Figure 3: Pcgf6 Enhances Induced Reprogramming Efficiency.

(A) Reprogramming efficiency of Oct4-EGFP MEFs transduced with OSKM and either Pcgf6 or DsRed (control) retroviruses. EGFP+ colonies were counted 2 weeks post-transduction, and the results are expressed as colony number relative to control DsRed-miPSCs. Results are the mean ± SEM of n ≥ 5. *p < 0.05 by Student’s t-test. (B) Reprogramming efficiency of human BJ fibroblasts transduced with OSKM and either Pcgf6 or DsRed (control) retroviruses. AP+ colonies were counted 3–4 weeks post-transduction, and the results are expressed as for (A). Results are the mean ± SEM of n ≥ 5. *****p < 0.000005 by Student’s t-test. (C) Reprogramming efficiency of Oct4-EGFP MEFs depleted of Pcgf6. Cells were treated with non-targeting (siNT) or Pcgf6-specific siRNA for 3–5 h and then transduced with OSKM retroviruses. EGFP+ colonies were counted 2 weeks later, and the results are expressed as for (A). Results are the mean ± SEM of . ****p < 0.00005 by Student’s t-test. (D–F) Images of Pcgf6-hiPSCs derived as in (B). Cells were cultured on feeders for at least 4 passages before imaging of: cell morphology (D), AP staining (E), and Tra-1-81, SSEA4, Tra-1-60, and Nanog immunofluorescence staining (F). Scale bars = 200 μm (D,E) or 50 μm (F). (G) Histological staining of mesodermal, endodermal, and ectodermal tissues in teratomas formed from Pcgf6-hiPSCs. Cells were injected under the kidney capsule of SCID mice, and teratomas were removed and analyzed ~10 weeks later. Scale bar = 50 μm. Images were taken using either phase contrast microscopy or fluorescent microscopy. (H) Normal karyotype of Pcgf6-hiPSCs. Cells were cultured for ~2 months on feeder cells before karyotype analysis. Chromosomes in the metaphase of 20 cells were counted, and 4 cells were examined for G-band staining with band resolution at 450–525. All cells contained normal karyotypes with 46 chromosomes including XY, and no clonal abnormalities were detected.