Figure 1: Increased activation of JAK/STAT3 overcomes the pre-iPS cell reprogramming block.

(a) phospho (p) -STAT3 western blot and Socs3 quantitative real-time PCR (qRT–PCR) demonstrating activation of GY118F by G-CSF in pre-iPS cells. Pre-iPS cells harbouring an empty vector were used as a control. Error bars indicate ±1 s.d. (n=3). (b–g) Reprogramming of female GY118F pre-iPS cells in serum and LIF culture conditions and subsequent analysis. (b) Appearance of GFP-positive colonies indicates activation of the Oct4 reporter transgene. White bars correspond to 178.8 μm. (c) Flow cytometry analysis of GY118F pre-iPS cells at day 7 and day 13 after addition of G-CSF. The percentage of Oct4-GFP-positive cells is indicated above the gate. Pre-iPS cells harbouring an empty vector were used as a control. 5-Azacytidine was used as a positive control for the reprogramming of pre-iPS cells. (d) Phase and fluorescent images of isolated GY118F Oct4-GFP-positive cells in serum plus LIF plus G-CSF. The white bar corresponds to 161 μm. (e) qRT–PCR analysis for pluripotency marker genes and retroviral (r) transgenes. iPS cells cultured in 2i plus LIF medium were used as a control. Gene expression and associated error bars representing s.d. (n=3) were normalized to the sample with highest expression. (f) Immunofluorescence for trimethyl(me3)H3K27 of GY118F iPS cells. Parental pre-iPS cells and female ES cells were used as controls. White bars correspond to 14.5 μm. (g) Fluorescent in situ hybridizations (FISH) for Xist RNA on GY118F iPS cells. Parental pre-iPS cells were used as a control for the presence of the inactive X chromosome. Male ES cells were used as a positive control for Xist pinpoint signal. White dashed lines outline examples of individual cell nuclei. White bars correspond to 8.8 μm. (h) Western blot analysis for phospho-ERK of GY118F or control vector pre-iPS cells stimulated with G-CSF for 24 h. Tubulin and total ERK were used as loading controls.