FIG. 3.

TW3R human feeders support long-term culture of undifferentiated human ESCs. H9 hESCs were cultured on TW3R or MEF for 12 consecutive passages. (A) Flow cytometry of TRA-1-60 staining showed that both feeders maintained similarly high percentages of undifferentiated hESCs at each passage. (B) After 12 passages on TW3R, the hESCs maintained undifferentiated morphology and expression of pluripotency markers such as SSEA4, TRA-1-60, OCT4, NANOG, and alkaline phosphatase (AP). (C) These cells were also capable of forming EBs with cell types expressing ectoderm (beta-3-tubulin), mesoderm (smooth muscle actin, SMA), or endoderm (alpha-fetoprotein, AFP) markers. (D) To further confirm pluripotency, these hESCs were injected into immuno-deficient mice for teratoma formation. H&E staining showed that tissue types representing all 3 germ layers were present, indicating the pluripotency of the cells. (E) After 12 passages on TW3R, the hESCs maintained a normal female karyotype (46,XX). Scale bar: 100 μm. EB, embryoid body; H&E, hematoxylin and eosin. Color images available online at www.liebertonline.com/scd