Pten-mediated Gsk3\u03b2 modulates the na\xefve pluripotency maintenance in embryonic stem cells

Wuming Wang,Gang Lu,Hsiang-Fu Kung,Zhiqiang Xiong,Zi-Jiang Chen,Wai-Yee Chan,Hongbin Liu,Jin-Long Ma,Man-Sze Wong,Chi-Kwan Leung,Chengcheng Tang,Hongjian Li,Hoi-Hung Cheung,Xianwei Su

doi:10.1038/s41419-020-2271-0

Abstract

Mouse embryonic stem cells (ESCs) are isolated from the inner cell mass of blastocysts, and they exist in different states of pluripotency—naïve and primed states. Pten is a well-known tumor suppressor. Here, we generated Pten−/− mouse ESCs with the CRISPR-Cas9 system and verified that Pten−/− ESCs maintained naïve pluripotency by blocking Gsk3β activity. Serum/LIF and 2i (MAPK and GSK3 inhibitors) conditions are commonly used for ESC maintenance. We show that the Pten-inhibitor SF1670 contributed to sustaining mouse ESCs and that Pten activation by the S380A, T382A, and T383A mutations (Pten-A3) suppressed the pluripotency of ESCs. The in vivo teratoma formation ability of SF1670-treated ESCs increased, while the Pten-A3 mutations suppressed teratoma formation. Furthermore, the embryoid bodies derived from Pten-deficient ESCs or SF1670-treated wild-type ESCs showed greater expression of ectoderm and pluripotency markers. These results suggest that Pten-mediated Gsk3β modulates the naïve pluripotency of ESCs and that Pten ablation regulates the lineage-specific differentiation.

Highlights

Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of the early mammalian embryo[1,2,3]
Loss of tumor suppressor Pten maintains ESC pluripotency and modulates ESC differentiation To explore the function of Pten in mouse ESCs, we generated Pten−/− ESCs using the CRISPR-Cas[9] system (Fig. S1a)
Our results indicate that phosphoinositide-3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) might be another signaling pathway regulated via Pten in modulating ESCs’ naïve pluripotency. β-catenin is a downstream factor of Gsk3β, and Wnt/

Summary

Introduction

Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of the early mammalian embryo[1,2,3]. ESCs possess the ability to self-renew and to differentiate into cells representative of all three embryonic germ layers[4]. In both mice and humans, ESCs exist in different pluripotent states such as the naïve and primed states, and these two states of pluripotency are distinguished by unique molecular and cellular features[5]. Self-renewing, pluripotent, and lineage primed states have been defined by multiple methodologies, including the recently reported method of single-cell expression profiling[6]. Small-molecule inhibitors have been used to maintain the pluripotency of mouse and primate ESCs7,8.

Methods

Results

Conclusion