Figure 4.

Direct Conversion of Conventional Human ESCs to Naive Pluripotency in 5i/L

(A) Strategy for assessing direct conversion of primed human ESCs into OCT4-ΔPE-GFP+ cells under optimized chemical conditions.

(B) Phase and fluorescence images of emerging naive colony and expanded cells from WIBR3 OCT4-ΔPE-GFP human ESCs treated with 5i/L for 10 days. Left and right panels are 40× magnification, and middle panel is 100×.

(C) Phase and fluorescence images and flow cytometric analyses of the proportion of GFP+ cells during conversion experiments in 5i/L supplemented with FGF and/or Activin A (FA). 40× magnification.

(D) Phase images of wild-type naive WIBR2 human ESCs converted in 5i/L supplemented with FGF and/or Activin A (FA). 40× magnification.

(E) Phase image of a primary human ESC line derived in 5i/L/FA from an explanted human blastocyst. Cell line is designated as Whitehead Institute Naive Human ESC line 1 (WIN1). 100× magnification.

(F) Top, green: strategy for generating secondary naive human iPSCs from secondary derived fibroblasts (Hockemeyer et al., 2008) harboring inducible OCT4, SOX2 and KLF4 transgenes and OCT4-ΔPE-GFP allele. Top, blue: cell culture media conditions used at representative stages of reprogramming experiment. Bottom: phase and fluorescence images of primary primed iPSCs and secondary derived fibroblasts and the reactivation of GFP in naive reprogrammed secondary iPSCs. 40× magnification.

(G) Flow cytometric analysis of the proportion of OCT4-ΔPE-GFP+ cells three passages after withdrawal of individual inhibitors and growth factors.

(H) Quantitative gene expression analysis for NANOG and KLF4 three passages after withdrawal of individual inhibitors and growth factors from 5i/L/FA control cells. Error bars indicate ± 1 SD of technical replicates.