Figure 5.

Evaluation of Alternative Culture Conditions for Naive Human Pluripotency

(A) Table comparing the components of four recent protocols for capturing naive-like human ESCs with 5i/L/A medium. Note that the protocol for naive conversion from Ware et al. (2014) involves preculture with the HDAC inhibitors butyrate and suberoylanilide hydroxamic acid.

(B) Phase and fluorescence images and flow cytometric analyses showing the response of OCT4-ΔPE-GFP− primed cells to recently reported protocols for naive human pluripotency (see Figure 5A) and 5i/L/A. 40× magnification.

(C) Quantification of the proportion of GFP+ cells in WIBR3 OCT4-GFP and OCT4-ΔPE-GFP human ESCs upon removal of DOX-inducible KLF2 and NANOG expression in primed human ES medium (hESM) and four alternative conditions for naive human pluripotency.

(D) Flow cytometric analysis of the proportion of OCT4-ΔPE-GFP+ cells in 5i/L/A and the JNK inhibitor SP600125 (6i/L/A) in serum-free N2B27 basal medium versus 20% KSR basal medium.

(E) Quantitative gene expression analysis for OCT4, SOX2, KLF2, and NANOG in human ESCs cultured in 6i/L/A and supplemented with 1%, 5%, 7.5%, or 10% FBS or KSR. Error bars indicate ± 1 SD of technical replicates.

(F) Phase and fluorescence images of induction of OCT4-ΔPE-GFP activity from the primed state in 6i/L/A, and 6i/L/A supplemented with 1%, 5%, 7.5%, or 10% KSR. 40× magnification.