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. 2014 Oct 2;15(4):471–487. doi: 10.1016/j.stem.2014.07.002

Figure 1.

Figure 1

A Reporter System for Naive Human Pluripotency Based on Endogenous OCT4 Distal Enhancer Activity

(A) Proximal enhancer (PE) targeting strategy in human ESCs containing a 2A-GFP sequence in frame with the 3′ UTR of OCT4.

(B) Southern blot analysis confirming disruption of PE in OCT4-2A-GFP ESCs. NdeI-digested genomic DNA was hybridized with 5′ and 3′ external probes. Expected fragment size: WT (wild-type) = 5.6 kb, T (targeted) = 6.4 kb.

(C) Images of OCT4-2A-GFP human ESCs before (left) and after (right) TALEN-mediated deletion of the PE. 40× magnification.

(D) Single-molecule RNA FISH analysis for OCT4 and GFP transcripts in OCT4-2A-GFP human ESCs before and after TALEN-mediated disruption of the PE.

(E) Flow cytometric analysis of the proportion of OCT4-ΔPE-GFP+ cells obtained after DOX induction of lentiviral KLF2, NANOG, or KLF2+NANOG. After primary infection WIBR3 human ESCs containing the OCT4-ΔPE-GFP reporter allele were trypsinized and treated with primed human ESC medium (hESM), 2i/L, or 2i/L/DOX for 1 week. R1, total proportion of GFP+ cells, includes weak GFP activity observed in primed human ESCs; R2, subset of cells with high GFP activity observed only upon combined overexpression of KLF2+NANOG.

(F) Phase and fluorescence images and flow cytometric analysis of a clonal line of WIBR3 OCT4-ΔPE-GFP+ cells derived in 2i/L/DOX (left). Phase and fluorescence images and flow cytometric analysis after replating in the absence of DOX for 1 week (right) are also shown. 40× magnification.

(G) Quantitative gene expression analysis for lentiviral FUW-KLF2, lentiviral FUW-NANOG, endogenous OCT4, and endogenous KLF4 in WIBR3 hESCs cultured in hESM and clonal OCT4-ΔPE-GFP+ derivatives generated in 2i/L/DOX. Error bars indicate ± 1 SD of technical replicates.

(H) Phase and fluorescence images of primitive neural stem cells (pNSCs) derived by treating WIBR3 hESCs containing the OCT4-ΔPE-GFP allele with 2i/L for three passages. 100× magnification.

(I) Immunofluorescence staining for OCT4 and NESTIN in a clonal line of OCT4-ΔPE-GFP+ cells derived in 2i/L/DOX and a clonal line of OCT4-ΔPE-GFP− pNSCs derived in 2i/L. 100× magnification.

(J) Model representing the distinct phenotypic responses of hESCs to treatment with 2i/L and 2i/L/DOX. OCT4-ΔPE-GFP+ cells generated in 2i/L/DOX do not maintain reporter activity upon transgene withdrawal. OCT4-ΔPE-GFP+ cells can revert back to the conventional “primed” hESC state by re-exposure to serum and FGF.