment of the more refractory or more
advanced cases of ovarian cancer.
Stem cell therapy and cerebral ischemia
Author(s)
Jean-Pyo Lee
Assistant Professor
Tulane University
Co-Author(s)
Lei Huang - Postdoctoral fellow, Tulane University
Topic
Basic Research
Problem
There is currently no proven stem cell-based therapy for stroke. Preclinical studies and clinical research are
needed to optimize the therapeutic benefit and minimize the risks of stem cells in stroke.
Background
Use of stem cells for stroke (the third leading cause of death in the U.S.) shows therapeutic promise. It has
been established that cytokines, including stromal-derived factor-1 alpha (SDF-1 alpha), facilitate migration of
stem cells to brain injury sites. Previously, we exploited the cytokine system by demonstrating stem cell
migration in mid-symptomatic Sandhoff-diseased mice (exhibits lethal gangliosidosis).
Hypothesis
Do stem cell-derived factors suppress proinflammatory genes involved in breakdown of the blood brain barrier
and blood cell extravasation into the brain caused by MCAO in a mouse model of stroke? Research
We employed a C57BL6 mouse model of stroke to transplant NSCs into the intracranium. After optimizing
stem cell growth conditions, both in vitro and at the transplantation site, mice were subjected to a MCAO and
reperfusion for 60 minutes to induce ischemia; transplantation transpired 24 hours later (during which time
upregulation of proinflammatory cytokines reportedly occurs).
Observations
Analysis 24 hours posttransplantation showed NSC-treated mice downregulated proinflammatory cytokines in
the brain, compared with controls. Transplanted cells dispersed widely across the injured brain, from the
implantation site into both cortical and subcortical regions. Assessment of mice by rotarod, beam walking, and
adhesive removal tests showed treated mice had improved physical behaviors compared with controls.
Stem Cell-Mediated Cancer Therapy: First-in-Human Brain Tumor Clinical Trial
Author(s)
Karen S. Aboody, MD
Associate Professor, Department of Neurosciences/Chief Scientific Officer and Director
City of Hope/TheraBiologics Inc.
Co-Author(s)
Timothy Synold, PhD - Associate Professor, City of Hope
Simon Lacey, PhD - Associate Professor, City of Hope
Alexander Annala, PhD - Associate Research Professor, City of Hope
Marianne Metz - Staff Scientist, City of Hope
Behnam Badie, MD - Professor, City of Hope
Jana Portnow, MD - Assistant Professor, City of Hope
Topic
Basic Research
Problem
Approximately 22,500 people a year in the United States are diagnosed with malignant primary brain tumors,
which are highly invasive, neurologically destructive, and considered to be among the deadliest human
cancers. Despite aggressive multimodal therapy and advances in chemotherapy, imaging, surgical and
radiation techniques, these tumors remain virtually incurable. Survival is typically measured in terms of
months for patients with recurrent glioblastoma, the most common and aggressive form of high-grade glioma
in adults. Treatment failure is primarily attributable to the diffuse and invasive nature of glioma cells, ineffective
delivery of chemotherapeutic agents across the blood-brain barrier (BBB), and associated dose-limiting
systemic toxicities. Therefore, novel tumor-selective therapies are critically needed to improve clinical
outcome.
Background
Neural stem cells (NSCs) have inherent tumor-tropic properties that can be exploited for targeted delivery of
anti-cancer agents to invasive and
brain tumors, and may offer an unprecedented advantage over conventional therapeutic approaches. NSCs
can overcome the major obstacles limiting the efficacy of current treatments through their abilities to cross the
BBB, target therapeutic agents to primary and invasive tumor foci throughout the brain, and minimize toxicity
to normal tissues by acting as a platform for tumor-localized treatment. For use as delivery vehicles, NSCs
have been engineered to express a variety of anti-cancer agents, including prodrug-activating enzymes,
apoptosis-inducing agents, antibodies, and oncolytic viruses. Intracerebral administration of NSCs engineered
to express various anti-cancer agents has demonstrated significant therapeutic efficacy in preclinical brain
tumor models of orthotopic glioma, medulloblastoma, melanoma brain metastases, and breast cancer brain
metastases. In addition, intravenously administered, genetically engineered NSCs demonstrated tumor
targeting and therapeutic efficacy in mouse models of disseminated neuroblastoma and primary and
metastatic breast cancer. These observations suggest that the potential for clinical application of NSCmediated cancer treatment may be quite extensive.
Pre-clinical characterization of the established cytosine deaminase (CD)-expressing, immortalized clonal
human NSC line, HB1.F3.CD, demonstrated chromosomal and functional stability, tumor-tropism, and
minimal immunogenicity (HLA Class II negative). The CD enzyme activates the conversion of the prodrug 5flurocytosine (5-FC) to the active chemotherapeutic agent 5-fluorouracil (5-FU). In vivo IND-enabling studies
in orthotopic glioma models demonstrated safety, non-tumorigenicity and efficacy when administered with 5FC.
Hypothesis
Based on pre-clinical data, we hypothesize that following injection of HB1.F3.CD NSCs into the patient brain
tumor site at the time of surgical resection or biopsy, the NSCs will localize to residual and invasive brain
tumor foci, and convert the orally administered prodrug 5-FC to the active chemotherapeutic 5-FU locally at
the tumor sites. This should result in tumor-localized chemotherapy production, and minimize toxicity to
normal tissues resulting in decreased treatment side effects.
Research
A genetically modified, clonal CD-expressing NSC line (HB1.F3.CD) has been established as a fully release
tested master cell bank at the COH Center for Applied Technology Devlopment. This first-in-human phase I
study is assessing the safety and feasibility of using these NSCs for tumor selective enzyme/prodrug therapy
(clinicaltrials.gov ID # NCT01172964). In this ongoing dose-escalation study, patients with recurrent highgrade gliomas undergo intracranial administration of HB1.F3.CD NSCs during resection or biopsy of tumor.
Four days later, 5-FC is administered orally every 6 hours for 7 days. Patients receive only one course of
NSCs and 5-FC treatment. Correlative studies include analysis of intracerebral levels of 5-FC and 5-FU (via
microdialysis cathether placed at the time of surgery), and corresponding blood levels. Immunogenicity
studies are also being performed on peripheral blood samples at various timepoints. Importantly, we have
recently received USFDA approval to include pre-labeling or our NSCs with Feraheme iron nanoparticles, in
order to track their migration and tumor distribution over time by MRI. We now present data from the first 3
cohorts, demonstrating safety and proof of concept. We are recruiting an additional 3 patients in cohort 3 for
our iron-labeling studies, for demonstration of NSC migration to residual tumor sites. We believe that
successful completion of these phase I studies will lay the foundation for development of additional NSCmediated treatments for other types of invasive cancers as well.
Observations
Correlative studies include analysis of intracerebral levels of 5-FC and 5-FU (via microdialysis cathether
placed at the time of surgery), and corresponding blood levels. Microdialysis data from the first 2 cohorts
demonstrate proof-of-concept of brain tumor localized NSC-mediated conversion of 5-FC to 5-FU. To date,
there has been no immune response detected to these allogeneic NSCs after first exposure.
Survival and Integration of Developing and Progenitor-Derived Retinal Ganglion Cells Following
Transplantation
Author(s)
Daniel A. Valenzuela
Undergraduate Student Researcher
Bascom Palmer Eye Institue
Co-Author(s)
Jonathan Hertz - Graduate Student, Bascom Palmer Eye Institue, Interdisciplinary Stem Cell Institue,
Bo Qu - Researcher, China Medical University, Ophthalmology
Roshni Patel - Undergraduate Researcer, Bascom Palmer Eye Institute, Ophthalmology
Jeffrey Goldberg, MD, PhD - Principal Investigator, Bascom Palmer Eye Istitue, Ophthalmology
Topic
Basic Research
Problem
Degenerative eye diseases like glaucoma have proven difficult to treat using traditional means. As such,
interest has increased in trying to use stem cells as a therapeutic avenue to heal damaged eyes. Before such
stem cell research can help with the problems faced in degenerative disorders, a feasible way of transplanting
the right kind of cells into the eye needs to be developed.
Background
Transplanting embryonic stem cells and adult neural progenitors has demonstrated only limited success at
integrating into various regions of the central nervous system. One of the greatest problems with getting cells
to integrate correctly in the eye is due to the complex circuitry of the eye. Retinal ganglion cells (RGCs) are
among the first neurons to arise from a population of multipotent progenitors during development, after which
they migrate across the retina to form the Ganglion Cell Layer (GCL), and eventually extend neurites toward
the optic nerve head. Several factors including injury reactivity, immune response, and a lack of crucial
developmental factors in the environment are likely to play a role in the failure of these cells to properly
differentiate and integrate.
Hypothesis
Retinal progenitors have thus far failed to integrate properly into the ganglion cell layer of the eye. We propose
that in-vitro differentiated retinal ganglion cells as well as primary cells can integrate into their respective
layers when transplanted under the appropriate conditions. We hypothesize that cells from different timepoints
in development may exhibit differing levels integration and functionality following transplantation.
Research
Using published techniques, we cultured retinal progenitors in differentiation media into retinal ganglion cells
as well as obtained primary retinal ganglion cells from rats of various ages. We then transplanted the neural
cells into the eye of anesthetized rats via intravitreal injection, or we injected RGCs onto a whole mount retina
taken from a rat and kept in culture. Finally, we waited variable amounts of time after transplantation. Using
imunohistochemical techniques, we stained for RGCs and neurites, and measured the number of transplanted
cells that found their way into the appropriate layers, as well as whether neurites were extended in a specific
orientation and made synapses.
Observations
Following the period after transplantation, we found that in-vitro differentiated progenitor cells as well as
primary retinal ganglion cells can survive, migrate, extend neurites, and form morphological synapses in vivo
and ex vivo. We also found that many of these cells made their way into the Ganglion Cell Layer. These cells,
except for those derived from adults, extended lengthy neurites in a direction slightly inclined toward the optic
nerve head. We demonstrated that progenitor derived RGCs are biologically comparable to developing
primary RGCs, although they formed fewer synaptic punctae. This work provides insight for further
understanding the integration of developing RGCs into their normal environment following injury.
Temporal monitoring of pO2 in stem cell cultures: a potential tool for directed differentiation.
Author(s)
simona maciotta rolandin
Simona Maciotta Rolandin PhD, Postdoctoral Fellow
Department of Pathophysiology and Transplantation, FondazioneCa' Granda IRCCS, Ospedale Maggiore
Policlinico, Centro Dino Ferrari, Universita' di Milano, Italy
Co-Author(s)
Christopher Fraker, Ph.D. - Co-Director Bioengineering Lab, University of Miami Diabetes Research Institute
Vita Manzoli, MS - Senior Research Associate, University of Miami Diabetes Research Institute
Chiara Villa, Doctoral Candidate - Scholar, FondazioneCa’ Granda IRCCS, Ospedale Maggiore Policlinico,
Centro Dino Ferrari, Universita’ di Milano, Italy
Alice Tomei, Ph.D. - Co-director , Bioengineering Laboratory, Diabetes Research Institute, University of Miami
Miller School of Medicine, Miami, FL
Yvan Torrente, Ph.D., M.D. - Director, Stem Cell Laboratory, FondazioneCa’ Granda IRCCS, Ospedale
Maggiore Policlinico, Centro Dino Ferrari, Universita’ di Milano, Italy
Luca Inverardi, M.D. - Co-director, Diabetes Research Institute, University of Miami Miller School of Medicine,
Miami, FL
Juan Dominguez-Bendala, Ph.D. - Director, Stem Cell Laboratory, Diabetes Research Institute, University of
Miami Miller School of Medicine, Miami, FL
Topic
Basic Research
Problem
Despite substantial progress over the last 14 yearson stem cell culture and differentiation, success at
efficiently guiding targeted development into functional, transplantable populations has been limited. A major
factor in this is the simplicity of in vitro culture systems and chemical protocols, which cannot approximate the
physiological complexity of metabolite and signal delivery of in vivo microenvironments.
Background
A recent paradigm shift has occurred in the stem cell field, where investigators are turning to critically
important physical variables (including tensile forces, ionic signaling and mechanical pressure) as key
determinants of cell differentiation. Our prior work has established that one such variable, oxygenation, plays
an important role in the specification of endocrine cell types (particularly β cells). Tissue oxygenation during in
vitro culture is critically dependent on the cellular oxygen consumption rate (OCR), proliferation/cell death,
plating density and medium depth. These variables are often overlooked and might lead to the variability
observed in stem cell cultures and differentiation outcomes. This is particularly true of OCR and proliferative
rates, as both can vary significantly from passage to passage. Current culture practices, therefore, are
inadequate for standardizing differentiation outcome.
