Figure 8: JAK/STAT3 induces naive pluripotency despite a culture environment that instructs and maintains the primed cell state. | Nature Communications

Figure 8: JAK/STAT3 induces naive pluripotency despite a culture environment that instructs and maintains the primed cell state.

From: JAK/STAT3 signalling is sufficient and dominant over antagonistic cues for the establishment of naive pluripotency

Figure 8

(a–d) Reprogramming of GY118F ΔPE Oct4-GFP EpiSCs to naïve pluripotency in N2B27 plus Activin plus FGF plus G-CSF. (a) Phase and fluorescent images showing the emergence of ΔPE Oct4-GFP-positive colonies. White bars correspond to 161.4 μm. (b) Flow cytometry analysis of GY118F EpiSCs at day 0 and 12 days after the addition of G-CSF. Parental ΔPE EpiSCs were used as a negative control. The percentage of ΔPE Oct4-GFP-positive cells is indicated above the gate. (c) GY118F ΔPE Oct4-GFP-positive cells isolated and maintained in N2B27 plus Fgf2 plus Activin plus G-CSF. White bars correspond to 141.54 μm. (d) qRT–PCR analysis for naïve pluripotency and EpiSC marker genes. Gene expression and associated error bars representing s.d were normalized to EpiSCs. (e) Phase and fluorescent images of GY118F iPS cells derived and maintained in N2B27 with Activin plus Fgf plus G-CSF cultured with or without inhibitors for Activin (Activinri) and Fgf (Fgfri) signalling or cultured in N2B27 with 2i or G-CSF. White bars correspond to 143.9 μm. Flow cytometry analysis (right panel) confirming continued expression of the ΔPE Oct4-GFP reporter transgene in all culture conditions. (f) qRT–PCR analysis for Lefty2 and Dusp4 expression on addition and subsequent withdrawal of A8301 (Activinri) and PD173074 (Fgfri) inhibitors or withdrawal and readdition of Activin and Fgf from GY118F iPS cells. Error bars indicate ± 1 s.d (n=3). (g) FISH for Xist RNA on GY118F ΔPE Oct4-GFP iPS cells derived and maintained in Activin plus FGF plus G-CSF. ΔPE Oct4-GFP EpiSCs were used as a control for presence of an inactive X chromosome. Male ES cells were used as a positive control for presence of pinpoint signal. White dashed lines outline examples of individual cell nuclei. White bars correspond to 8.8 μm. (h) Contribution of GY118F ΔPE Oct4-GFP iPS cells derived and maintained in N2B27 plus Activin plus FGF plus G-CSF to chimaeras as reflected by agouti coat colour.

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