S74 Poster Abstracts
216
A HUMAN PLATELET LYSATE-BASED CULTURE SUPPLEMENT
FOR THE SUCCESSFUL ISOLATION AND SCALABLE EXPANSION
OF UMBILICAL CORD MATRIX-DERIVED MESENCHYMAL
STEM/STROMAL CELLS
A. Source1, A. Fernandes-Platzgummer1, F. Moreira1, S. Liu2, C. Ku2,
Y. Huang2, W. Milligan2, J.S. Cabral1, C.L. da Silva1
1
Department of Bioengineering and IBB, Institute for Bioengineering and
Biosciences, Lisboa, Portugal, 2R&D Department, AventaCell Biomedical
Co., Ltd., New Taipei City, Taiwan
Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSC)
are considered promising therapeutic agents. However, low cell yields per um-
bilical cord unit and high cell doses required in clinical settings emphasize the
need for ex vivo expansion of UCM MSC.
We recently demonstrated the ability to isolate and ex-vivo expand
UCM MSC under serum-/xeno-free culture conditions using static systems.
Also, we have previously established a xeno-free microcarrier-based system for
the scalable expansion of human MSC from bone marrow and adipose tissue,
combining the use of plastic microcarriers with a commercially available xeno-
free medium formulation. However, the scalable expansion of UCM MSC under
dynamic conditions, using plastic microcarriers combined with different com-
mercially available serum-/xeno-free media, remains challenging. Here, we
demonstrate the ability to use a human platelet lysate (HPL)-based
culture supplement (UltraGROTM, AventaCell) at 5%(v/v) for the successful
isolation and scalable expansion of UCM MSC. MSC-like cells were isolated
from UCM explant cultures (n = 3) after 11 ± 2 days and were shown to
express CD73, CD90 and CD105. After five passages in static culture,
UCM MSC retained their multilineage differentiation potential and
immunophenotype. In addition, UCM MSC expanded using UltraGROTM-
supplemented medium expanded faster compared to UCM MSC
expanded using the previously established protocol (μapp of 1.0 day−1 and 0.54
day−1, respectively). Importantly, UCM MSC were successfully expanded under
dynamic conditions on plastic microcarriers using UltraGROTM-supplemented
medium in spinner flasks. Upon an initial 60% cell adhesion to the beads,
UCM MSC were able to expand by >12 fold after 4–6 days in culture. Upon
dynamic culture, cells maintained their immunophenotype and multilineage
differentiation ability, as well as the capacity to support the ex-vivo expansion
of umbilical cord blood-derived hematopoietic progenitors. To our knowl-
edge, this is the first study reporting the successful expansion of UCM MSC
in a fully scalable xeno-free microcarrier-based system, representing an impor-
tant advance in obtaining safer and clinically meaningful MSC numbers for
Cell Therapy.
217
HYPOXIC CULTURED UMBILICAL CORD DERIVED SUB-
EPITHELIAL CELLS: MOVING TOWARDS AND OPTIMAL CELL
SOURCE AND CULTURE METHOD
A. Patel, D. Atkinson, R. Walker, C. Bartlett, F. Silva
University of Utah, Salt Lake City, Utah, United States
Background: The therapeutic use of umbilical cord derived mesenchymal
stem cells offers a promising cell-based therapeutic for various disorders and
as a result has gained a lot of attention as a potent allogeneic cell source for
regenerative medicine. Traditionally stem cells have been cultured under
normoxia (20% O2), recently it has been demonstrated that culture under
hypoxia (<10% O2) may change the basic biology of the cell. This study
aimed to investigate the effects of hypoxia on the proliferation and differenti-
ation of umbilical cord derived sub-epithelial cells and to determine if this
results in a more potent cell.
Methods: Cells from the sub-epithelial lining of umbilical cord tissue
were isolated and cultured using xeno-free media and cultured under
normoxia (20% O 2 ) and hypoxia (<10% O 2 ). Cells were analyzed to
compare markers, proliferation rates and differentiation potential under both
conditions.
