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. 2021 Jun;54(6):e13049.
doi: 10.1111/cpr.13049. Epub 2021 May 7.

Retinoic acid induces NELFA-mediated 2C-like state of mouse embryonic stem cells associates with epigenetic modifications and metabolic processes in chemically defined media

Yanqiu Wang  1   2 , Qin Na  1   2   3 , Xihe Li  1   2   4 , Wee-Wei Tee  5   6 , Baojiang Wu  1   2   4 , Siqin Bao  1   2
Affiliations

Retinoic acid induces NELFA-mediated 2C-like state of mouse embryonic stem cells associates with epigenetic modifications and metabolic processes in chemically defined media

Yanqiu Wang et al. Cell Prolif. 2021 Jun.

Abstract

Objectives: Mouse embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos and cultured in different culture media with varied pluripotency. Sporadically, a small population of ESCs exhibit 2-cell stage embryonic features in serum containing medium. However, whether ESCs can transit into 2-cell embryo-like (2C-like) cells in the chemically defined media remains largely unknown.

Materials and methods: We established a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase the subpopulation of 2C-like cells. Further test the pluripotency and 2C features of ESCs cultured in RA. 2C reporter-positive cells were selected by FACS; the level of protein was detected via immunofluorescence staining and western blot; the level gene expressions were measured by RNA-seq.

Results: Retinoic acid drives a NELFA (negative elongation factor A)-mediated 2C-like state in mouse ESCs, characterized with 2C-specific transcriptional networks and the ability to contribute trophectoderm (TE) when injected into developing embryos. In addition, RA treatment triggers DNA hypomethylation, active histone modification, suppressed glycolysis metabolism and reduced protein synthesis activity of ESCs.

Conclusions: We showed that RA has a broader role in 2C-like cells state, not only is one of the upstream regulators of the 2C-like state in chemically defined media but also illuminates genetic and epigenetic regulations that govern ESCs to 2C-like transition.

Keywords: 2C-like cells; NELFA; embryonic stem cell; epigenetic; mouse; retinoic acid.

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Conflict of interest statement

