Epigenetic integrity of paternal imprints enhances the developmental potential of androgenetic haploid embryonic stem cells

Hongling Zhang,Yuanyuan Li,Guangyong Shi,Chongping Lai,Qian Yu,Jinsong Li,Yongjian Ma

doi:10.1007/s13238-021-00890-3

Abstract

The use of two inhibitors of Mek1/2 and Gsk3β (2i) promotes the generation of mouse diploid and haploid embryonic stem cells (ESCs) from the inner cell mass of biparental and uniparental blastocysts, respectively. However, a system enabling long-term maintenance of imprints in ESCs has proven challenging. Here, we report that the use of a two-step a2i (alternative two inhibitors of Src and Gsk3β, TSa2i) derivation/culture protocol results in the establishment of androgenetic haploid ESCs (AG-haESCs) with stable DNA methylation at paternal DMRs (differentially DNA methylated regions) up to passage 60 that can efficiently support generating mice upon oocyte injection. We also show coexistence of H3K9me3 marks and ZFP57 bindings with intact DMR methylations. Furthermore, we demonstrate that TSa2i-treated AG-haESCs are a heterogeneous cell population regarding paternal DMR methylation. Strikingly, AG-haESCs with late passages display increased paternal-DMR methylations and improved developmental potential compared to early-passage cells, in part through the enhanced proliferation of H19-DMR hypermethylated cells. Together, we establish AG-haESCs that can long-term maintain paternal imprints.

Highlights

Culture conditions with 2i supplemented with leukemia inhibitory factor (LIF) (2i/L) enhance the derivation of embryonic stem cells (ESCs) with naïve ground-state pluripotency (Ying et al, 2008)
We showed that H3K9me3 deposition and ZFP57 binding are preserved at H19 and IG DNA methylated regions (DMRs) and highly correlated with DMR methylation, which are likely involved in the stable maintenance of paternal imprints in Two-step a2i/L (TSa2i/L)-derived AG-haploid ESCs (haESCs)
We further demonstrated that TSa2i/Ltreated AG-haESCs are a heterogeneous cell population regarding H19-DMR methylation levels and cells with hypermethylated H19-DMR display better proliferation potential compared to cells with hypomethylated H19-DMR, partially accounting for long-term maintenance of paternal imprints

Summary

Introduction

Culture conditions with 2i (inhibitors of Mek1/2 and Gsk3β) supplemented with leukemia inhibitory factor (LIF) (2i/L) enhance the derivation of embryonic stem cells (ESCs) with naïve ground-state pluripotency (Ying et al, 2008). Recent studies indicate that prolonged application of 2i results in a widespread loss of DNA methylation, including repetitive elements and imprinted genes, and impaired developmental potential in ESCs (Choi et al, 2017b; Yagi et al, 2017). The longterm culture of female ESCs in a2i/L induced a reduction of ICR (imprinting control region) methylation (Yagi et al, 2017). The conventional serum plus LIF (S/L) medium induced loss of DNA methylation at ICRs in mouse ESCs upon prolonged culturing (Dean et al, 1998; Humpherys et al, 2001; Yagi et al, 2017).

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Conclusion