LIF dependent mitochondrial activation increases reprogramming efficiency in EpiSC.Figure 6(A) Gene expression analysis of EpiSCs (GOF18) cultured in FGF+Activin (grey bars) and ES cells cultured in 2i+LIF (orange bars) shows a general reduction of both nuclear LIF targets (Socs3, Stat3) and mitochondrial markers (Nd1, Nd4, Nd4l) in EpiSCs. Data were normalized to EpiSCs in F/A and mean and s.e.m. of two technical replicates of two independent experiments (light and dark bars) is shown. (B) Experimental scheme for testing the effect of rotenone on EpiSCs reprogramming. EpiSCs were plated in FGF+Activin (Day0) and cultured for 24hours. Cells were then treated with or without LIF and rotenone at different concentrations as indicated (Day1). Cells were either harvested after 48hours (Day3) for gene expression analysis or left in 2i media and Oct4-GFP positive colonies were scored at Day 6 or 8.(C) Quantification of Oct4-GFP positive iPS colonies at day7 of reprogramming generated from GOF18 EpiSCs subjected to 48hours treatment (at D1-D3) with LIF and two doses of rotenone. Note that rotenone treatment reduces the number of iPS colonies generated. Mean and s.e.m of two technical replicates of three independent experiments is shown. See also Appendix Figure S10B. (D) Left: number of Oct4-GFP positive colonies at day8 of reprogramming upon 48hours treatment with rotenone at day1-2 or at day5-6. Note that rotenone treatment given at day5-6 does not reduce the number of iPS colonies. Mean and s.e.m of two technical replicates is shown. See also Appendix Figure S10C. Right: representative Alkaline Phosphatase (AP) staining of iPSc colonies generated from GOF18 EpiSCs at day7 of reprogramming treated with DMSO (top) or with 48h of rotenone at days 1-2 (middle) or 5-6 (bottom). (E) Gene expression analysis of iPS cells generated from GOF18EpiSCs and cultured in 2i+LIF for at least 2 passages. Note that iPS cells show high expression of naïve pluripotency markers (grey bars on left panel) and low expression of EpiSC markers (green bars on right panel). Data were normalized to the highest value for each gene and mean and s.e.m of three technical replicates is shown. Right panel shows representative bright field and Pou5f1-GFP+images of iPS colonies at day 8 of reprogramming. Scale bar, 50µm. (F) and (G) Gene expression analysis of GOF18 EpiSCs cultured in FGF+Activin or treated for 48hours with 2i+LIF in absence or presence of decreasing concentrations of rotenone.Note that both nuclear (F) and mitochondrial (G) genes expression is induced upon LIF treatment and rotenone did not impair LIF effect. Data were normalized to F/A and mean and s.e.m of three independent experiments is shown. (H) Number of iPS colonies generated from GOF18 EpiSCs transfected transiently with a piggyBac vector containing GFP or Klf4 and treated with LIF for 48hours and rotenone as indicated. Mean and s.e.m. of two technical replicates of a representative experiment is shown. Unpaired t-test: ** P<0.01 *** P<0.001. See also Appendix Figure S10H. (I) Left panel shows the number of GFP positive iPS colonies generated from OEC2 EpiSCs transiently transfected with a piggyBac vector containing Klf4 (1st and 2nd bar), Tfcp2l1 (3rd and 4th bar) or a combination of both (5th and 6th bar) and treated with LIF for 48hours or rotenone as indicated. Note that rotenone abolished the effect of LIF on iPS formation. Each bar shows the cumulative number of colonies from four independent experiments and mean and s.e.m. of two technical replicates for each experiment is shown. Unpaired t-test: * P<0.05. Right panel shows representative Alkaline Phosphatase (AP) staining of iPSc colonies generated from OEC2EpiSCs at day7 of reprogramming transfected with a piggyBac vector containing Klf4 and Tfcp2l1 and treated with 2i (top), 2i+LIF (middle) or with 2i+LIF and rotenone for 48h at days 1-2 (bottom). (J) Model depicting the dual role of Stat3 as an inducer of nuclear Transcription Factors critical for maintenance and induction of Naïve Pluripotency and, at the same time, as an activator of mitochondrial transcription and activity responsible for the high levels of respiration observed in Naïve pluripotent cells.