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. 2021 Feb 19;31(5):526–541. doi: 10.1038/s41422-021-00477-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a The morphology of typical naïve mESCs (left panel), sEpiblastoids (middle panel), and EpiSCs (right panel); scale bars, 50 μm (left panel), 20 μm (middle panel), and 300 μm (right panel). b The immunofluorescent staining of sEpiblastoids for Oct4, Sox2, or Nanog (green) and Ezrin or F-actin (magenta). DNA was stained with Hoechst 33342 (blue). Scale bar, 20 μm. c The lysates of mESCs (2i/lif or S/lif), 48 h EpiLCs, sEpiblastoids and EpiSCs were collected, and analyzed by western blot assay with specific antibodies for Oct4, Sox2, Nanog, Otx2, T, E-Cadherin and N-Cadherin. β-actin served as the loading control. d The expression of putative formative pluripotency markers (Otx2, Dnmt3b, Sox4 and Fgf5) was examined for naïve mESCs, EpiLCs, sEpiblastoids and EpiSCs by qRT-PCR with specific primers of these genes. The gene expression level was measured relative to β-actin (100%). Data were represented as means ± SD (n = 3). e sEpiblastoids were stained with specific antibodies for Otx2 (green), E-Cadherin (magenta), and Hoechst 33342 for DNA (blue). Scale bar, 20 μm. f Female fPSCs were stained with specific antibody for H3K27me3 (green) and Phalloidin for F-actin (magenta). DNA was stained with Hoechst 33342 (blue). Scale bar, 40 μm. g Oct4-ΔPE-GFP reporter mESCs were cultured in fPSC medium for 4 days (fPSCs passage 1, P1, top). P1 cells were propagated (P2, bottom) in the same medium. The cells of days 1–4 were stained with Phalloidin for F-actin (magenta) and Hoechst 33342 for DNA (blue). The enhancer utilization of Oct4 was indicated by GFP reporter (green). Scale bar, 40 μm.