Combined transcriptomic and phosphoproteomic analysis of BMP4 signaling in human embryonic stem cells.

Angelos Papadopoulos,Theodore Fotsis,Olga Mikhaylichenko,Carol Murphy,Debbie L Cunningham,John Heath,Aditi Kanhere,Marko Hyvönen,Varvara Chalmantzi,Dimitris Stellas

doi:10.1016/j.scr.2020.102133

Abstract

Human embryonic stem cells (hESCs) are an invaluable tool in the fields of embryology and regenerative medicine. Activin A and BMP4 are well-characterised growth factors implicated in pluripotency and differentiation. In the current study, hESCs are cultured in a modified version of mTeSR1, where low concentrations of ActivinA substitute for TGFβ. This culture system is further used to investigate the changes induced by BMP4 on hESCs by employing a combination of transcriptomic and phosphoproteomic approaches. Results indicate that in a pluripotent state, hESCs maintain WNT signaling under negative regulation by expressing pathway inhibitors. Initial stages of differentiation are characterized by upregulation of WNT pathway ligands, TGFβ pathway inhibitors which have been shown in Xenopus to expand the BMP signaling range essential for embryonic patterning, and mesendodermal transcripts. Moreover, BMP4 enhances the phosphorylation of proteins associated with migration and transcriptional regulation. Results further indicate the vital regulatory role of Activin A and BMP4 in crucial fate decisions in hESCs.

Highlights

Due to their pluripotent profile, entailing limitless in vitro selfrenewal potential and the ability to adopt nearly any cell identity, human embryonic stem cells have countless applications in regenerative medicine (Dupont et al, 2019)
To elucidate the transcriptional changes induced by BMP4, H1 cells cultured in ActA-mTeSR were treated with the cytokine for 1 h or 4 h and RNA-seq was carried out
Results indicate that in a pluripotent state, Human embryonic stem cells (hESCs) maintain WNT signaling under negative regu lation by expressing pathway inhibitors

Summary

Introduction

Due to their pluripotent profile, entailing limitless in vitro selfrenewal potential and the ability to adopt nearly any cell identity, human embryonic stem cells (hESCs) have countless applications in regenerative medicine (Dupont et al, 2019) Harnessing their therapeutic potential requires an extensive understanding of the elabo rate signaling pathways implicated in the maintenance of pluripotency and control of differentiation. Of GSK3β by ERK and activation of the WNT/β-catenin cascade (Singh et al, 2012) In this context, TGFβ-phosphorylated SMAD2/3 proteins cooperate with WNT components to drive differentiation towards endoderm (Chen et al, 2012). The same factor has a prominent role during gastrulation, acting as a morphogen in concert with BMP and WNT This process starts with BMP ligands originating from the extra-embryonic ectoderm acti vating WNT in both the epiblast and hypoblast (Rivera-Perez et al, 2005). We provide in sights into the initial transcriptional changes occurring using RNA-seq, and identify novel phosphopeptides and map their location by mass spectrometry (MS)

Results and discussion

Transcriptomic landscape of hESCs post 1 h BMP4 treatment

Transcriptomic landscape of hESCs post 4 h BMP4 treatment

Phosphoproteomic changes induced by BMP4 in hESCs

Conclusion