An optimized small molecule inhibitor cocktail supports long-term maintenance of human embryonic stem cells.

A major challenge in stem cell-mediated regenerative medicine is the development of defined culture systems for the maintenance of clinical-grade human embryonic stem (hES) cells. Here, we identify, using a feedback system control scheme, a unique combination of three small molecule inhibitors that enables the maintenance of hES cells on a fibronectin-coated surface through single cell passaging. After 20 passages, the undifferentiated state of the hES cells was confirmed by OCT4, SSEA4 and NANOG expressions, whereas their pluripotent potential and genetic integrity were demonstrated by teratoma formation and normal karyotype, respectively. Our study attests to the power of the feedback system control scheme to quickly pinpoint optimal conditions for desired biological activities, and provides a chemically defined, scalable and single cell passaging culture system for hES cells.