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Extracellular matrix free microcarrier cultures of human pluripotent stem cells induced by inhibition of rock-myosin II signaling

      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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