Hypothesis
The use of biosensors for in-process control of the culture environment could allow for the elimination of
current differentiation protocol variability and for targeted differentiation standardization. As an example, we
propose the use of fluorescent oxygen sensors to monitor and modulate pO2 at the cell culture surface in the
differentiation of pancreatic precursors into insulin producing tissue for the treatment of Type I Diabetes
Mellitus.
Research
In this work, we implement 2D spatio-temporal fluorescence-based oxygen sensing across the cell culture
surface to monitor pO2changes/patterns as they develop during both proliferation and differentiation and to
better maintain target pO2.
Observations
The feasibility and utility of the sensors to control and modulate patterned oxygenation in real time is
demonstrated. We envision that in-process corrections of oxygenation during extended culture protocols could
allow for the development of more standardized and efficient target cell differentiation methods, particularly for
insulin producing cell aggregates to treat Type I Diabetes Mellitus.
The Effect of Mesenchymal Stem Cells and Platelet Rich Plasma Treatments on injured Rat Medial
Collateral Ligaments
Author(s)
Danica D. Vance
Medical Student
University of Miami
Co-Author(s)
David Ajibade, MD - Department of Orthopaedics, Division of Sports Medicine, 1. University of Miami
Lauren Vernon - Department of Biomedical Engineering, Stem Cell and Mechanobiology Lab, 1. University of
Miami
Rosemeire i Kanashiro-Takeuch - Interdisciplinary Stem Cell Institute, Miami, 1. University of Miami
Andrew Rosenberg, MD - Department of Pathology, 1. University of Miami
Joshua Hare, MD - Interdisciplinary Stem Cell Institute, Miami, 1. University of Miami
Lee D. Kaplan, MD - Department of Orthopaedics, Division of Sports Medicine, 1. University of Miami
Bryson Lesniak, MD - Department of Orthopaedics, Division of Sports Medicine, 1. University of Miami
Topic
Basic Research
Problem
The objective of this in-vivo study is to investigate the histologic effects of MSC and PRP therapy on ligament
healing in a rat medial collateral ligament (MCL) injury model.
Background
Cell therapy for cartilage and ligament injuries has become a widely debated topic in the orthopaedic
literature. Adult mesenchymal stem cells (MSCs) are of interest given their high capacity for self-renewal and
multipotency to differentiate into chondrocytes and tenocytes. Platelet rich plasma (PRP) has been reported
to promote collagen synthesis and cell proliferation, influencing the healing of ligaments and cartilage.
Hypothesis
We hypothesize that the addition of MSCs to an injured Rat MCL will show increase collagen regeneration
when compared to controls and those MCLs treated with PRP.
Research
MCLs of 20 female Sprague rats were bilaterally transected and treated with either saline or 1 of 3 treatment
groups; (1) MSCs (10^5cells), (2) MSCs + PRP, and (3) PRP. Ratswere sacrificed 16 days post-surgery and
the MCLs harvested, fixed in formalin, processed routinely and stained with H&E and Alcian blue. Histological
analysis was performed by a pathologist blinded to the treatment groups, and the tissue specimens were
graded based on the degree of cellularity (0-+3), change in collagen representing new or regenerative
collagen fibers (0-+3), vascularity (0-+2) and inflammation (0-+3). Statistical analysis was performed using
Analysis of variance (ANOVA) and Fischers exact test.
Observations
The degree of cellularity and change in collagen fibers were statistically significantly increased between the
four groups (p<.0032). Paired comparisons between groups demonstrated that all treatment groups had
increased cellularity and collagen changes when compared to controls but not between the experimental
groups. Alcian blue staining revealed increased extracellular matrix within treatment groups (MSCs= avg.
grade of 1.33, MSCs & PRP = avg. grade of 2.67 and PRP = avg. grade of 2) when compared to the control
(avg. grade of 1).
The addition of MSCs and/or PRP to an injured MCL increases cellularity and the regeneration of collagen
fibers in the setting of an acute MCL injury when compared to controls. While not significant, there was a
trend towards even higher cellularity and collagen fiber regeneration in the treatment group treated with MSC
and PRP, indicating a possible potentiation effect between the two treatments. This study suggests improved
ligament healing with MSC and PRP treatment and that biomechanical testing is warranted to explore whether
this treatment will result in improved biomechanical properties of MCL injuries.
The effect of pancreatic mesenchymal stem cells on differentiation of human cord blood CD133+ cells
into insulin secreting cells
Author(s)
Fazel Sahraneshin Samani
Research assistant
Royan Institute
Co-Author(s)
Topic
Basic Research
Problem
Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient
number of pancreatic endocrine cells that function similarly to primary islets. CD133+ cells are population of
stem cells in bone and cord blood which express some embryo specific markers such as SSEA-4 and OCT4
and suggested that can differentiate into other cells including insulin secreting cells
Background
Epithelium-mesenchyme interactions play a crucial role during organogenesis and differentiation is controlled
by the microenvironment surrounding stem cells . Evidence points to a crucial role for epithelial mesenchymal
interactions in cell proliferation and differentiation during pancreatic development .They are mediated at least
in part by soluble factors produced by the mesenchyme and acting on the epithelium and rat pancreatic
extracts (RPE) could stimulate MSC differentiation into IPCs and increase insulin secretion
Hypothesis
here we attempt to evaluate the effect of rat pancreatic MSCs on Differentiated Insulin secreting cells from
UCB CD133+ cells.
Research
: CD133+ cells were isolated from healthy human cord blood, by magnetic activating cell sorting(MACS).
subsequently, cells differentiated using activin A, retinoic acid, bFGF, nicotine amide, B27 and N2 in co culture
with pancreatic mesenchymal cells directly or indirectly or without pancreatic mesenchyme cells. The
expression of Insulin and c-peptide protein was detected by Immonocytochemistry. ELISA was performed to
analysis the function of differentiated cells in different glucose concentrations, and the expression of
insulin,Nkx6.1,Pdx1,Glocagon genes was analyzed by RT-PCR
Observations
Our results showed that UCB-CD133+ cells has the ability to differentiate into insulin cells. direct and indirect
Co-culture with pancreatic MSCs didn’t increased percentage of insulin positive cell and c-peptide positive cell
in comparison with control group .The insulin,Nkx6.1,Pdx1,Glocagon genes were not expressed in all
experimental groups ,and they were could not responsed to different concentrations of glucose.
The Isolation and Differentiation of Human Adipose-Derived Stem Cells Using Membrane Filtration
Author(s)
Akon Higuchi
Distinguished Professor
National Central University
Co-Author(s)
Topic
Basic Research
Problem
Huamn adipose-derived stem cells (hADSCs) can be isolated from liposuction-derived adipose tissue by
centrifugation followed by cultivation in cell culture dishes for at least one passage. The cultivation of cells
derived from adipose tissue is necessary to purify hADSCs (i.e., “the culture method” for the purification of
hADSCs) because the adipose tissue also contains adipose cells and other cell types. Purifying hADSCs via
the culture process requires 5-12 days. If hADSCs could be purified from adipose tissue in a shorter time
period (i.e., less than 30 min) using a cell purification device such as that used in the membrane filtration
method, cell therapy and tissue engineering applications using autologous hADSCs might become more
efficient.
Background
Stem cells are an attractive source of cells for tissue engineering and cell therapy (i.e., regenerative medicine
using stem cells) because of their unique biological properties. Stem cells have been isolated from a variety of
somatic tissues. Adipose-derived stem cells (ADSCs) hold promise for regenerative medicine, particularly for
cell therapies and tissue engineering applications. Human ADSCs (hADSCs) express specific surface markers
such as STRO-1 (a stromal cell surface antigen), CD29 (integrin β1), CD44 (receptor for hyaluronic acid and
matrix proteins), CD73, CD90, CD105 (endoglin, receptor for transforming growth factor-β (TGF-β) and
integrins), and CD166 (cell adhesion molecule) but are negative for CD14 (monocyte surface antigen) and
CD45 (leukocyte surface antigen) [5]. These and other cell surface markers, although not unique to hADSCs,
are used to isolate and/or characterize hADSCs using flow cytometry. hADSCs can be isolated from
liposuction-derived adipose tissue by centrifugation followed by cultivation in cell culture dishes for at least
one passage [9, 10]. The cultivation of cells derived from adipose tissue is necessary to purify hADSCs (i.e.,
“the culture method” for the purification of hADSCs) because the adipose tissue also contains adipose cells
and other cell types. Purifying hADSCs via the culture process requires 5-12 days.
Hypothesis
Membrane filtration method though porous polymeric membranes having specific nanosegments should be
useful to purify hADSCs from adipose tissue. The membrane filtration method is a good candidate for the
purification of stem cells because it is a rapid, simple method, and sterility can easily be maintained during the
filtration process. Furthermore, the membrane filtration method does not necessitate the use of antibodies,
which are required for purifications that utilize magnetic-activated cell sorting (MACS) or fluorescenceactivated cell sorting (FACS). Antibodies are generally produced using animal-derived proteins and cells,
which might lead to contamination with viruses or prions. Therefore, hADSCs should not be prepared using
animal-derived materials, such as antibodies, to maintain the safety of these cells in clinical applications.
Research
The base membranes used for the chemical modification were polyurethane (PU) foam membranes with a
pore diameter of 11 um. U foam membranes containing an epoxy group (PU-epoxy) were prepared by plasma
polymerization with glycidyl methacrylate after the membranes has been plasma discharged at 200 W for 300
s under 0.2 Torr of Ar gas. Several functional groups were introduced via a reaction between the epoxy group
on the PU-epoxy membranes and various chemicals. Briefly, the surface modification of the PU membranes
was performed as follows: (a) To prepare PU membranes with a carboxylic acid group (PU-COOH
membranes), (b) To prepare PU membranes with a sulfonic acid group (PU-SO3H membranes), (c) To
prepare PU membranes with a hydroxide group (PU-OH membranes), and (d) To prepare PU membranes
with an amino acid group (PU-NH2 membranes). We purified hADSCs from a digested solution of human
adipose tissue by the conventional cell culture method and by the membrane filtration method using
unmodified polyurethane (PU) membranes and surface-modified PU membranes, and we compared the
purities and the osteoblast differentiation abilities of the hADSCs after each purification.
Observations
hADSCs were successfully purified from a human adipose tissue cell solution by the conventional culture
method and by the membrane filtration method. hADSCs can be obtained via the membrane filtration method
in a short period of time (under 30 min), whereas the culture method requires 5-12 days. Surface marker
analysis suggests that stem cells expressing the mesenchymal stem cell markers CD44, CD73, and CD90
can be concentrated 3-4.5 fold in the recovery solution obtained after passing the cell solution through one
sheet of an unmodified or a surface-modified PU membrane by perfusion-type filtration compared with the
levels of cells expressing CD44, CD73, and CD90 in primary adipose tissue cell (SVF) solution. Cells
expressing the stem cell-associated marker CD34 can be successfully isolated from a primary adipose tissue
cell (SVF) solution by filtration through the PU membranes, after which these cells are present in the recovery
solution, but these cells cannot be purified by the conventional culture method. The hADSCs that were
recovered after passing the cell solutions through the unmodified PU and the PU-FN membranes displayed a
higher capacity for osteogenic differentiation than did the cells in the primary adipose tissue cell (SVF)
solution. These results suggest that hADSCs with the ability to undergo osteogenic differentiation adhere to
PU membranes. Linear correlations between the expression rates of CD44, CD73, and CD90 and the
osteogenic differentiation ability were identified (r=0.854-0.964). Cells expressing higher levels of the
mesenchymal stem cell-specific surface markers were shown to have a greater capacity to differentiate along
the osteogenic lineage.
Transdifferentiation of Macrophages into Neuronal-Like-Cells as a potential model for treatment
prediction in schizophrenia
Author(s)
Alfredo Bellon, MD, PhD,
Assistant Professor
University of Miami
Co-Author(s)
Michael Valente - PhD student, INSERM
Julien Matricon - Postdoctoral fellow, INSERM
Florence Gbahou - Postdoctoral fellow, INSERM
Robert Gardette, Researcher - Researcher, INSERM
Seung-Kwon Yang - Postdoctoral fellow, INSERM
Joshua Hare - Professor, University of Miami
Charles Nemeroff - Professor, Univeristy of Miami
Therese Jay - Research Director, INSERM
Marie-Odile Krebs - Professor, INSERM
Anne Hosmalin - Professor, INSERM
Vincent Feuillet - Researcher, INSERM
Topic
Basic Research
Problem
Presents a model of transdifferentiation of blood cells (monocytes) into pluripotential macrophages and then
into neuronal-like cells with electrical activity that allows the researcher to have neuronal-like cells in culture
directly from patients with mental illness or neurological illnesses without the need to radically change the cells
genome. One of the process that can be tested in this model is treatment response in patients with
schizophrenia.
