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. Author manuscript; available in PMC: 2017 Sep 1.
Published in final edited form as: Cell Stem Cell. 2016 Jun 23;19(3):355–369. doi: 10.1016/j.stem.2016.05.025

Figure 3. Zfp281 is Critical for Primed Pluripotency. See also Figure S3.

Figure 3

(A) Proliferation analysis of WT and Zfp281 KO ESCs cultured in SL, 2iL or FA. Error bars indicate average ± SEM (n=3).

(B) Colony formation assay for WT and Zfp281 KO ESCs. Colonies were stained for alkaline phosphatase (AP) activity at day 6 of culture in specified conditions, and positive colonies were scored into three categories (compact, mixed and diffuse as indicated). Error bars indicate average ± SEM (n=6). Student's unpaired t test (***P<0.001, **P<0.01) relative to WT ESCs was performed.

(C) Immunostaining of Nanog and Oct4 at day 8 under FA. Scale bars represent 100 μm.

(D) Representative bright-field images at day 6 of Zfp281 KD (shZfp281) EpiSCs and rescued (+R) line compared with control luciferase knockdown (shLuci) EpiSCs.

(E) Zfp281 KD facilitates reprogramming of EpiSCs towards naive-like cells (Epi-iPSCs). Two different EpiSC lines were transfected with three independent shRNAs against Zfp281, and Epi-iPSC colony numbers were quantified at day 6 in 2iL. Error bars indicate average ± SEM (n=3). Student's unpaired t test (***P<0.001) relative to shLuci was performed.

(F) Contribution of Epi-iPSCs to the germline (top) and chimeric E13.5 mouse embryos (bottom). Epi-iPSCs were generated by reprogramming EpiSCs with pTRIPZ-shZfp281. EpiiPSCs were labeled with a Tomato reporter transgene and maintained without Dox (OFF).

(G) ZNF281 dependency of conventional hESCs. Representative colony morphology under bright-field (top) and fluorescent (for OCT4-GFP; middle) microscope as well as AP-stained colonies (bottom) are shown. ZNF281-depleted and control shLuci O4G hESCs were passaged twice in primed culture conditions before microscopy.

(H) A strategy to test effects of ZNF281 depletion on converting primed hESCs to a naive state using the published 5iL condition.

(I) Quantification of AP-positive colony numbers of hESCs at day 10 of primed human ES medium (FA) or naive culture conditions (5iL) upon control or ZNF281 KD. Error bars indicate average ± SEM (n=3). Student's unpaired t test (***P<0.001, **P<0.01) relative to shLuci hESCs.