Figure 4: The defined culture system is suitable for de novo derivation of stable biologics-free hESCs. (A) The embryos were thawed and allowed to develop through 8 cell (day 3) and morula (day 4) stages to blastocyst stage (day5). (B) The blastocysts hatched from the zona pellucida either spontaneously or with acid Tyrode’s solution treatment were placed in the defined culture to allow the ICM to expand while the trophoblast degenerated. (C) The ICM was further dissected and placed in the defined culture system for expansion. The cells were maintained in this defined system for >50 passages during which time hESCs in the colonies retained their undifferentiated compact morphology and expressed undifferentiated markers including alkaline phosphatase (red), Oct-4 (green), SSEA-4 (green), Tra-1-60 (red), Tra-1-81 (red). (D) The de novo derived biologicsfree representative hESCs retained pluripotent as suggested by forming teratomas when transplanted into SCID mice, and (E) maintained a stably normal diploid karyotype for >50 passages.