Background
The mechanisms by which antipsychotics elicit a range of therapeutic or undesired effects remain to be fully
elucidated. One of the main problems faced when attempting to clarify this dilemma is the impossibility to work
with neurons from patients.
Hypothesis
Blood cells such as monocytes can be transdifferentiated into neuronal-like cells that express neuronal
markers, present electrical activity and structurally resemble neurons, without the need to radically transform
the genetic information the cells carry by inserting viruses.
Research
In order to overcome this obstacle we are transdifferentiating multipotential macrophages obtained from blood
circulating monocytes into neuronal-like cells. Recent evidence indicates that a population of monocytes can
be differentiated into macrophages with multipotential properties (Zhao et al, PNAS 2003;100(5):2426-31;
Kuwana et al, J Leukoc Biol 2003;74(5):833-45) and in turn transdifferentiated into neuronal-like cells.
However, the differentiation rate reported so far is low, and the structure of cells obtained with those protocols
do not resemble that of neurons and more importantly do not present electrical activity (Zhao et al, PNAS
2003;100(5):2426-31; Kodama et al, Immunol Cell Biol 2006;84(2):209-17). We have developed a protocol to
transdifferentiate such macrophages into neuronal-like cells that provide differentiation rates of up to 60%.
These cells express a wide variety of neuronal markers including Nestin, GAP-43, MAP-2, NeuN,
Neurofilament M&H and PSD-95, and most importantly the structure of these neuronal-like cells resembles
actual neurons (Figure 1B, C and D). In addition, these macrophage-derived-neuronal-like cells (MDNs)
present spontaneous action potentials as well as postsynaptic inhibitory and excitatory currents.
Observations
To our knowledge this is the first protocol described that allows transdifferentiation of cells obtained from a
simple blood sample into neuronal-like cells with electrical activity and without the need to drastically altered
the cell's genome. Other models, like induced pluripotent stem cells (iPS cells) rely on altering the cell's
genome. In illnesses such as schizophrenia where the genetic load is significant but still not well understood,
the impact of drastic genotypic changes cannot be controlled and therefore can become a significant
confounder. In order to establish the reliability of our protocol, we have tested it in over 50 individuals (patients
and controls included) with consistent differentiation rates determined by structural similarity with neurons and
by decreases in expression of CD14 a marker of monocytes/macrophages. Based on data indicating
antipsychotics can alter neuronal structure in vitro and in vivo (Harrison PJ, Schizophr Res 1999;40(2):87-99)
we aim to use MDNs obtained directly from patients with schizophrenia and controls to determine first if
antipsychotics alter its structure and then determine if these structural changes relate to either antipsychotics
therapeutic or undesired effects.
Transplantation of stem cells from the human umbilical cord to repair perinatal brain injury: a preclinical study.
Author(s)
Jeremie Dalous
PhD / Post-doctoral research fellow
UMR676 - INSERM (French National Institute of Health and Medical Research) - Paris Diderot University PremUp
Co-Author(s)
Julien Pansiot - Ing, UMR676-PremUp
Hoa Pham - MD-PhD, UMR676
Laura Charlotte Bruneau - Master student, UMR676
Paul Chatel - Master student, UMR676
Celine Nadaradja - Master student, UMR676
Guillaume David - Master student, UMR676
Irene D'Agostino - MD, UMR676
Gaelle Vottier - MD, UMR676
Leslie Schwendimann - Technician, UMR676
Valerie Vanneaux - MD, Cell Therapy Unit, St Louis Hospital, Paris
Christiane Charriaut-Marlangue - PhD, UMR676
Luigi Titomanlio - MD-PhD, UMR676
Pierre Gressens - Prof, MD-PhD, UMR676-PremUp
Jerome Larghero - Prof, MD-PhD, Cell Therapy Unit, St Louis Hospital, Paris
Olivier Baud - Prof, MD-PhD, UMR676-PremUp
Topic
Basic Research
Problem
Cerebral palsy (CP) is a neurological disorder that affects the developing brain causing motor and cognitive
deficits. Major brain lesions associated with CP are white matter (WM) damage in preterm infants and corticosubcortical lesions in term newborns. To date, there is no treatment for these perinatal brain lesions. Cell
therapy seems very promising to repair brain damages. The human umbilical cord is a rich source of various
stem cells that have many advantages over other stem cell sources, including few ethical issues. We want to
test the potential of stem cells from the human umbilical cord to prevent or repair cerebral neonatal lesions in
a pre-clinical model.
Background
Cerebral palsy (CP) is a neurological disorder that affects the developing brain causing motor and cognitive
deficits. Major brain lesions associated with CP are white matter (WM) damage in preterm infants and corticosubcortical lesions in term newborns. There is no treatment for these perinatal brain lesions.
Cell therapy seems very promising to repair brain damages. The human umbilical cord is a rich source of
various stem cells that have many advantages over other stem cell sources, including few ethical issues.
Some interesting results were obtained using cells from the human umbilical cord to repair neonatal brain
lesions in animal models. These works showed histological and/or behavioral improvements (Meier et al.,
2006; Pimentel-Coehlo et al., 2010; Yasuhara et al., 2010; Kim et al., 2012). However, other groups showed
no beneficial effect using similar cell types and animal models (de Paula et al., 2009) and there is still much
controversy about the potential of cells from the human umbilical cord to protect against or repair neonatal
brain lesions.
Hypothesis
Our hypothesis is that human umbilical cord blood mononuclear cells (hUCB-MNCs) or mesenchymal stem
cells from the Wharton’s jelly of the human cord (hUC-MSCs) can prevent or repair white and grey matter
lesions in an animal model of neonatal excitotoxic brain injury.
Research
The animal model of perinatal brain lesions consisted of an intracranial injection of ibotenate, a glutamate
agonist, in 5-day-old Sprague-Dawley rats. Cells (from 106 to 107) were injected either intraperitoneally (i.p.),
intracranially (i.c.) or intravenously (i.v.). They were injected at an early or at a late time point after ibotenate
injection. Cell fate and effects of transplantation on brain lesions and surrounding tissue were studied using
molecular biology (PCR, Luminex) and histological techniques including immunohistochemistry.
Thus, we used various routes of administration, various cell amounts and various injection delays after injury.
Observations
Using the i.p. route, hUCB-MNCs could not enter the systemic circulation. However, we showed that the
injection of 107 hUCB-MNCs entailed a deleterious increase in the lesion size exclusively in the WM that was
associated with perilesional microgliosis and with the increase in serum concentrations of several cytokines.
On the contrary, the i.p. injection of 106 hUCB-MNCs decreased the cortical lesion size.
hUCB-MNCs injected i.v. soon or 24 h after the insult did not affect lesion size, serum cytokine concentrations,
microglial activation, astroglial cell density, nor cell proliferation at any concentration used (Dalous et al., 2012,
Stem Cells Dev).
Injections of hUC-MSCs were also performed as hUC-MSCs present many very interesting features (for a
review, see Dalous et al., 2012, Ped Res). These first i.c. injections of hUC-MSCs entailed a decrease in
astrogliosis in the perilesional WM.
Conclusion:
We showed that in most conditions tested, hUCB-MNCs could not integrate into the developing brain or
promote subsequent repair. The i.p. injection of high amounts of hUCB-MNCs aggravated WM damage and
was associated with systemic inflammation, while small amounts decreased the cortical lesion size (Dalous et
al., 2012, Stem Cells Dev).
Preliminary results with hUC-MSCs are interesting and support their possible protective role.
Tumor stem/germ cell proteins as a potential cause of chromosomal instability
Author(s)
Mark Eller
Associate Scientist
University of Miami Miller School of Medicine/Melanoma Program
Co-Author(s)
Julia Escandon, MD - Graduate Student, University of Miami Miller School of Medicine
James Grichnik, MD, PhD - Melanoma Program Director, University
Scott Lindsey, BS - research assistant, University of Miami Miller School of Medicine
Topic
Basic Research
Problem
This work examins the expression ofgerm/stem cell proteins in cancers and investigates their potential role in
genomic instability.
Background
Melanomas, like other cancers, are best viewed as dysregulated
developmental systems (a heterogeneous collection of cells, attempting to
differentiate toward pigment cells).
These tumors include a sub-population of cells with stem-cell-like
features. Stem cells and cancer cells
both exhibit chormosomal instability and shared pathways, particularly germ cell pathways, may be playing a
role in driving this instability. One such protein is the meiotic cohesion, REC8. REC8 is involved in sister chromatid
adhesion and homologousproducing genomic variation during meiosis.
Hypothesis
we hypothesize that expression of the meiotic cohesion REC8 may play a role in
chromosomal instability
Research
To examine whether REC8 expression may play a role in
chromosomal instability, we studied REC8 expression in melanomas and normal
tissue as well as its sub-cellular distribution in melanoma and normal cell
lines using qRT-PCR Similarly, in vitro melanoma cells were examined for Rec8 expresion by western blot.
Furthermore, an expression vector containing GFP under the
control of the REC8 promoter measured Rec8 promoter activity and to further explore the effect of REC8
expression
in non-meiotic cells, we constructed a REC8-GFP fusion protein. Expression of
this construct lead to DNA bridging and polyploidy. Immunofluorescence studies of melanoma cells
and normal melanocytes and fibroblasts examined Rec8 intracellular localization.
Observations
Using qRT-PCR, we found a statistically significant higher level of REC8 mRNA in melanoma compared to
normal tissue. Similarly, in vitro melanoma cells revealed higher REC8 protein levels than normal
melanocytes or fibroblasts. Furthermore, an expression vector containing GFP under the control of the REC8
promoter showed higher expression, as determined by GFP fluorescence, in melanomas cells than in normal
human melanocytes and fibroblasts. Expression in non-meiotic cells of a REC8-GFP fusion protein lead to
DNA bridging and polyploidy. Immunofluorescence studies of melanoma cells and normal melanocytes and
fibroblasts revealed foci of REC8 in the melanoma cells but not in the normal cells. Furthermore, these foci colocalized with pericentrin, a marker for centrosomes, in both mitotic and non-mitotic cells. The presence of
REC8 in centrosomes instead of the mitotic cohesion Rad21, known to be a centrosomal component, has
implications in centrosome cohesion and the formation of centrosome splitting forming multi-polar spindle
assemblies and improper chromosome segregation that could drive chromosomal instability. In conclusion, we
have determined that a meiotic protein known to be involved in chromosome adhesion and meiotic
recombination is inappropriately expressed in melanoma. The localization of this protein to centrosomes in the
tumor but not normal cells suggests that REC8 may also affect chromosome segregation through effects on
the mitotic spindle. Thus aberrant expression of the germ cell protein REC8 may directly contribute to
chromosomal instability in melanoma and other cells in which stem/germ cell proteins are expressed.
Use of Mesenchymal Stem Cells for promoting nerve regeneration in an axonotmesis rat model: in
vitro and in vivo analysis
Author(s)
Andrea Gartner
Ph.D Student
Food and Agrarian Sciences and Technologies Institute (ICETA)/Institute of Biomedical Sciences Abel
Salazar (ICBAS), PortoUniversity (UP)
Co-Author(s)
Tiago Pereira - Ph.D student, Institute of Biomedical Sciences Abel Salazar (ICBAS)/Food and Agrarian
Sciences and Technologies Institute (ICETA), Porto University (UP), Portugal
Jorge Ribeiro - Ph.D student, Institute of Biomedical Sciences Abel Salazar (ICBAS)/Food and Agrarian
Miguel França - Fellowship, Institute of Biomedical Sciences Abel Salazar (ICBAS)/Food and Agrarian
Raquel Gomes - Ph.D student, Institute of Biomedical Sciences Abel Salazar (ICBAS)/Food and Agrarian
Irina Amorim - Ph.D student, Institute of Biomedical Sciences Abel Salazar (ICBAS), Porto University (UP),
Portugal
Beatriz Porto - Professor, Institute of Biomedical Sciences Abel Salazar (ICBAS), Porto University (UP),
Portugal
Rosa Sousa - Lab technician, Institute of Biomedical Sciences Abel Salazar (ICBAS), Porto University (UP),
Portugal
Paulo Armada da Silva - Professor, Faculty of Human Kinetics (FMH), Technical University of Lisbon (UTL),
Portugal
Ana Lúcia Luis - Professor, Institute of Biomedical Sciences Abel Salazar (ICBAS)/Food and Agrarian
Stefano Guena - Professor, Neuroscience Institute of the Cavalieri Ottolenghi Foundation/Department of
Clinical and Biological Sciences, University of Turin, Italy
Ana Colette Maurício - Professor, Institute of Biomedical Sciences Abel Salazar (ICBAS)/Food and Agrarian
Topic
Basic Research
Problem
With the world wide global increase in life expectancy, a variety of disabling diseases with large impact on
human population are arising. This includes cardiovascular, neurological, musculoskeletal, and malignancies.
