Activin A and BMP4 Signaling Expands Potency of Mouse Embryonic Stem Cells in Serum-Free Media.

Baojiang Wu,Baojing Zhang,Yanglin Chen,Bojiang Li,Siqin Bao,Zhiqing Yang,Junpeng Gao,M Azim Surani,Fuchou Tang,Mengyi Wei,Xihe Li,Changshan Wang,Lin Li,Shudong Li,Kexin Li

doi:10.1016/j.stemcr.2020.01.004

Abstract

SummaryInhibitors of Mek1/2 and Gsk3β, known as 2i, and, together with leukemia inhibitory factor, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency (2i/L-ESCs). However, recent reports show that prolonged Mek1/2 suppression impairs developmental potential of ESCs, and is rescued by serum (S/L-ESCs). Here, we show that culturing ESCs in Activin A and BMP4, and in the absence of MEK1/2 inhibitor (ABC/L medium), establishes advanced stem cells derived from ESCs (esASCs). We demonstrate that esASCs contributed to germline lineages, full-term chimeras and generated esASC-derived mice by tetraploid complementation. We show that, in contrast to 2i/L-ESCs, esASCs display distinct molecular signatures and a stable hypermethylated epigenome, which is reversible and similar to serum-cultured ESCs. Importantly, we also derived novel ASCs (blASCs) from blastocysts in ABC/L medium. Our results provide insights into the derivation of novel ESCs with DNA hypermethylation from blastocysts in chemically defined medium.

Highlights

Mouse embryonic stem cells (ESCs) were originally derived by coculture with a feeder layer of mitotically inactivated fibroblasts in medium containing fetal calf serum (Evans and Kaufman, 1981; Martin, 1981)
We recently developed a culture system without serum and feeder, to derive advanced embryonic stem cells (ASCs), and demonstrated that such pluripotent stem cells possess enhanced in vivo developmental potential and unique self-renewing features when compared with mouse 2i/L-ESCs (Bao et al, 2018)
ABC/L Converts ESCs into esASCs with High Genomic Stability We previously reported that ABC/L medium converted blastocyst-derived AFSCs into ASCs (Bao et al, 2018)

Summary

Introduction

Mouse embryonic stem cells (ESCs) were originally derived by coculture with a feeder layer of mitotically inactivated fibroblasts in medium containing fetal calf serum (Evans and Kaufman, 1981; Martin, 1981). It was later shown that feeder cells could be replaced by the cytokine leukemia inhibitory factor (LIF) (Smith et al, 1988), and that serum could be substituted by bone morphogenetic protein (BMP) (Ying et al, 2003). ESCs cultured in BMP plus LIF medium show low single-cell clonogenic capacity, and are difficult to maintain in a homogeneous state (Ying et al, 2003). Inhibitors of Mek1/2 (PD0325901, PD) and Gsk3b (CHIR99021, CH), known as 2i, enhanced the derivation of ESCs (hereafter termed 2i/L-ECSs) and promoted ground-state pluripotency (Ying et al, 2008), known as ‘‘naive pluripotency’’ (Nichols and Smith, 2009), when LIF was added. Defects that are efficiently rescued by serum (Choi et al, 2017; Yagi et al, 2017) return mouse ESCs to their original culture condition and original state

Results

Discussion

Conclusion