Figure 3.
Synthetic factors increased the efficiency of human induced pluripotent stem cell generation. (A) Numbers of AP-positive colonies with iPSC-like morphology obtained from 1 × 105 HFF cells transduced with lentiviral constructs for three or four reprogramming factors, as indicated. Error bars, s.d. (n=4). (B) Representative images of human XYKZ primary colony (in situ) and derived iPSC line colonies (P4) using phase contrast or AP staining as before. Scale bar, 200 μm. (C) Immunostaining for hiPSC line XYKZ 6 using antibodies against OCT4, NANOG, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. Scale bar, 200 μm. (D) RT–PCR assays for the endogenous pluripotency markers indicated at the left in different hiPSC lines compared with HFFs or ESCs. Representative data for two independent experiments are shown. (E) Immunocytochemistry of differentiated human iPSCs with antibodies against markers for the three germ layers (FOXA2 and SOX17 for endoderm; SMA and BRACHYURY for mesoderm; and GFAP and β-TUBULIN for ectoderm). Scale bar, 100 μm. Nuclei were stained with DAPI (blue). (F) Hematoxylin and eosin-stained sections from teratomas derived from human iPSCs 8 weeks after transplantation into immunodeficient mice. Representative images show tissues from all three germ layers: gland (endoderm), cartilage (mesoderm) and neural tissue (ectoderm). Teratomas were obtained from the two iPSC lines (3 and 6) examined. Scale bar, 100 μm. AP, alkaline phosphatase; ESC, embryonic stem cell; HFF, human foreskin fibroblast; hiPSC, human induced pluripotent stem cell; iPSC, induced pluripotent stem cell.