Therefore, it is imperative that new and more effective treatment methods are developed to correct for these
changes. After Central Nervous System (CNS) lesions, Peripheral Nervous System (PNS) injuries are the
ones with minor successes in terms of functional recovery. These kinds of injuries are frequent in clinical
practice. About two centuries ago it was assumed that these nerves would never regenerate. Indeed, scientific
and clinical knowledge has greatly increased in this area. A full understanding of nerve regeneration process,
especially what concerns a complete functional recovery and organ reinnervation after nerve injury, still
remains the principle goal of regenerative biology and medicine. The rat sciatic nerve has been the most
commonly experimental animal model used in studies concerning the peripheral nerve regeneration and
possible therapeutic approaches. Although sciatic nerve injuries themselves are rare in humans, this
experimental model provides a very realistic testing bench for lesions involving plurifascicular mixed nerves
with axons of different size and type competing to reach and reinnervate distal targets.
Background
Recent advances in Regenerative Medicine are remarkable and in the last years the scientific community has
witnessed the arrival of many new concepts and discoveries. Until a few years ago, biological tissues were
regarded as unable of extensive regeneration, but nowadays organs and tissues appear as capable to be
reconstructed, based on “stem cells”. Mesenchymal stem cells (MSCs) have become one of the most
interesting targets for tissue regeneration due to their high plasticity, proliferative and differentiation capacity
together with their attractive immunosuppressive properties. Cellular systems implanted into an injured nerve
may produce growth factors or extracellular matrix molecules, modulate the inflammatory process and
eventually improve nerve regeneration. It has been speculated that once MSCs have the potential to
differentiate into several tissues, they might be responsible for turnover and maintenance of adult tissues. it
was believed that after local infiltration or effusion of MSCs, these cells would migrate to the damaged site
(homing capacity), differentiate into specific cell types or might modulate the microenvironment promoting the
regenerative process. So. MSCs could mediate tissue repair through there multi-lineage capacity replacing
damaged cells. Subsequent studies have suggested that the mechanism used by MSCs for tissue repairing is
not really this way. Recent studies demonstrated that transplanted MSCs are able of modifying the
surrounding tissue microenvironment, promoting repair with functional improvement by secretion of growth
factors (known as paracrine effect), by stimulating the preexisting stem cell populations and by decreasing the
inflammatory and immune response.
Hypothesis
In the present study, we evaluated the therapeutic value of human umbilical cord matrix (Wharton’s jelly)
MSCs (HMSCs) on rat sciatic nerve after axonotmesis injury associated to a Poly(DL-lactide-ε-caprolactone)
PLC Vivosorb® membrane, a flexible bioresorbable polymer film, which presents retention of mechanical
strength throughout the critical healing period.
Research
MSCs expansion and differentiation in neuroglial-like cells, was performed. Culture medium was collected at
48, 72 and 96h for nuclear magnetic resonance (NMR) analysis in order to evaluate the metabolic profile.
Immunocytochemistry was done to identify typical specific neuroglial markers such as the GFAP, the GAP-43
and NeuN, after differentiation. In vivo testing was carried out in Sasco Sprague adult rats (Charles River
Laboratories, Barcelona, Spain) which were divided in groups of 6 animals each: A group of 6 animals was
used as control without any sciatic nerve injury (Group 1 – Control). In Group 2 the crushed sciatic nerve did
not have any other intervention (Group 2 - Crush). In Group 3, the axonotmesis lesion of 3 mm was
enwrapped with a PLC (Vivosorb®) membrane (Group 3 – CrushPLC). In Group 4, the crushed sciatic nerve
was infiltrated in the lesion area with a suspension of 1500 HMSCs (in a total volume of 50 μl) (Group 4 –
CrushCell), in Group 5, the crushed sciatic nerve was encircled by a PLC (Vivosorb®) membrane covered
with a monolayer of non differentiatedHMSCs (Group 5 – CrushCellNonDifPLC) and in Group 6 the
axonotmesis lesion of 3 mm was enwrapped with a PLC (Vivosorb®) membrane covered with a monolayer of
differentiated HMSCs (neuroglial-like cells) (Group 6 – CrushCellDifPLC). A standardized crush injury was
carried out with the animals placed prone under sterile conditions and the skin from the clipped lateral right
thigh scrubbed in a routine fashion with antiseptic solution. After injury and treatment of animals, follow-up
results are very important for analysis of functional recovery. Animals are tested preoperatively (week 0), and
every week during 12. Motor performance and nociceptive function are evaluated by measuring extensor
postural thrust (EPT) and withdrawal reflex latency (WRL), respectively. For EPT test, the affected and normal
limbs are tested 3 times, with an interval of 2 minutes between consecutive tests, and the 3 values are
averaged to obtain a final result. The normal (unaffected limb) EPT (NEPT) and experimental EPT (EEPT)
values are incorporated into an equation (Equation (1)) to derive the percentage of functional deficit. For
Sciatic Functional Index (SFI), animals are tested in a confined walkway that they cross, measuring 42 cm
long and 8.2 cm wide, with a dark shelter at the end. Several measurements are taken from the footprints: i)
distance from the heel to the third toe, the print length (PL); ii) distance from the first to the fifth toe, the toe
spread (TS); and iii) distance from the second to the fourth toe, the intermediary toe spread (ITS). In the static
evaluation (SSI) only the parameters TS and ITS, are measured. For SFI and SSI, all measurements are
taken from the experimental (E) and normal (N) sides. Prints for measurements are chosen at the time of
walking based on precise, clear and completeness of footprints. Stereological analysis was carried out on
regenerated nerve fibers. Nerve samples were processed for quantitative morphometry of myelinated nerve
fibers.
Observations
The HMSCs exhibited a mesenchymal-like shape with a flat and polygonal morphology. During expansion the
cells became long spindle-shaped and colonized the whole culturing surface. After 96 hours of culture in
neurogenic medium, we observed a morphological change. The cells became exceedingly long and there was
a formation of typical neuroglial-like cells with multi-branches and secondary branches. The differentiation was
tested based on the expression of typical neuronal markers such as GFAP, GAP-43 and NeuN in neurogliallike differentiated HMSCs. Undifferentiated HMSCs were negatively labeled to GFAP, GAP-43 and NeuN.
After 96 hours of differentiation the attained cells were positively stained for glial protein GFAP and for the
growth-associated protein GAP-43. All nuclei of neuroglial-like cells were also labeled with the neuron specific
nuclear protein NeuN demonstrating successful differentiation of MSCs in neuroglial-like cells.
Undifferentiated HMSCs exhibited a normal star-like shape with a flat morphology. After in vitro differentiation,
HMSCs morphology changed into typical neuroglial-like pattern with multi-branches and secondary branches.
NMR showed clear evidence that HMSCs expansion is glycolysis-dependent but their differentiation requires
the switch of the metabolic profile to oxidative metabolism. Our data obtained from in vivo testing,
demonstrated that PLC does not deleteriously interfere with the nerve regeneration process, as a matter of
fact, the information on the effectiveness of PLC membranes and tube-guides for allowing nerve regeneration
was already provided experimentally and with patients. PLC becomes hydrophilic by water uptake, which
increases the permeability of the polymer. This is important in the control of nutrient and other metabolite
transport to the surrounding healing tissue. A few weeks after implantation, the mechanical strength gradually
decreases and loss of molecular weight occurs as a result of the hydrolysis process. In approximately 24
months, PLC degrades into lactic acid and hydroxycaproic acid which are both safely metabolized into water
and carbon dioxide and/or excreted through the urinary tract. In contrast to other biodegradable polymers,
PCL has the advantage of not creating an acidic and potentially disturbing micro-environment, which is
favorable to the surrounding tissue. Our in vivo results showed that the use of either undifferentiated or
differentiated HMSCs enhanced the recovery of sensory and motor function. In both cell-enriched
experimental groups myelin sheath was thicker, suggesting that HMSCs might exert their positive effects on
Schwann cells, the key element in Wallerian degeneration and the following axonal regeneration. We
conclude that HMSCs delivery through PLC membranes might thus be regarded a potentially valuable tool to
improve clinical outcome especially after trauma to sensory nerves, such as digital nerves.
Valproic acid enhances/primes the migration of cord blood mesenchymal stromal cells
Author(s)
Anna Janowska-Wieczorek, MD, PhD
Professor of Medicine
University of Alberta
Co-Author(s)
Yuanyuan Qiu, PhD - Post-doctoral Fellow, Canadian Blood Services
Leah Marquez-Curtis, PhD - Research Assistant, Canadian Blood Services
Topic
Basic Research
Problem
Mesenchymal stromal cells (MSC) have great potential for cellular therapies of many disorders. The
interaction between the chemokine stromal cell-derived factor (SDF)-1 and its receptors (CXCR4 and CXCR7)
plays a pivotal role in migration of cells to the injury site where the local concentration of SDF-1 increases.
However, the low surface expression of CXCR4 on MSC limits their ability to migrate towards SDF-1.
Background
We recently demonstrated that valproic acid (VPA), an inhibitor of histone deacetylase that plays an important
role in transcriptional regulation, increases the CXCR4 expression and chemotaxis of cord blood (CB)
hematopoietic stem/progenitor cells (Stem Cells Dev 2009 18(6):831). However, it has also been suggested
that VPA may decrease the differentiation of MSC.
Hypothesis
As the ability of MSC to migrate towards injury sites is important for their clinical application, in this study we
hypothesized that VPA increases the migration of CB-derived MSC. In addition to migration, we also
investigated the effect of VPA on proliferation and differentiation of CB MSC.
Research
CB MSC (characterized by their morphology, differentiation into osteocytes and chondrocytes, surface
expression of CD90 and CD105, and the lack of hematopoietic markers CD34 and CD45) were exposed to
VPA at various concentrations (1mM, 5mM, 10mM) and times of exposure (3 h or 6 h). CB MSC were
evaluated for: i) the expression of CXCR4 and CXCR7 using quantitative real-time PCR and flow cytometry; ii)
invasion/migration across the reconstituted basement membrane Matrigel towards SDF-1 in Boyden
chambers; iii) expression of matrix metalloproteinases (MMPs), especially those known to regulate the
migration of MSC, namely membrane type 1 (MT1)-MMP and MMP-2, using real-time PCR and zymography;
iv) proliferation using LUMENESC-96 assay (a gift from HemoGenix Inc.); and v) their differentiation
capabilities.
Observations
We found that short-term (3 h and 6 h) treatment with VPA increased in a dose-dependent manner the gene
expression of CXCR4 and CXCR7, but surface expression was not affected. VPA primed/enhanced the
migration of MSC towards a low gradient (20 ng/ml) of SDF-1 by nearly three-fold, which was similar to
migration observed towards a high SDF-1 gradient (100 ng/ml). This priming effect was inhibited by the
CXCR4 antagonist AMD3100 (p
VEGF treatment increases the expression of angiogenesis, pluripotency and cell adhesion factors in
stem cells from the canine yolk sac
Author(s)
Paula Fratini
Researcher post doc
University of Sao Paulo veterinary school FMVZ-USP
Co-Author(s)
Dayane Alcântara - post graduate student, FMVZ-USP
Sonia Will - post graduate student, FMVZ-USP
Rose Eli Rici - researcher, FMVZ-USP
Maria Angelica MIglino - principal researcher, FMVZ-USP
Topic
Basic Research
Problem
Since the yolk sac (YS) is the first site of blood cell formation during early embryogenesis, it was hypothesized
that pluripotent stem cells, capable to generate red and white blood cells as well as endothelial tissues, could
be present therein. Indeed, such stem cells have been isolated from the YS during early gestation, i.e. before
the formation of blood islets, and have been identified to be mesenchymal cells. They may represent a
scource of mesenchymal stem cells that may differentiate into endothelial and haematopoietic cells, indicating
a promising value for clinical applications in stem cell therapy, for example to repair vascular alterations. The
aim of this study is to firstly characterize the differentiation of canine yolk sac cells and to infere their potential
as stem cells and/or hematopoietic progenitor cells that may form endothelium. Based on that, we aim to
develop protocols in regard to their therapeutic value for regenerative veterinary medicine.
Background
The YS is important for embryonic and fetal life. This membrane is associated with intrauterine nutrition and
works as a source of multipotential hematopoietic precursor cells situated within erythropoietic islands. These
cells have similar characteristics than embryonic mesenchymal stem cells in regard to their pluripotency and
differentiation capability. In this study we firstly analyzed the differentiation potential of cells derived from the
canine yolk sac with or without treatment with VEGF (vascular endothelial growth factor) and other substances
that may increase cell growth and differentiation in order to establish protocols for cell therapy in veterinary
medicine.
