UC San Diego

The differentiation potential of human pluripotent stem cells (hPSCs) promises to treat degenerative diseases with cell replacement therapies. Current culture media and substrates are not only expensive, but are undefined for clinical use because of the various known and unknown animal components that are required to promote proliferation and differentiation. Chemically defined materials are ideal substitutes for hPSC culture. This study focused on the use of PMVE-alt-MA, a synthetic polymer that was previously identified by arrayed screening technology for ex vivo hPSCs maintenance. Synthetic hydrogels and microcarriers endowed with PMVE- alt-MA moieties were used to evaluate hPSC expansion, adhesion, proliferation, colony formation and maintenance. Flow cytometry and realtime PCR were used to characterize the expression of pluripotency markers in both hydrogel and microcarrier cultures. Protein adsorption to PMVE-alt- MA was found necessary for initial cell adhesion. Enzymatic dissociation with Accutase TM was optimized to passage the embryonic stem cell line Hues9 on 9.7% semi- IPN hydrogel for 8 passages and the result was comparable to Matrigel culture. However, polymer coatings on polyacrylamide hydrogel and microcarriers were found to be insufficient for stem cell self-renewal and attachment respectively. Embryoid bodies (EBs) with uniform size and shape were observed in microcarrier suspension but no difference was found in the differentiation pattern when compared to normal suspension EB formation. Our experimental results demonstrated the potential to use PMVE-alt-MA in hPSC expansion, as expected from the arrayed screening technology