Results: We isolated and expanded cells from the sub-epithelial lining of
umbilical cord tissue. Hypoxia enhanced the derivation and proliferation rate
(P < 0.05) and differentiation potential. Differentiation under hypoxia exhib-
ited greater cardiac-like cells and increased chondrogenesis compared to
normoxia.
Conclusions: These results indicate that the isolation, expansion and
differentiation of umbilical cord derived sub-epithelial cells under xeno-free
and hypoxic condition, results in more robust cell types for cell-based
therapies.
218
MINIMAL CRITERIA OF EXPANDED MESENCHYMAL STEM
CELLS FOR CLINICAL APPLICATION: HYPOTHESIS AND
EXPERIMENTS
P.V. Pham
Lab of Stem Cell Research and Application, University of Science, VNU-
HCM, Ho Chi Minh, Viet Nam
Mesenchymal stem cells (MSCs) were widely used in clinical applications. To
date, nearly 1000 clinical trials were registered in clinicaltrial.gov. In some
initial studies, all non-expanded MSCs were clinically used to treat some
diseases, while recent years expanded MSCs were used. Although expanded
MSCs hold several useful properties such as high purity, more homogenous
cell population, and plentiful source; they also faced to some risks, especially
some changes during in vitro culture. This study aimed to evaluate some
changes during in vitro culture that can be recorded as minimal criteria. The
results showed that MSCs could change in surface marker expression, karyo-
type, oncogene expression, tumor suppression gene expression, telomere
length, in vitro differentiation potential, in vivo tumorigenesis capacity. These
findings suggested some essential assays to evaluate the expanded MSCs
before clinical application.
219
EFFECT OF CO-MEDICATIONS AND ENDOTOXINS ON
MESENCHYMAL STEM CELL CHARACTERISTICS
A.L. Russell, A. Zubair
Human Cell Therapy Laboratory, Mayo Clinic, Jacksonville, Florida, United
States
The impact of immunosuppressive medications and endotoxemia on mesen-
chymal stem cell (MSC) therapy have not been established. We examined the
effects of calcineurin inhibitors cyclosporin (Cyc) & tacrolimus (Tac), cortico-
steroids hydrocortisone (Hyd) & dexamethasone (Dex), rapamycin (Rap),
mycophenolic acid (MPA) and lipopolysaccharide (LPS) endotoxins on bone
marrow MSC secretion of cytokines and growth factors that contribute to MSC
activation, anti-inflammatory and regenerative functions. We used Luminex
bead platform to assess expression of 41 cytokines (Table 1). We showed se-
cretion of cytokines that activate MSC such as IFNγ and TNFα was increased
in response to all compounds tested. However, other cytokines with similar
effect such as IL-1α and IL-17 were largely unaffected. Factors known to play
a role in MSC-induced immunosuppression and cell differentiation such as
IL-10, EGF and TGFα were variably affected. LPS and Dex increased secre-
tion of IL-10, while effects of other medications were concentration dependent.
TGFα secretion was increased by LPS and MPA with variable effects induced
by other drugs. EGF secretion was largely decreased, yet was increased by
MPA and Tac at high concentration. We evaluated changes in secretion of
factors known to induce angiogenesis, neurogenesis, hematopoiesis and epi-
thelial proliferation such as VEGF, IL-6, GM-CSF and G-CSF. LPS, Tac,
and Cyc exposure increased secretion of VEGF-A. Contrarily, corticosteroids
and Rap (except 1 ng/ml) decreased VEGF-A secretion. IL-6 secretion was
increased by LPS, Rap, Tac and Cyc and decreased by MPA and corticoste-
roids. With few exceptions, LPS increased and immunosuppressive drugs
decreased GM-CSF secretion. G-CSF was increased by LPS, but unaffected
by the drugs evaluated. In summary, MSC characteristics are influenced by
immunosuppressive medications and endotoxins. Further studies are neces-
sary to evaluate how these variations might affect the intended therapeutic
function of MSC.