FIG. 5.

Generation of iPS cells from mouse wild-type CECs with two reprogramming factors. (A) Brightfield and AP staining of representative 2F iPS colony. Scale bar, 50 μm. (B) Genotyping of 2F iPS cells. Genomic PCR showed only Oct4 and Klf4 transgenes in 2F iPS cells as expected. (C) RT-PCR of pluripotency-associated genes and GAPDH. 2F iPS cells express all tested endogenous pluripotency genes, and parental wild-type CECs express Sox2, Klf4 and c-Myc. (D) Immunostaining of pluripotency markers Oct4, Sox2, Nanog, and SSEA-1 in 2F iPS cells. Scale bar, 100 μm. (E) Representative 2F iPS cell line cultured in N2B27+ 2i/LIF medium on gelatin-coated plate without feeder cells. Scale bar, 50 μm. (F) RT-PCR of typical lineage markers after differentiation of EBs derived from 2F iPS clones. (G) Teratoma formation. Hematoxylin and eosin staining of teratoma sections derived from 2F iPS clones showed all three embryonic germ layers. Immunostaining of teratoma sections showed tubulin-β III-positive neural epithelium, α-smooth muscle actin-positive muscle, and E-cadherin-positive endodermal cells. Scale bar, 100 μm. Color images available online at www.liebertpub.com/scd