Large quantities of human pluripotent stem cells (hPSC) needed for therapeutic applications
can be obtained in scalable suspended microcarrier cultures. However, these microcarriers
have to coated with animal or human extracellular matrix (ECM) proteins which can
present safety risks, and/or are very expensive for large scale use. This study demonstrates
that human embryonic stem cells (HES-3, H7) and induced pluripotent stem cell (IMR90)
can be propagated on non-coated positively charged cellulose microcarriers in serum
free medium containing ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin.
Dephosphorylation of myosin phosphotase 1 (MYPT1) and myosin light chain 2 (MLC2)
were observed in the presence of these two inhibitors suggesting that reduced myosin
contractility is responsible for hPSC survival and growth on ECM-coating free microcarriers.
Cells were propagated on the non-coated microcarriers for at least 15 passages while
maintaining pluripotency and karyotype stability. Scalability of this platform was
demonstrated in 100 ml spinner flask resulting in cell yields of 2.3 × 106 cells/ml
(HES-3) after 5 days of growth. The capability of these cells to differentiate into
the three primary lineages was demonstrated in in-vitro embryoid bodies and in-vivo
teratoma formation studies. Moreover, directed differentiation to PSA-NCAM+ neural
progenitor cells was demonstrated, high cell yields (9.1 ± 1.2 × 106 cells/ml) and
expression levels (91 ± 1.1% cells expressing polysialylated neuronal cell adhesion
molecule (PSA-NCAM)) were obtained. This defined serum- and coating- free scalable
microcarrier culturing system can serve as a safe and less expensive method for generating
large amounts of human pluripotent stem cells for cell therapies.
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© 2013 Published by Elsevier Inc.