Figure 2: Sufficient JAK/STAT3 activation eliminates the need for pluripotency culture requisites in somatic cell reprogramming.

(a) Female aNS cells containing the GY118F transgene showing activation of direct STAT3 target Socs3 on stimulation with G-CSF for 1 h. Error bars indicate ±1 s.d. (n=3). (b,c) Reprogramming of aNS cells. The cells were infected with retroviral Oct4, Klf4 and c-Myc and subsequently stimulated with G-CSF in N2B27 medium only. (b) GFP expression indicates activation of the Oct4 reporter transgene. N2B27 plus 2i plus LIF medium was used as a positive control for reprogramming. White bars correspond to 195 μm. (c) Phase and fluorescent images depicting the derived GY118F iPS cells maintained in N2B27 plus G-CSF. The white bar corresponds to 182.6 μm. (d–i) Analyses of the GY118F iPS cells derived and maintained in N2B27 plus G-CSF. (d) qRT–PCR analysis for pluripotency marker genes and retroviral (r) transgenes. iPS cells cultured in 2i plus LIF were used as a control. Endo, endogenous. Gene expression and associated error bars representing s.d. (n=3) were normalized to the sample with highest expression. (e) Immunostaining of GY118F iPS cells for me3H3K27. aNS cells show a me3H3K27 focus, unlike iPS or control female ES cells. White bars correspond to 6.4 μm. (f) FISH for Xist RNA on GY118F iPS cells. aNS cells and male ES cells were used as controls. White dashed lines outline examples of individual cell nuclei. White bars correspond to 8.8 μm. (g) Phase and fluorescence images of GY118F iPS cells after withdrawal of G-CSF from N2B27. White bars correspond to 184 μm. (h) Flow cytometry analysis for Oct4-GFP reporter expression after G-CSF withdrawal from GY118F iPS cell culture. Percentage is indicated above the gate. (i) Agouti coat colour reflects contribution of GY118F iPS cells on blastocyst injection.