The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance

The majority of methodologies for maintaining human embryonic stem cell (hESC) pluripotency require the use of human or animal feeder cell layers, the most common being murine embryonic fibroblasts. In this study, we applied a protocol aimed at maintaining hESCs in culture without exposure to animal cells or proteins. hESCs were encapsulated in 1.1% (w/v) calcium alginate hydrogels and grown in basic maintenance medium for a period of up to 260 days. Investigation of the cell aggregates formed within the hydrogels yielded no evidence of the formation of any of the three germ layers, although the hESCs retained their pluripotency and could differentiate when they were subsequently cultured in a conditioned environment. Immunohistochemistry and RT-PCR showed that the hESC aggregates expressed protein and gene markers characteristic of pluripotency including Oct-4, Nanog, SSEA-4, TRA-1-60 and TRA-1-81. At the ultrastructural level, the cells were arranged in closely packed clusters and showed no cytoplasmic organelles, suggesting an undifferentiated state. These data show that it is possible to maintain hESCs in an undifferentiated state, without passaging or embryoid body formation, and without animal contamination.