Hypothesis
Does canine yolk sac cells have pluripotential capacity in order to form endothelial cells? What are the
best media and groth facter treatments for cell culture?
Research
YS samples were obtained from pregnant dogs (n = 6), of 30-35 days of gestation, during castration
campaigns in São Paulo-SP, Brazil. Initially, a surgical incision in the uterus, to expose the extra-embryonic
membranes as such as the canine embryos or fetuses, were performed. The fetal membranes were
separated, and the yolk sacs were isolated, collected and placed in Petri dishes with a solution of PBS 1X and
1% antibiotic solution. The gestational age was estimated according to Sack and Evans (1973).
The yolk sac tissue samples were washed with PBS (0.1%) and streptomycin / penicillin (5%). They were
macerated manually with a scalpel blade to obtain a tissue homogenate of about 3 mm diameter. The
fragments were placed in cell culture flasks, 25 cm², which were previously filled with 1 ml of fetal bovine
serum. After 24 hours, the bottles were filled with 5 ml of culture medium Alpha-MEM supplemented with 10%
FBS (fetal bovine serum) and 1% streptomycin / penicillin and incubated at 37 °C in a gaseous atmosphere
with 5 % CO2. To improve cell culture techniques,tests were performed with culture media to assess the
conditions for optimal cultivation of the cells from canine yolk sac.
Treatment with growth factors and interleukins: after adequate cell growth, cells were plated (5 x 104 cells)
and, after 24 hours, factors which induce cellular differentiation and proliferation, VEGF (25ng/ml, Sigma), IL3
(50ng/mL, Sigma) and SCF (50ng/mL, Sigma), respectively, were added to the culture medium. The
supplemented culture medium was replaced each 3 days and the cells were maintained in culture for 21 days.
The treated cells were periodically photodocumented to observer the morphology during cell growth and
proliferation through an inverted microscope Nikon Eclipse TS100.
Flow Cytometry: after treatment, cell culture was trypsinized and inactivated with fetal bovine serum. The cells
were sedimented by centrifugation at 1500rpm for 10 minutes and the supernatant was discarded. The pellet
was resuspended in 5 ml of 0.9% saline solution and centrifuged at 1500rpm for 10 minutes. After the
supernatant was discarded and the FACS buffer added, the suspension was transferred to cytometry tubes.
The antibodies CD34, CD44, CD105, STRO-1, OCT4, Cyclin D1, VEGF were added and incubated for 15
minutes at 4°C. The expression analyzes were performed on a FACSCalibur flow cytometer, 10,000 events,
and analyzed by WinMid 2.8 Program.
Observations
Canine yolk sac stem cells had the potential to differentiate into endothelial progenitor cells, hematopoietic
and mesenchymal cells. After treatment with VEGF enhanced development and morphological changes were
observed. Embryoid bodies and colonies of endothelial-like cells could be identified after 72 hours of
treatment. An increased VEGF expression, demonstrated by flow cytometry assays, was verified. Canine yolk
sac samples treated with VEGF and SCF (stem cell factor) showed stimulation in regard to endothelial cell
diferentiation. Cells derived from the canine yolk sac represent a new source of regenerative cells for cell
therapy in veterinary medicine with high potential for applications in neovascularization.
Voltage-dependent spontaneous calcium activity critically regulates neural progenitor differentiation
Author(s)
Paola Rebellato
PhD student
Karolinska Institutet
Co-Author(s)
Per Uhlén - Associate Professor, Karolinska Institutet
Topic
Basic Research
Problem
The onset of spontaneous calcium activity during early neural development has been investigated for long
time, but mechanisms and consequences remain poorly understood.
Background
Embryonic stem (ES) cells cultured in a definite medium follow a default differentiation path towards anterior
neuroectoderm. Cells assemble in polarized structures that exhibit spontaneous calcium and electrical activity,
forming neural networks.
Hypothesis
We hypothesized that voltage dependent calcium channels (VDCCs) represent the basic mechanism for
spontaneous calcium activity during early neural development.
Research
We tested our hypothesis following ES cells differentiation with calcium imaging, real time PCR,
immunocytochemistry, and pharmacological treatments. To further investigate the impact of calcium signaling
on neural development, lentiviral vectors were used to knock-down different VDCCs during differentiation.
Observations
We found that from day 6 of differentiation, cells started to respond to depolarizing stimuli (50mM KCl) and
showed increased spontaneous calcium activity. Real time-PCR analysis revealed a progressive increase in
expression of low and high VDCCs. Spontaneous calcium and electrical activity were inhibited by nickel in a
concentration (50µM) that is considered specific for T channels, whereas the response to the depolarizing
stimuli was not affected. Mibefradil, an L and T channels inhibitor, completely blocked the spontaneous
calcium activity and the response to KCl. Nickel treatment also decreased the expression of early stage neural
markers and knocking down of T channels confirmed the role for these channels for PAX6 expression.
Furthermore, back-tracing the calcium experiments allowed identification of single cells with low or high
spontaneous activity. After confirming that VDCCs expression was similar between differentiating ES cells and
developing embryonic forebrain, ex-utero electroporation with shRNA against VDCCs showed a variation in
cell number and in migration towards the cortical plate.To conclude, our data demonstrate that VDCCs have a
critical role for intrinsically driven neuronal differentiation.
Can Health Care Outcomes be Quantified following Stem Cell Therapy?
Author(s)
Dennis M. Lox
Dr. Dennis Lox
Florida Spine and Sports Medicne Center
Background
Since 1990, Dennis M. Lox, M.D has applied his personal interests in sports medicine and chronic pain
management to helping patients increase their quality of life by reducing their pain. He specializes in
platelet-rich plasma and stem cell therapy for musculoskeletal conditions.
Bio
Since 1990, Dennis M. Lox, M.D has applied his personal interests in sports medicine and chronic pain
management to helping patients increase their quality of life by reducing their pain. He specializes in
platelet-rich plasma and stem cell therapy for musculoskeletal conditions.
Co-Author(s)
Topic
Ethics, Law and Society
Problem
Implementation of stem cell therapy faces numerous challenges from basic research, federal regulation, and
challenges in a new health care paradigm. These challanges must be overcome and stem cell therapy must
be viewed as a beneficial societal need to flourish in many diverse fields. The treatment of degenerative
arthritis with stem cell therapy will need quantified favorable outcomes to become a frequently utilized
procedure.
Background
In order for stem cell therapy therapy to be a viable treatment option in degenerative arthritis, it must contend
with traditional therapeutic regimes entrenched in the current medical model. Pharmocologic options, surgical
and hospital procedures, and patient expectations of what stem cell therapy entails, await the interpretation of
a successful health care outcome.
Hypothesis
Defining a positive health care outcome for treating degenerative arthritis with stem cell therapy may prove
elusive. Prior treatments not held to the same standards of success that will be required of stem cell therapy
in todays medical enviroment, may render objective quantification of health care outcomes difficult to
determine.
Research
Quantification of health care outcomes must be undertaken to reflect cost effectiveness. Defining cost
effectivenes, will need to be adjusted as more research becomes available over longer time frames.
Quantification can be evaluated by arthritis diagnosis related health care expenditures and serial
measurements of Quality of Life (QoL)
scales.
Observations
The interpretation of cost effectiveness will need to be further defined based upon future experiences with
improvement in QoL.
Quantification of successful health care outcome following stem cell therapy for degenerative arthritis may not
be uniformly agreed upon by patients, physcians, and health care reimbursement parties.
Ethics of translational stem cell research: moving pluripotent stem cells to the clinic
Author(s)
Annelien L. Bredenoord
Assistant Professor of Medical Ethics
University Medical Center Utrecht, dept Medical Ethics
Co-Author(s)
Johannes JM van Delden, MD PhD - Professor of Medical Ethics, University Medical Center Utrecht, dept
Medical Ethics
Topic
Problem
Pluripotent stem cells (PSCs) are capable of self-renewal and have the capacity to differentiate into any cell
type of the human body. Two types of PSCs exist: human embryonic stem cells (hESCs) and induced
pluripotent stem cells (iPSCs). Though PSC research has great potential for novel approaches in regenerative
medicine, it at the same time is accompanied by a fierce ethical and political debate. This debate, however,
has almost exclusively focused on the contested moral status of the human embryo. Whereas iPS cells
bypass the ethical debates associated with embryo destruction, they are by no means free of ethical
concerns. There is a set of ethical questions relevant to both hESC and iPSC research that are not related to
the moral status of the embryo that received scant attention: the ethical issues associated with moving PSCs
from bench to bedside. Although many of these ethical challenges do also arise in other innovative early
interventions, they become particularly cogent in translational PSC research due to the unique risks and
characteristics of pluripotent stem cells, the sensitivity of the preclinical research, the governance of PSC
banks, the potential vulnerability of research participants, the normative choices underlying first-in-human trial
design, the tremendous scientific and commercial stakes and the high public and political attention associated
with stem cell research.
Background
At this moment, attempts to translate basic PSC research into cellular treatments are well under way. The
world’s first hESC-based trials have been launched and iPSC-based trials could be expected in the future.
The process of translating basic research into clinical applications that could potentially lead to larger clinical
trials and implementation in the health care system begins with preclinical research and subsequently moves
into first-in-human (or phase I) studies. First-in-human trials are ethically challenging by nature, particularly
because the needed evidence to reliably predict risk and benefit (testing in humans) is missing. First-in-human
PSC trials will be replete with uncertainties and safety concerns, among others because PSC-based
treatments will be highly variable and heterogeneous due to the origin of the cells, degree of manipulation,
degree of differentiation and manufacturing process, the inherent genetic instability of PSCs, the risk of
insertional mutagenesis and/or teratomas and the irreversibility of the transplantation.
Hypothesis
Whereas many steps in translational science are uncertain and ethically contentious, this will particularly be
the case for first-in-human PSC trials: the inherent uncertainty of first-in-human trials combined with the
technical complexity of PSCs make early-phase PSC trials ethically very challenging.
Research
In our project we will combine empirical research with ethical analysis. We are currently setting up in-depth,
semi-structured interviews (N=40) to examine the views and experiences of relevant stakeholders in at least
four countries (The Netherlands, United Kingdom, United States and Singapore). Two groups of respondents
will be selected: (1) professionals (stem cell researchers, clinicians and members of research ethics
committees / Institutional Review Boards) and (2) potential research participants and patients. By having
interviews with a variety of relevant respondents in different countries, we aim to collect an array of insights
and information regarding PSC research as broad as possible (contrast maximization). This will inform us
about what relevant stakeholders experience to be the major ethical issues in translational PSC research. The
empirical data obtained by these interviews will be used as input for the ethical analysis.
Observations
In this paper we first briefly highlight the ethical specifics of translational PSC research. Subsequently we
present our project in which we examine how we can ethically justify and launch early-phase pluripotent stem
cell trials. We more specifically explore the question of how we should respond to the uncertainty inherently
linked to first-in-human iPS cell-based interventions. After all, early iPS cell trials will be characterized by a
high degree of uncertainty combined with a low chance of medical benefits for research participants. Five
strategies of risk minimization as proposed in the research ethics literature will be discussed, i.e. (1) choice of
intervention strategies, (2) participant selection, (3) trial design, (4) ethical review and (5) monitoring of study
participants.
Oocytes & Blood as Gifts & Commodities: A Transformative Case Study For Undergraduates That
Promotes Ethical Reasoning
Author(s)
Alexa Riggs, BA Candidate
Research Assistant , Stem Cells Across the Curriculum
Eugene Lang College The New School for Liberal Arts
Co-Author(s)
Katayoun Chamany, Ph.D. Molecular and Cell Biology - Associate Professor of Biology, Eugene Lang College
The New School for Liberal Arts
Topic
Problem
Should human tissues and cells be treated as sacred gifts, commodities that result from performed labor, or
products whose retrieval places the provider at significant risk requiring compensation? As stem cell and
fertility research advances, there is a need for democratic deliberation concerning the status of tissues and
cells, and the cost of “labor” surrounding their retrieval and manipulation. In the United States, the history of
assisted reproductive technology (ART) suggests that without government regulation a range of practices
persists including compensation, payment, and gifting. But even in countries where public funds are used in
the context of reproductive technology and stem cell research (SCR), we continue to see a dynamic range of
possibilities. One example is the “egg sharing” option in the United Kingdom where individuals interested in
procuring eggs for fertility purposes can reduce the cost of subsequent cycles of IVF if a portion of the first
cycle is “donated” to public funded SCR that utilizes the egg to reprogram adult nuclei or to create embryos
via IVF for research purposes. In both examples the practices and policies are a result of human egg scarcity
and increasing demand in both the ART and SCR sectors. With the identification of ovarian stem cells by John
Tilly’s research team in 2012, stem cell researchers may be able to create a large number of human eggs in
vitro thus shifting the supply-demand ratio. Will this advance in science and technology eliminate or
exacerbate human egg markets? Given economic disparities and the rising cost of tuition, will students find
themselves being targeted to create this initial supply of ovarian stem cells? If fewer egg providers are needed
for SCR, will this shift the risk-benefit ratio for human research subjects that are providing the ovarian stem
cells, and will this affect compensation?