The authors declared no potential conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Retinoic acid induces the 2C‐like state in chemically defined media. A, Experimental outline of the MERVL::tdTomato‐labelled ESCs was treated with RA. B, MERVL::tdTomato reporter ESCs were cultured in 2i/L and 2i/L+RA medium. 2i/L+RA condition was significantly increasing the MERVL::tdTomato‐positive cells. Scale bars, 100 μm. C, tdTomato based Fluorescence‐activated cell sorting (FACS) on 2i/L and 2i/L+RA cultured MERVL::tdTomato reporter ESCs. D, Relative expression levels of 2C genes (Mervl, Zscan4c, Dux‐coding, Eil1a‐like, Tcstv3 and Cdx2) measured by qPCR in 2i/L and 2i/L+RA cultured MERVL::tdTomato reporter ESCs. Error bars are mean ± SD (n = 4). P values were calculated by two‐tailed Student's t test, P < .05. E, Immunostaining of ZSCAN4 and OCT4 in 2i/L and 2i/L+RA cultured ESCs. Scale bars, 50 μm. F, Quantification of the ZSCAN4‐positive cells in 2i/L and 2i/L+RA cultured ESCs. Error bars are mean ± SD (n = 30). P values were calculated by two‐tailed Student's t test, P < .05
FIGURE 2
FIGURE 2
Retinoic acid drives NELFA‐mediated 2C‐like state in ESCs. A, Experimental outline of the NELFA‐GFP reporter ESCs were treated with RA. B, NELFA‐GFP reporter ESCs were cultured in 2i/L and 2i/L+RA medium. 2i/L+RA condition was significantly increasing the NELFA‐GFP‐positive cell number. Scale bars, 100 μm. C, FACS quantification of the proportion of GFP‐positive (GFP+) cells upon activation by RA. D, qPCR of 2C‐specific genes Nelfa, Zscan4c, Tcstv3, Mervl, Dppa2 and Dppa4 expression level in 2i/L and 2i/L+RA cultured NELFA‐GFP reporter ESCs. Error bars are mean ± SD (n = 4). P values were calculated by two‐tailed Student's t test, P < .05. E, Western blotting analysis for ZSCAN4 in 2i/L and 2i/L+RA cultured NELFA‐GFP reporter ESCs and the band intensity of western blotting was quantified in control. F, Immunostaining of ZSCAN4 and OCT4 in 2i/L, 2i/L+RA cultured NELFA‐GFP reporter ESCs. Scale bars, 50 μm. G, Quantification of the ZSCAN4‐positive cells in 2i/L and 2i/L+RA cultured ESCs. Error bars are mean ± SD (n = 30). P values were calculated by two‐tailed Student's t test, P < .05. H, mCherry‐labelled NELFA‐positive cells were injected into 8‐cell stage embryos, which were then cultured 48 h in vitro. mCherry‐labelled NELFA‐positive RA‐2i/L‐ESCs were able to contribute to both TE or ICM, whereas 2i/L‐ESCs were only contribute to ICM. Scale bars, 50 μm
FIGURE 3
FIGURE 3
Retinoic acids regulate transcriptional networks related to 2C‐like state. A, Hierarchical clustering of transcriptomes data from pre‐implantation embryos (E1.5, E2.5 and E3.5), 2i/L‐ESCs and RA‐2i/L‐ESCs. B, Principle component analysis (PCA) of transcriptomes data from pre‐implantation embryos (E1.5, E2.5 and E3.5), 2i/L‐ESCs and RA‐2i/L‐ESCs. C, Heat map showing differentially expressed genes (mean log2 (normalized read counts) > 2, log2 (fold change) > 2, adjusted P value < .05) in RA‐2i/L‐ESCs compared with 2i/L‐ESCs. Significantly enriched GO terms and representative genes in each cluster are listed on the right. D, We use Short Time‐series Expression Miner (STEM) software program to analysis the gene expression trend on E1.5, E2.5, E3.5, 2i/L‐ESCs and RA‐2i/L‐ESCs. There are 1632 genes highly expressed in both RA‐2i/L‐ESCs and E1.5. E, Venn Diagram indicated that total of 52 genes were overlaped between previous reported 2C genes (126 genes) versus E1.5 and RA‐2i/L‐ESCs upregulated genes (1632 genes), such as Zscan4b, Zfp352, Zscan4c, Tdpoz3, Gm4027 and Gm8994
FIGURE 4
FIGURE 4
Epigenetic modifications enhance retinoic acid‐induced 2C‐like state of ESCs via NELFA. A, NELFA‐GFP reporter ESCs were cultured in 2i/L, 2i/L+RA, 2i/L+5aza and 2i/L+RA+5aza medium. 2i/L+RA+5aza condition was significantly increasing the NELFA‐GFP‐positive cell number. Scale bars, 100 μm. B, FACS quantification of the proportion of GFP‐positive (GFP+) cells upon activation by 2i/L, 2i/L+RA, 2i/L+5aza and 2i/L+RA+5aza. C, DNA methylation related genes heat map in 2i/L‐ESCs and RA‐2i/L‐ESCs. D, Western blotting analysis for DNMT1, DNMT3A, DNMT3L and β‐CATENIN in 2i/L‐ESCs and RA‐2i/L‐ESCs. E, Histone deacetylation related genes heat map in 2i/L‐ESCs and RA‐2i/L‐ESCs. F, Western blotting analysis for H3K37ac and histone H3 in ESCs cultured in different culture conditions
FIGURE 5
FIGURE 5
Retinoic acid regulates 2C‐like state by metabolic process. A, RA‐2i/L‐ESCs show global downregulation of glycolysis gluconeogenesis‐related genes by gene set enrichment analysis (GSEA). Normalized enrichment score (NES) and nominal P values are shown. B, Heat map showing expression levels of metabolic process and signalling pathway genes in 2i/L‐ESCs and RA‐2i/L‐ESCs. C, NELFA‐GFP reporter 2i/L‐ESCs and RA‐2i/L‐ESCs were treated with protein synthesis inhibitor cycloheximide (CHX). Scale bars, 100 μm. D, RA‐2i/L‐ESCs show global downregulation of ribosome‐related genes by gene set enrichment analysis (GSEA). Normalized enrichment score (NES) and nominal P values are shown. E, RA‐2i/L‐ESCs show global upregulation of WNT signalling pathway‐related genes by gene set enrichment analysis (GSEA). Normalized enrichment score (NES) and nominal P values are shown. F, RA‐2i/L‐ESCs show global upregulation of Hippo signalling pathway‐related genes by gene set enrichment analysis (GSEA). Normalized enrichment score (NES) and nominal P values are shown. G, RA‐2i/L‐ESCs were removed CHIR99021 and cultured with WNT signalling inhibitor IWR1 or XAV939 containing medium. Scale bars, 100 μm
FIGURE 6
FIGURE 6
Retinoic acid induces NELFA‐mediated 2C‐like state of ESCs in chemically defined media. 2C‐like cells arise spontaneously in ESCs upon serum and leukaemia inhibitory factor (LIF) containing culture condition and some genetic modified ESCs, and therefore, 2C‐like cells refer to totipotent. Intriguingly, naïve ESCs culture medium fail to induce 2C‐like state of ESCs. In this study, we report a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase 2C‐like cells population in culture without any genetic modification and serum
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References

    1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292(5819):154‐156. - PubMed
    1. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA. 1981;78(12):7634‐7638. - PMC - PubMed
    1. Macfarlan TS, Gifford WD, Driscoll S, et al. Embryonic stem cell potency fluctuates with endogenous retrovirus activity. Nature. 2012;487(7405):57‐63. - PMC - PubMed
    1. Riveiro AR, Brickman JM. From pluripotency to totipotency: an experimentalist's guide to cellular potency. Development. 2020;147(16):dev189845. - PubMed
    1. Bao S, Tang WWC, Wu B, et al. Derivation of hypermethylated pluripotent embryonic stem cells with high potency. Cell Res. 2018;28(1):22‐34. - PMC - PubMed