DNMTs Play an Important Role in Maintaining the Pluripotency of Leukemia Inhibitory Factor-Dependent Embryonic Stem Cells.

Baojiang Wu,Yunxia Li,Chen Chen,Shudong Li,Siqin Bao,Baojing Zhang,Yuting Fu,M Azim Surani,Junpeng Gao,Lin Li,Yanqiu Wang,Bojiang Li,Xihe Li,Fuchou Tang

doi:10.1016/j.stemcr.2021.01.017

Abstract

SummaryNaive pluripotency can be maintained in medium with two inhibitors plus leukemia inhibitory factor (2i/LIF) supplementation, which primarily affects canonical WNT, FGF/ERK, and JAK/STAT3 signaling. However, whether one of these three supplements alone is sufficient to maintain naive self-renewal remains unclear. Here we show that LIF alone in medium is sufficient for adaptation of 2i/L-ESCs to embryonic stem cells (ESCs) in a hypermethylated state (L-ESCs). Global transcriptomic analysis shows that L-ESCs are close to 2i/L-ESCs and in a stable state between naive and primed pluripotency. Notably, our results demonstrate that DNA methyltransferases (DNMTs) play an important role in LIF-dependent mouse ESC adaptation and self-renewal. LIF-dependent ESC adaptation efficiency is significantly increased in serum treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike epiblast stem cells, L-ESCs contribute to somatic tissues and germ cells in chimeras. L-ESCs cultured under such simple conditions as in this study would provide a more conducive platform to clarify the molecular mechanism of ESCs in in vitro culture.

Highlights

Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo (Martin, 1981)
Sv, and C57BL/6J strains) 3 129/sv F1 mouse (Yoshimizu et al, 1999) ESC lines (W1, W2, W4, W5, W6, SQ3.3, and X/GFP; the sex of the cell lines is indicated in Figure S1A), which were directly derived in 2i/leukemia inhibitory factor (LIF) medium and switched to chemically defined LIF (1000 IU/mL)-alone medium based on N2B27 (L medium) (Figures 1A and S1A)
After two or more repeated fluorescence-activated cell sorting (FACS) assays for each L-ESC line (Figure S1B), GOF/GFP+ LESCs reached nearly 98% purity, which was similar to the control 2i/L-ESCs (Figure S1B). These results indicate that LIF alone can maintain FACS-purified GOF/GFP+ L-ESCs in an undifferentiated pluripotent state (Figures 1B and S1B), with stable growth over 40 passages (Figure S1C) and high alkaline phosphatase (AP) activity (Figure S1D)

Summary

Introduction

Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass (ICM) of the pre-implantation embryo (Martin, 1981). It is generally believed that the optimal culture conditions for groundstate ESCs comprise the three-additive 2i/LIF supplement, which affects canonical WNT, FGF/ERK, and JAK/STAT3 signals, respectively (Ohtsuka et al, 2015). Since LIF was detected in an extract from feeder cells and has been used for most mouse ESC media, it has been fully demonstrated to be an important supplement for ESC self-renewal and pluripotency (Gao et al, 2019; Williams et al, 1988; Yang et al, 2017; Ying et al, 2003, 2008). Essential LIF/STAT3 functions can be compensated for by activation of canonical WNT signaling and inhibition of FGF/ERK in the established culture system for self-renewal of ESCs (Ohtsuka et al, 2015).

Results

Discussion

Conclusion