Background
With financial support from the New York State Empire State Stem Cell Board, faculty across the natural
sciences, humanities, social sciences, and design collaborated to develop and implement a semester-length
non-majors interdisciplinary course focused on the scientific, social, ethical, and legal dimensions of stem cell
research (SCR). The course utilizes case-based teaching and learning methods that promote the
development of an informed citizenry and future scientists that practice ethically responsible research. Casebased reasoning requires students to actively learn and experience the stages of ethical development as
outlined by William Perry to address stem cell related controversies such as those surrounding egg
procurement. To understand the scientific and social dimensions of this evolving industry, students explore
resources spanning literature, government reports, scientific animations, timelines, and infographics to identify
and understand the intersections of basic science, health psychology, feminist and disability rights discourse,
social justice, bioethics, and health policy. Oocytes & Blood: Gifts & Commodities is one such case study
designed to highlight the shift in practices and policies surrounding egg procurement, and provide students
with an opportunity to build an appreciation for different points of view. With social egg freezing no longer
considered an experimental procedure and news of ovarian stem cells, there will be an increasingly large
number of excess eggs that can be sold, bartered, or donated for fertility purposes and SCR. Thus, the egg is
a mobile entity transitioning between the clinic and the scientific lab, while the woman becomes more and
more invisible. The case requires students to role-play various stakeholders and submit position statements
and policy briefs that move them from an uninformed position of duality, for or against, to one that
demonstrates ethical reasoning and draws on evidence from reproductive biology and utilizes a social justice
framework.
Hypothesis
We hypothesize that if cases require a student to adopt the role of a specific person and engage in dialogue
with those that might hold very different positions regarding a fractious/wicked problem, students develop an
appreciation for a range of worldviews and recognize instances of political empathy. Having students move
beyond statements to conversations in which they construct knowledge together and seek to identify a
strategy that respects multiple value systems is in line with the constructivist approach to learning. Paulo
Freire proposed that critical pedagogy is one that acknowledges power hierarchies, questions normative
assumptions, and is more authentic to the lived experience of the learner. John Dewey argued that if we are to
meet students where they are and make learning meaningful, learning has to move beyond the traditional
classroom experience of informational delivery to one that requires students “to do something” in the name of
civic action. Lev Vygotsky’s theories of education advocate for the fundamental role of social interaction in the
development of cognition and the role that community and culture play in the process of “making meaning.”
Thus, our use of dialogue that requires different stakeholders to converse and exchange with one another
builds off the work of these educational reformers. Research in cognitive science regarding motivation led us
to hypothesize that if we can move students from situational interest (the case and problem) to personal
interest (allowing them to identify with a person and juxtapose this position with their own personal value
system), we will improve long-term learning retention. The goal is to develop contributive members of civic
society who are capable of using evidence to arrive at informed decisions, recognize that there are multiple
value systems at play, and acknowledge the benefits and trade offs involved in social policy making.
Research
There has been a call to action to apply the problem-based and case-based pedagogies to solve “wicked” or
“fractious” problems, as it might accelerate the development of ethical reasoning skills in the early years of
college. These kinds of problems involve unavoidable policy decisions that affect wide sectors of the
population and do not have one “right” answer. Though many STEM educators have taken up this reform
effort, most case simulations result in placing students in stakeholder groups that represent a single position
and require students to engage in evidence-based debate. We found that this approach does not help
students develop a tolerance for other points of view, and does not fairly represent the pluralistic society in
which we live. Having students represent and debate the view of a stakeholder group, such as “ feminists,” “
industry,” “scientists,” etc. suggests that there is a unified stance for stakeholders’ groups resulting in
polarization that blocks democratic participation. During debates students get so caught up in “winning” that
they are unwilling to prioritize their values in ways that would allow for compromise. Based on initial pilots
conducted in a liberal arts college seminar based course, we revised the case study assignment to include
more scaffolding to guide students and move them through the 7E learning cycle (elicit, engage, explore,
explain, elaborate, evaluate, and extend) as they complete five learning activities. Learning Activity 1
introduces students to the topic through a discussion based on short readings and video clips using a
progressive disclosure approach that prompts students to ask questions about how oocyte provision is
practiced, regulated, and valued. In this discussion, novices are introduced to biological concepts such as the
culturing of gametes and embryos for embryonic stem cells (ESCs) and feminist theories such as “women’s
autonomy,” “choice,” and transnational organizations such as “Hands Off Our Ovaries,”(elicit and engage).
Learning Activity 2 asks student to dive deep into the literature to explore and explain to their peers a
particular perspective by adopting a specific stakeholder’s persona and crafting a position statement, two
questions that they would pose to other stakeholders, and a counterargument in preparation for conversation
with those that may hold a position that differs from their own (explore and explain). To aid students in this
activity, we have identified textual and visual resources to explore the unique biological characteristics of
oocytes and menstrual blood and the social context from which they’re obtained. Learning Activity 3 is a
simulated role-play of a conference session at a bioethics conference that focuses on communication and
allows students to engage in a 60-80 minute dialogue (explore, explain, and elaborate). Learning Activity 4 is
a debriefing session that provides instructor and students space and time to exercise metacognitive reflection
to fill in gaps in understanding, provide alternative conceptions, and/or address behaviors that did not promote
inclusive discussion. Learning Activity 5 asks students to write a critical essay in which they revisit their
personal stance on the case after having completed the role-play and to consider the ways in which their
personal values intersect with those of the stakeholders (evaluate and extend). The newly revised case
assignment was administered in a variety of formats at The New School, SFSU, Vassar College, and UC
Davis. We are collecting feedback on student learning and affect from these pilots. Course evaluations and
student essays are providing data on which resources are most useful and informative for non-majors, the
degree to which students can engage with views that differ from their own, and the range of ethical issues that
students recognize as associated with SCR.
Observations
Students in the pilot non-majors seminar course at The New School (n-=14) were able to recognize more than
one ethical issue related to stem cell research. Specifically, when asked to identify two ethical issues
associated with SCR, eight students remarked on the moral status of the embryo as it relates to ESCR, while
a third identified health risks in the context of human research subjects. Taken together their responses span
oocyte provider’s health, social justice, animal rights, disability rights, and exploitation of vulnerable
populations in the context of human subjects research. When students were given the opportunity to write an
additional essay on a SCR related topic for this non-majors course, more than half the class focused on
oocyte procurement. With respect to ethical reasoning or case-based reasoning development, students in the
seminar based non-majors course were able to provide evidence from the course resources to support their
stance and address their opponents concerns suggesting a shift to personal commitment. The readings span
the benefits and risks associated with egg “donation,” the laws and practices currently in place for egg
retrieval for research purposes, and the issues surrounding the private and public sector management of
payment for these cell sources, and commercialization and regulation of access to potential stem cells made
from these sources. Perhaps most notable was their acquisition of a more comprehensive understanding of
the oocyte procurement process and the ways in which current practices and policies intersect with race,
class, and gender disparities. Students in the seminar-based course also saw value in collecting data on
health risks of hormone stimulation. In the one day session in the “Feminist Thought and Action” course at
The New School, where the case study was used without the role-play component, many students chose to
write critical essays that revealed their personal value system as it relates to social policy in the context of
recruitment of oocyte donors from college-age populations that may be economically vulnerable, as well as
other populations that may not be recognized as privileged or able to exercise autonomy and agency. Using
content analysis, we found that the citations in these essays mapped back to the resources that were
immediately relevant to the students and fell into the categories of those that addressed college-age students
specifically as targets of oocyte procurement, or merged theory and practice in concrete ways. In this lecturebased course, we found that the truncated nature of the case study and the lack of the role-play component
left students somewhat confused about the similarities and differences of oocyte procurement as applied to
the reproductive versus stem cell research context. Thus, pilot studies suggest that students who engage in
role-play and deliberation benefit from a clearer understanding of the ethical issues of oocyte procurement
and are better equipped to argue for a particular position using evidence. However, given the number of
variables at play (student background, pedagogy, course format, and length of case module) more studies are
required to investigate our hypothesis. We are currently evaluating the choice of resources that will
accompany this case study to address a range of student backgrounds and provide instructors with flexibility
in choosing resources and activities and offer substantive material appropriate for students who may be
situated in different positions within the zone of proximal development. That is to say, how much a learner
can achieve on their own versus what they can achieve with the guidance or assistance of an instructor or
structured assignment. To that end, instructors piloting the case assignment find the organization of resources
by topic area and the rubrics for the role-play and critical essay to be useful, suggesting that the development
of these instruments will aid data collection on learning outcomes regarding ethical and case-based
reasoning.
The Final Version of Japanese Guidelines on Ensuring Quality and Safety of Products Derived from
Processing of Various Human Stem Cells
Author(s)
Takao Hayakawa
Director and Professor
Pharmaceutical Research and Technology Institute, Kinki University
Role: Author
Yoji Sato
Head, Division of Cellular & Gene Therapy Products
National Institute of Health Sciences
Role: Author
Background
Development and validation of assay systems for evaliation of safety and quality of cell/tissue-based
products
Bio
Development and validation of assay systems for evaliation of safety and quality of cell/tissue-based
products
Co-Author(s)
Takashi Aoi - Professor, Department of Regulatory Science, Center for iPS Cell Research and Application,
Kyoto University
Akihiro Umezawa - Department Head and Chairman, Department of Reproductive Biology, National Institute
for Child Health and Development
Keiya Ozawa - Professor, Division of Hematology, Department of Medicine, Jichi Medical University
Yoshiki Sawa - Professor, Division of Cardiovascular Surgery, Department of Surgery, Osaka University
Graduate School of Medicine
Akifumi Matsuyama - Head, Development and Support Division of Regenerative Medicine, Foundation for
Biomedical Research and Innovation
Shinya Yamanaka - Director and Professor, Center for iPS Cell Research and Application, Kyoto University
Masayuki Yamato - Professor, Advanced Biomedical Science Center, Tokyo Women’s Medical University
Topic
Problem
Regenerative medicine using relevant products derived from various human stem cells are being keenly
anticipated in Japan due to limited resources of human organs and tissues transplantation and difficulty of
treatments to cure various severe diseases. The more advances in research in this field, the more people are
hoping that such novel products should be translated effectively and efficiently into actual therapy. Identifying
points to consider for ensuring quality, safety and efficacy of products derived from the various types of stem
cells is crucial through product development for their rapid application in patients.
Background
Technical requirements for manufacturing and marketing authorization of cells/tissue-based products under
the Japanese Pharmaceutical Affairs Law, are described in the Notification No. 1314 of Secretary-General of
Pharmaceutical and Food Safety Bureau (PFSB), Ministry of Health, Labour and Welfare (MHLW), dated
December 26, 2000. The 2006/2007 Scientific Research Committee of the MHLW looked into preparing a
revised version of “Guideline on Ensuring Quality and Safety of Products Derived from Processing of Human
Cells/Tissue”, which is Appendix 2 in the PFSB/MHLW 1314, in response to requests that Japan should push
forward with appropriate regulations for regenerative medicine through the revision of assessment criteria that
reflects rapidly developing science and technology, ethical viewpoints, and international trends. This revision,
splitting the original guideline up into 2 different guidelines in order to make clearer the specific technical
requirements for products derived from autologous cells and allogenic cells, led to the publication of the
“Guideline on Ensuring Quality and Safety of Products Derived from Processing of Human Autologous
Cells/Tissue” (PFSB/MHLW Notification 0208003) in February 2008, and the “Guideline on Ensuring Quality
and Safety of Products Derived from Processing of Human Allogenic Cells/Tissue” (PFSB/MHLW Notification
0912006) in September 2008, respectively.
Hypothesis
However, further studies became necessary to identify specific issues related to products derived from
processing of somatic stem cells, embryonic stem cells or induced pluripotent stem cells (iPSCs), all of which
have been attracting a lot of attention in recent years.
Research
At this point, the MHLW decided to form in fiscal year 2008. a panel of experts (the authors) for drafting
guidelines on ensuring the quality and safety of products derived from processing of various human stem
cells.
