Figure 4.

Nanog Is Sufficient to Reprogram Epiblast-Derived Stem Cells to Naive Pluripotency

(A) Strategy for examining the sufficiency of Nanog in reprogramming of epiblast-derived stem cells (EpiSCs). A piggyBac (PB) Nanog-dsRed transgene was used to generate constitutive Nanog expressing EpiSCs, which also contain an Oct4-GFP reporter transgene. After selection in EpiSC culture conditions, PB-Nanog-dsRed EpiSCs were cultured in serum-free medium with LIF or serum-free medium alone.

(B) Phase and Oct4-GFP images of emerging Epi-iPS colonies that were resistant to puromycin selection for the Oct4 reporter transgene.

(C) qRT-PCR analysis of PB-Nanog EpiSCs in Fgf2 and Activin (F+A) and passage 1 PB-Nanog transfectants in serum-free medium with LIF. Error bars indicate the range of fold change relative to the sample with highest expression.

(D) qRT-PCR analysis of PB-Nanog Epi-iPS cells derived in serum-free medium alone. Error bars indicate the range of fold change relative to wild-type EpiSCs.

(E) Immunostaining for me3H3K27 of PB-Nanog EpiSCs cultured in Fgf2 and Activin, and PB-Nanog Epi-iPS cells derived in serum-free medium with LIF or serum-free medium alone.

(F) Time course flow cytometry analysis of Oct4-GFP reporter activity after transferring wild-type (WT) EpiSCs and PB-Nanog Epi-iPS cells that were derived in serum-free medium or serum-free medium with LIF to 2i/LIF.

(G) Fluorescence images showing Oct4-GFP reporter activity in blastocysts 52 hr after morula aggregation of PB-Nanog Epi-iPS cells derived in serum-free medium with LIF and serum-free medium alone.

(H) Western blot analysis for p-Erk1/2 and total Erk1/2 protein expression in PB-Nanog-EpiSCs cultured in Fgf2 and Activin or for 1 day in 2i/LIF, LIF or serum-free medium alone.