Generation of iPSCs from fibroblasts by Oct4 and small molecules. (A) GFP+/iPS-like colony numbers induced from OG-MEFs transduced with Oct4/Sox2/Klf4. VPA and CHIR99021 greatly improved the efficiency of GFP+/iPS-like colony generation (approximately 30-fold). Approximately 30 iPS colonies were generated from 1 × 10⁴ MEFs at day 15 after infection. Similar results were obtained in three independent experiments. “V” stands for VPA. “VC” stands for the combination of VPA and CHIR99021. (B) GFP+/iPS-like colony numbers induced from OG-MEFs transduced with Oct4/Klf4, in combination with VPA, CHIR99021 and 616452 (VC6). Approximately 5-20 GFP+/iPS-like colonies were generated from 5 × 10⁴ MEFs at day 15 after infection. Similar results were obtained in three independent experiments. (C) GFP+/iPS-like colony numbers induced from OG-MEFs transduced with Oct4/Sox2/Klf4. Tranylcypromine significantly promoted iPSC generation (approximately 20-fold), with an efficiency similar to that of VPA. When VPA and tranylcypromine were added together (VT), iPSC generation efficiency was further increased. The number of iPS colonies generated from 1 × 10⁴ MEFs was counted on day 15 after infection. Similar results were obtained in three independent experiments. “V” stands for VPA. “T” stands for tranylcypromine. (D) A typical GFP+/iPS-like colony generated from MEFs (left) and adult fibroblasts (right) after 30 days of Oct4 and VC6 treatment. (E) Timeline of Oct4-iPSC generation using one single factor Oct4 and small molecule treatment (VC6T: VPA, CHIR99021, 616452 and tranylcypromine). Culture medium containing small molecules was changed every four days. (F) GFP+ colonies appeared 18 days after OG-MEFs were transduced with Oct4 and treated with VC6T. Bars, 500 μm. (G) Genome PCR showed that these Oct4-iPSCs had only the exogenous Oct4 insertion and were free of other exogenous factors (1a,1b,1c: Oct4-iPSC lines from MEFs of ICR×OG genetic background; 2a,2b: Oct4-iPSC lines from MEFs of 129×OG genetic background; 3a,3b: Oct4-iPSC lines from MEFs of C57×OG genetic background; 4f-iPS: iPSCs induced by the original four transcriptional factors, Oct4, Sox2, Klf4 and c-Myc as positive control).

Differentiation potential of Oct4-iPSCs. (A) Teratoma formation of Oct4-iPSCs. The tissues of all three germ layers, such as pigmented retinal epithelium, cartilage, neural tube-like epithelium and gut-like epithelium, were detected in these Oct4-iPSC-derived teratoma sections. Bars, 200 μm. (B) When the Oct4-iPSC aggregated embryos were transplanted into mice, GFP+ cells were detected in the gonad tissues of 17 days post copulation embryos, which suggests that the iPS cell contribution to the germ line. (C) Adult mice with a high degree of chimerism were developed from Oct4-iPSC lines generated from MEFs (left) and adult fibroblasts (rigtht), after blastocyst transplantation. (D) Genome PCR showed that these Oct4-iPSCs contributed to brain, lung, tail, liver, heart, stomach, testis, kidney, spleen and skin in a chimeric mouse. Tail fibroblasts from ICR and 129 mice were used as negative control. Tail fibroblasts from OG2 mice and MEFOG, which have pOct4-GFP alleles in their genomes, were used as positive control.

Oct4 induced iPSCs to express pluripotency markers and resemble ESCs. Oct4-induced iPSCs maintained ESC-like morphology, AP activity and GFP expression in mESC growth media (bars, 500 μm), while expressing pluripotency markers Oct4, Nanog, Utf1, Rex1 and SSEA1, detected by immunofluorescence (bars, 100 μm). (A) RT-PCR analysis showed the pluripotent marker gene expression of these Oct4-iPSCs (1a,1b,1c: three separate Oct4-iPSC lines from MEFs of ICR×OG genetic background; 3a,3b: two separate Oct4-iPSC lines from MEFs of 129×OG genetic background). (B) DNA methylation analysis of several CpG sites in the Nanog and Oct4 promoters, indicating that the demethylation of Nanog and Oct4 promoters in Oct4-iPSCs was similar to that of mESCs. Nanog and Oct4 promoters of MEFs were hypermethylated. (C) Microarray analyses showed a similar global gene expression profile among Oct4-iPSCs, 4F-iPSCs and mESCs (R1), which were quite different from that of OG-MEFs.

The efficiency of iPSC generation with DOX-inducible Oct4 expression and VC6T treatment. (A) iPSC colony numbers generated by different induction time of Oct4 using the tet-on system. Doxycycline (dox) was added at different time periods during the reprogramming process as shown on the x axis. The various shaded bars indicate the days on which colonies appeared in the culture. Similar results were obtained in three independent experiments. (B) iPSC colony numbers generated by different treatment times. The numbers on x axis stand for the days with VC6T treatment from day 0 after transduction. Dox was added during the entire process to induce Oct4 expression. Colony numbers were calculated at days 12, 15, 18, 21 and 24, shown by various shaded bars. Results were obtained in three independent experiments.