Observations
At the presentation, we introduce the final version of the guidelines concerning the basic technical
requirements for ensuring the quality and safety of products derived from processing of various human stem
cells, which the MHLW released on September 7, 2012. The guidelines reflect the results of the study on the
current situation and future outlook, from a scientific and technological perspective, of the manufacture and
clinical application of human stem cell-based products. In particular, the sources of supply of human stem
cells, the definitions of “iPSC-like cells”, the genetic modifications of cells, the establishment, standardization
and control of cell banks as raw materials for cell-based products, and the other factors and measures
required in order to achieve specified objectives of the final products are presented.
The Molecular biology reagents and services for stem cell research field provided by TAKARA BIO.
Author(s)
Masanari Kitagawa
General Manager of Dragongenomics Center
TAKARABIO INC.
Co-Author(s)
Keiji Tanaka - Senior Scientist, TAKARABIO INC.
Yoshimasa Tsujimoto - Senior Scientist, TAKARABIO INC.
Topic
Industry Infrastructure
Problem
At Takara Bio, some tools for stem cell reseach, such as the vectors for constructing iPS cells, or antibodies
for stem cell markers, has been provided.
Background
Then, using the ES cell materials supplied by our NEDO project, we have developed a set of real-time RTPCR primers for the analysis of the expression of genes associated with ES cell pluripotency.
Hypothesis
This set can be used to analyze the gene expression in differentiated and undifferentiated ES cells. Each
array contains 96 primer pairs representing 88 pathway-related genes and 8 housekeeping genes.
Research
When comparing an unknown sample to a control sample, gene expression differences can be expressed
using the relative quantification method.
Observations
In addition, expression levels of multiple genes can be screened simultaneously.
Stem Cell Advocacy - Role of Patient Advocates
Author(s)
SAI VEMULA
STUDENT
UC BERKELEY
ASHKAN AHMADIAN
STUDENT
UC BERKELEY
Role: Author
Co-Author(s)
Topic
Patient Advocacy and Communications
Problem
The field of stem cells has great potential for treating and curing many illnesses and diseases. However, along
with the promise that it holds, stem cell research also comes with a myriad of accompanying ethical, legal,
religious, and policy-related issues. In particular, the matter of federal funding for human embryonic research
is consistently met with substantial opposition, which has consequently lead to a history of conflicting and
shifting policies. Even now with President Obama’s lift on restricting federal funding for human embryonic
stem cells (hESCs), research in this field still faces many other legal and political obstructions that significantly
hinder the work being done.
Background
With opposition from an array of different sources routinely imposing constrictions on the funding of stem cell
research, research is obstructed from progressing and ‘going forward’ at a desired pace due to major financial
disruptions. Consequently, delays in obtaining significant results could inadvertently lead to diminished public
hope in the curing potential of stem cells and motivation for future investment. Thus, it has become imperative
to not only fight resistance at the national level to preserve federal investment for stem cell research, but to
also seek out other, decentralized sources of capital that could potentially keep a more stable inflow of funding
for stem cell projects.
Spearheading the front of this other approach are patient advocates. Many of them have found creative
ways of attaining revenue to help finance many stem cell research programs throughout the country. Many
patient advocate groups have chosen to principally focus on lobby efforts at more local levels, promoting state
governments to pass crucial legislation that financially support stem cell scientists working at universities and
other institutions.
To gain support for new government initiatives, advocates have used various means to reach out to the
general public and to demonstrate to them the power that the ‘prospective future’ of this field holds. Through
these methods, advocates are able to ultimately mobilize large patient communities to promote awareness of
stem cell science.
Hypothesis
The purpose of this study is to address the role of patient advocates as leaders of the stem cell movement,
examining their efforts in creating local influence in social, political, and business realms. We propose that
through a well-coordinated, prudent, and concerted endeavor on all these fronts, patient advocacy groups can
have a profound impact on establishing non-centralized funding sources that are critical in supporting the
progress of stem cell research, especially for project dealing with more controversial topics.
Research
In this study, we used the Roman Reed Foundation as our focal point in modeling a successful patient
advocacy organization, while utilizing other patient advocacy groups—Christopher Reeve Foundation, Sabrina
Cohen Foundation, Michael J. Fox Foundation, Gwendolyn Strong Foundation and T. J. Atchison Program—
as comparison points. First, we did a comprehensive analysis of the Roman Reed Foundation’s past efforts in
promoting stem cell research, identifying the decisive factors critical to their success or failure. On the social
front, we looked at how the foundation established strong, local conglomerates of people to support its cause
by reaching out to the public and raising awareness via the use of social media—such as Facebook, Twitter,
Reddit, News Channel interviews, and funding events—and close relationships with educational institutions.
On the political front, we looked at the lobbying strategies that the foundation utilized in order to further
legislation in different states that’d benefit its cause. On the business front, we looked at how the foundation
delegated funds to stem cell research organizations and how it took advantage of its business influences to
further its other endeavors.
After establishing the nature of its network of influence, we explored the extent of the impact of the
Roman Reed Foundation’s work, in terms of its success in garnering increased investments to financially
support its stem cell research of interest and promoting overall progress in discovering significant findings in
the realm of stem cell related research.
Finally, using a broadened perspective, we proposed on how patient advocacy programs and the
different resources they have can most effectively be utilized in order to further the progress of stem cell
research as a whole.
Analyses employed in this study relied on various sources of information. Most of the information was
collected through Internet research and via phone and in-person interviews with some of the patient
advocates and their team members. Statistical information was also collected from data tabulated from the
patient advocates’ websites, Facebook comments, followers on Twitter, and Reddit. We used this data to
analyze the demographics of people who followed these foundations.
Observations
In terms of the social aspect, with regard to raising awareness, we discovered that the key factor is to achieve
a delicate balance between trying to reach a large audience and being selective. We found that reaching out
to a greater number of people won’t only create more support for a cause but also increase the opposition,
especially in populations with highly mixed opinions. It is in this way that patient advocacy groups can utilize
social media, by taking advantage of its malleability. Forums such as Facebook, Twitter, and Reddit allow
patient advocates to garner supporters and communicate with a far-reaching audience, even international
ones. However, at the same time, these sites have a selective component to them, where news of upcoming
events and promotions are geared towards a patient advocate’s friends on Facebook, followers on Twitter,
and sub-communities on Reddit—those who would be the most willing to help in the first place. Additionally,
patient advocate groups and educational institutions tend to have an intimate relationship, where ‘professor
advocacy’ for a cause can selectively raise awareness among the intellectual elite and students—
demographics that tend to share a progressive view towards these topics.
From a political standpoint, although private funding is important for facilitating research, public funding,
along with the legislation that directs it, is of paramount importance in order to promote almost any field. Thus,
lobbying strategies are needed at all levels of the political system to be successful: increasing constituent
support to promote petition filings, applying political pressure via phone calls and letters, forming good
relationships with state Senate leaders to obtain partisanship support, and endorsing progressive governors in
favor of such reform. Going from micro to macro, each level possesses its own significance, and thus in
conglomeration, all these levels will invariably increase the likelihood of obtaining a beneficial political
platform.
From a business aspect, patient advocate foundations, as non-profit revenue-allocating businesses, play
critical roles in forwarding stem cell research as ‘money lenders.’ It is undeniable that progression from the
first breakthrough in research to mature clinical trials is a long, arduous, and incredibly expensive process.
Many early research findings with great potential but immature empirical results are stalled solely because of
lack of funding. These foundations become the backbone for the researchers by providing this ‘early money’
to accelerate research that can have high-impact final results. Additionally, we also found that as businesses,
patient advocate foundations can, in turn, use their financial position to increase their social and political
influences.
An important caveat must be mentioned when discussing the utilization of patient advocates’ influence
in promoting stem cell research, where the range of research each group is supporting is limited to their own
interests. The Roman Reed Foundation primarily funds stem cell research related to spinal cord injury; the
Michael J. Fox Foundation is similarly fixated on stem cell research pushing Parkinson’s treatments. Thus,
how can such varied interests seeking the same limited public resources, which are inadequate to satisfy
them all, be accounted for in creating a strong, unified front promoting funding policies for stem cell research?
The solution to this question is similar to that of raising awareness, which is to strike a balance between
collaboration and self-promotion—collaborating by cross-promoting each other’s platform on social media
sites and co-sponsoring the election of progressive political leaders, while self-promoting individual lobbying
efforts. Furthermore, through this process of promoting some stem cell research, they also may inadvertently
lead to the progress of research in other disease fields that use stem cell research.
Survey says: Engagement and Interaction are Needed to Avoid Swaying the General Public against
Embryonic Stem Cell Research
Author(s)
Aradhana Verma, UC Berkeley Undergraduate Student
UC Berkeley SSSCR President
Student Society for Stem Cell Research
Eshaan Kashyap, UC Berkeley Undergraduate Student
UC Berkeley SSSCR Member
Student Society for Stem Cell Research
Role: Author
Co-Author(s)
Laurel Barchas, B.A. Integrative Biology, UC Berkeley - Associate Director, Student Society for Stem Cell
Research
Topic
Patient Advocacy and Communications
Problem
It is difficult to generate a population that is steadfastly in support of embyronic stem cell research due to
general inadequate understanding of the science and protocols that are accompanied by it. Primarily seen at
the polls, passing legislation tends to be tricky due to opinions that are easily swayed, especially with mediaoriented controvesial topics. It is advantageous for the scientific community to consider if further education is
required to solidify opinions on stem cell research past what is provided by the media and directly given at
polls.
Background
There are many ways to educate the public on real-world scientific issues (Barchas, Thompson, and Conboy
2006), and this tends to fractionate information and introduce misinformation, which is undesirable for a united
stem cell advocacy effort. Thus, there needs to be a common understanding in the desired outcome of our
education and outreach efforts.
Hypothesis
Our hypothesis is that the ballot-style survey will encourage opinions of ESC research to be swayed more
readily than when respondents are given time to process and weigh the pros and cons of the topic. This is
because we believe that furthering stem cell research requires consistent, in-depth explanations of what stem
cells are, where they come from, and the ethical and biological pros and cons.
Research
In November 2012, we surveyed two groups: 1) a public group (n = 300) advertised through facebook that
was given a short reading list (“ballot-style”) of the pros and cons of embryonic stem cells and 2) UC Berkeley
undergraduate students (n = 40) taking the authors’ 2012 “Stem Cells: Science and Society” DeCal
(Democratic Education at Cal) course presented with the same pros and cons but in the style of a
lecture/discussion. We measured the degree to which responders were swayed after reading a very brief
description about pros/cons of stem cell research or when receiving this information via lectures and
discussion regarding stem cell research.
Observations
When the survey was given to 300 people within the general population, there were two significant results:
66% had no change in their opinion favoring embryonic stem cell research, and 16% were swayed negatively,
reducing or retracting their support. When the same survey was given to 40 students in the undergraduate
course, which emphasizes regular discourse and debate with students, lecturers, and facilitators of the class,
85% had no change in favoring stem cell research while 5% of the people were swayed away from supporting
ESC research. Using a chi-squared goodness of fit test, we showed that the two distributions of responders
were significantly different and that those who participated in the DeCal were more equipped to hold to their
opinions despite the arguments against stem cell research.
A style of education that involves "ballot style" teaching, as opposed to discussion and engagement, allows
those who are on the fence to be swayed against stem cell research due to the dearth of information and
disincentivization of in-depth processing; we must seek better ways to foster public understanding and
engagement, so we conclude that lecture (formal or informal) and open discourse must be emphasized in high
school and college biotechnology and biology courses in order to reach our goal of a world that understands
the importance of ESC research.
(99) Submission
ID#OL00001
Title: Cellular Clinical Trial Services (CCTS) - A much needed solution
Authors: Kenneth L. Harris1, Venkatesh Ponemone2, Dalip Sethi2
Affiliations: 1TotipotentRX Corporation, Los Angeles, CA, U.S.A., 2TotipotentRX Cell Therapy
Pvt. Ltd., Gurgaon, India
Introduction
In the last decade, the clinical industry has witnessed great advancement in the field of cellular
therapies, but the translation into clinical success has been mixed both clinically and
commercially. Questions continue to be raised about whether adequate understanding and
controls for cell quality (potency), cell dose, and cell delivery are considered for most of the
studies. We hypothesize that successful clinical results require not only the correct cell
therapeutic but a radically different approach to study design and trial execution. Traditional
Clinical Research Organizations (CRO's) which have expertise in conducting pharmaceutical
drug or medical device trials generally lack the skills, experience, infrastructure and resources
required to successfully complete cellular therapy (CT) clinical trials. Using the house design
metaphor - the sponsor may properly supply the base of a successful treatment including cell
formulation, dose and administration, but are the pillars of the CRO robust enough for the cell
manufacturing, cell shipping/logistics, physician training and management, and patient
enrollment/retention?
A successful cellular trial should be conducted by an integrated clinical research and
manufacturing organization and team. Such an organization is rare, and the approach is cost
prohibitive for most small biotech companies in the G-8 countries let alone elsewhere in the
world. An option is conducting CT trials outside the US and Europe in order to reduce the cost however, the prospective sponsor faces various challenges which include (1) understanding the
local/national CT trial Regulations; (2) finding an organization sufficiently equipped with local
cellular production suites/facilities compliant with international Good Manufacturing Practices
(GMP) and Good Tissue Practices (GTP); (3) proper control and security for intellectual
property; (4) sufficient collaboration and vetting of health-care providers having superlative
clinical credentials, (5) world-class in-patient services.
As with small molecules and biologicals, India continues to rise to the top of the list for locations
meeting high quality healthcare guidelines in a cost effective study execution model. However,
in the case of Indian cell therapy trials - partnering with an organization competent in both the
local Indian bureaucracy and international regulatory and oversight groups such as the U.S.
Food and Drug Administration and European Medicines Agency is essential. The question for
the biotech industry (and academia) is how to find a cost sensitive option that successfully
integrates the five essentials mentioned above?
Solution:
Driven by its population, low healthcare costs, physician training and English skills, and vast
numbers of cardiovascular, orthopedic and diabetic patients, India is undeniably the leading
destination for CT trials. Multi-national organizations considering cost-effective clinical trials in
India should select a Cellular Clinical Trial Services (CCTS) partner with access or partnerships
in Joint Commission International (JCI) accredited hospitals, having world-class GMP
laboratory and GLP analytical facilities, having state-of-the art documentation systems and a
technical, medical and administrative team competent in ensuring all data is secure and
compliant to international standards including procedures for safeguarding intellectual property.
Cellular Clinical Trial Services (CCTS)
Study Type
Study Update
Sponsor
CRO
GMP
Facilities
JCI Hospital
Medical Device
Indication: CLI
Thermogenesis
Corp (USA)
TotipotentRX
(New Delhi)
Yes / Licensed /
US FDA Reg.
Yes
(Fortis
Healthcare)
Autologous
Therapy: AMI
TotipotentRX
(USA)
TotipotentRX
(New Delhi)
Yes / Licensed /
US FDA Reg
Yes (Fortis
Healthcare &
Care Hospital)
Study Underway
Autologous
Therapy: SCI
Max Healthcare
TotipotentRX
(New Delhi)
Yes / Licensed /
US FDA Reg
Yes (Max
Healthcare)
Study Underway
Autologous
Therapy: Ortho
MK Alliance (USA)
TotipotentRX
(USA & India)
Yes / Licnesed /
US FDA Reg
Yes (Fortis
Healthcare)
Study Completed
Published May 2012
Autologous
Therapy: Neuro
Apollo Heathcare
TotipotentRX
(New Delhi)
Yes / Licnesed /
US FDA Reg
Yes (Apollo
Medicity)
Study Under
Submission
Harvest
Technologies (USA)
LifeCell
International
(Chennai)
Yes / Licensed
No
Stempeutics
Stempeutics
Yes / No
Registraion
Details Avail.
No
Medical Device
Indication: CLI
Allogenic Therapy:
CLI
Study underway
Open Enrollment
Closed Enrollment
CE CLI Approval
Granted 11/2012
Closed
Table 1: Internationally Sponsored Cell Therapy Clinical Trials in India
At present, sixteen cell therapy trials are underway or concluding in India as reported by the
Indian Council of Medical Research. Seven of these trials have international sponsors and are
designed with the rigor of achieving advanced IND status in the G-8.
(100) Submission
ID#OL00002
GENERATION OF RECOMBINANT HUMAN GROWTH FACTORS FOR TISSUE ENGINEERING AND
REGENERATIVE MEDICINE.
Carreira ACO, Belchior GG, Halcsik E, Lojudice FH, Astorino-Filho R, Degaki TL, Sá PM, Spindola-Junior
AS, Granjeiro JM, Sogayar MC
Chemistry Institute, Biochemistry Department, NUCEL – Cell and Molecular Therapy Center, University of
São Paulo, 05508-000 SP, Brazil
Tissue engineering is a promising approach for both soft tissue repair and bone healing. Wound
repair is a complex process involving three different phases, namely: blood clotting and inflammation,
proliferation (new tissue formation) and tissue remodeling. By manipulating the growth factors
composition, it is possible to modify and accelerate this process. During inflammation, neutrophils and
macrophages are recruited to produce two essential growth factors, namely: TGF-β1 and G-CSF. Other
growth factors, such as PDGF and TGF-β1 play critical roles in recruiting fibroblasts during the
proliferative phase and VEGF is essential for angiogenesis. During tissue remodeling, epidermal
proliferation is mediated by GM-CSF and TGF-β3. Neovascularization and granulation tissue formation is
promoted by GM-CSF. TGF-β1 has a direct involvement in cutaneous scarring, while TGF-β3
antagonizes this effect, preventing excessive scarring. Bone remodeling, induced by bone morphogenetic
proteins (BMPs), involves inflammation, vascularization and matrix deposition. BMPs 2 and 7 are crucial
for human bone formation in fracture repair and in non-union spinal fractures.
We set out to produce several recombinant human peptide growth factors involved in the wound
healing and bone repair processes, namely: G-CSF, GM-CSF, PDGF-B, FGF, TGF-β1, TGF-β3,
VEGF121, VEGF165, VEGF-C, BMPs 2 and 7. In order to ensure proper protein glycosylation and correct
conformational folding and to prevent undesirable immunogenicity, we elected two mammalian cell
expression systems. Our platform includes: to amplify the human cDNAs from a Human Full-Length
cDNA Bank; to subclone the cDNA into the pGEM®-T-Easy vector for validation by DNA sequencing; to
subclone the cDNA into an adequate mammalian expression vector to yield the desired human
recombinant proteins. Mammalian cell lineages were transfected with PDGF-BB, CSFs, VEGFs, BMPs 2
and 7 constructs for selection of over-expressing cellular clones. The constructs were successfully
expressed, as confirmed by qRT-PCR and Western blotting, displaying high biological activity in vitro and
some of them also in vivo. This initiative to produce recombinant growth factors (biopharmaceuticals) in
mammalian cells is likely to be useful not only to better understand the wound healing and bone repair
processes, but, also, for development of future therapeutic approaches and tissue engineering protocols
for regenerative medicine.
Support: FAPESP, CNPq, CAPES, FINEP, BNDES, MS-DECIT, MCTI.
Keywords: wound healing, tissue bioengineering, inflammation, recombinant peptide growth factors,
biopharmaceuticals, mammalian expression systems.
(102) Submission
ID#OL00003
EMBRYONIC STEM CELLS (ESCs) DIFFERENTIATION INTO INSULIN-PRODUCING β-LIKE
CELLS: ROLE OF PURKINJE CELL PROTEIN 4 (PCP4).
Kossugue, PM; Grazioli, G; Lojudice, FH; Sogayar, MC.
New cellular sources for Diabetes mellitus type 1 treatment have been investigated, the most
promising of which seems to be the insulin producing cells (InsPCs) obtained upon stem cells
differentiation. Some reports show that mESCs are able to form islet-like structures, however,
their insulin production is insufficient to induce normoglycemia in diabetic mice.
In order to obtain an adequate protocol, we subjected mESCs to a set of endoderm
differentiation inducers, followed by β-cell differentiation factors. We also investigated the role of
the PCP4 gene in the process of differentiation of ESCs into InsPCs.
Early on during mESCs differentiation, we detected the expression of INS1, INS2, NGN3, ISL1,
GLUT2, NES and ARX genes. During the differentiation process, INS2 expression increases,
NGN3 levels are higher when progenitor cells proliferation is higher, and ISL1 expression
follows the pattern observed during embryogenesis. At the end of the differentiation process,
the islet-like clusters positively stained for the insulin-specific dithizone and expressed INS2,
NGN3, ISL1, GLUT2, NES and ARX genes. These cell clusters were microencapsulated in
Biodritin® (alginate + chondroitin sulphate) microcapsules, and then transplanted
intraperitoneally into diabetic mice. Although the levels of insulin production were insufficient to
achieve normoglycemia, mice which received InsPCs displayed improved conditions, when
compared to the control group, as judged by a better glycemic control, body weight gain and
healthy fur appearance, in the absence of apathy.
We also investigated the functional role of the PCP4 gene, whose expression increases during
ESCs differentiation into InsPCs, being 3,700 more expressed at the end of the process. To this
end, lentiviral particles containing PCP4 RNAi sequences were produced and transduced into
differentiating ESCs. The results show that PCP4 knock-down of mESCs subjected to
differentiation resulted in upregulation of genes related to immature cells, such as INS1, NGN3,
PDX1, and downregulation of the GLUT2 gene, which is related to mature cells.
In conclusion, we developed a new protocol for ESCs differentiation into InsPCs, based on
some steps of pancreatic organogenesis. This protocol allows mESCs to differentiate into
InsPCs clusters, which induce improved glycemic control in diabetic mice. In addition, we
identified PCP4 as a new gene expressed by β cells, which seems to be related to β cell
maturation.
Support:
FAPESP,
CNPq,
BNDES,
MS-DECIT,
MCTI.
(103) Submission
ID#OL00004
Pancreatic stem cells reside in niches of the adult human biliary tree, whereas committed
progenitors are located in the pancreas.
Giacomo Lanzoni1,2, Yunfang Wang1,3, Guido Carpino4, Cai-Bin Cui3, Eliane Wauthier3,
Domenico Alvaro5, Eugenio Gaudio6, David Gerber7, Mark E. Furth8, Juan Dominguez-Bendala1,
Lola M. Reid3,9, Luca Inverardi1,9
1
Co-primary authors
2
Diabetes Research Institute, University of Miami Miller School of Medicine; 1450 NW 10th
Avenue, Miami FL 33136, USA
3
Department of Cell and Molecular Physiology and Program in Molecular Biology and
Biotechnology, University of North Carolina School of Medicine; Glaxo Research Building, Rm.
34, Chapel Hill NC 27599, USA
4
Department of Health Sciences, University of Rome “ForoItalico”; Rome, Italy
5
Department of Scienze e Biotecnologie Medico-Chirurgiche, Fondazione Eleonora Lorillard
Spencer Cenci, Universita` di Roma “Sapienza”; Rome, Italy
6
Department of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences,
Universita` di Roma “Sapienza”; Rome, Italy
7
Department of Surgery, University of North Carolina School of Medicine; 4025 Burnett
Womack Building Chapel Hill, NC 27599, USA
8
Comprehensive Cancer Center/ Wake Forest Baptist Medical Center/ Winston Salem, NC
27510, USA
9
Co-senior authors
The quest for therapies than can normalize blood glucose control and provide independence
from exogenous insulin could impact the life of millions of patients with diabetes. Given the
scarcity of human islets, we need to identify an alternative source of transplantable insulinproducing cells. The existence of adult human pancreatic stem cells is actively debated. We
screened the human pancreas and its connections to the biliary tree in search of anatomical
niches where markers of stem and progenitor cells could be identified. We cultured site-specific
cell isolates under conditions designed for stem and progenitor cells. We then assessed the
differentiation potential of the isolated cells in vitro and in vivo. We identified stem cell signatures
in peribiliary glands (PBGs) of the biliary tree and progenitor cell signatures in pancreatic duct
glands (PDGs). Stem cell populations were found in PBGs and committed progenitors in PDGs,
as assessed by the expression of markers of pluripotency (NANOG, OCT4, SOX2, SALL4),
proliferation (Ki67), early hepato-pancreatic commitment (SOX9, SOX17, PDX1, LGR5),
pancreatic endocrine commitment and maturation (NGN3, INSULIN). Stem and progenitor cell
cultures were selected using serum-free Kubota's Medium (KM), then differentiated in a serumfree hormonally defined medium (HDM-P) combined with extracellular matrix components
tailored for islet differentiation. Precursors matured into glucose-regulatable, insulin-producing
cells both in culture and after transplantation in vivo. Bile ducts and the associated PBGs,
pancreatic ducts and their PDGs comprise a ramified network of cells organized in maturational
lineages from stem cell populations transitioning to committed progenitors and to mature cells.
Proximal-to distal lineages start in PBGs near the duodenum with cells expressing markers of
pluripotency, proliferation and early hepato-pancreatic commitment. The lineages progress to
committed progenitors in PDGs with loss of pluripotency, hepatic and proliferation markers and
increased expression of pancreatic endocrine maturational markers. Adult biliary tree stem cells
have the potential to differentiate into pancreatic committed progenitors and subsequently into
functional insulin producing cells.

2012 Accepted Poster Abstracts

Transcription

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