Single-cell qPCR demonstrates that Repsox treatment changes cell fate from endoderm to neuroectoderm and disrupts epithelial-mesenchymal transition.

Qiuhong Li,Qingsong Huang,Gregory M Kelly

doi:10.1371/journal.pone.0223724

Abstract

A definitive endodermal cell lineage is a prerequisite for the efficient generation of mature endoderm derivatives that give rise to organs, such as the pancreas and liver. We previously reported that the induction of mesenchymal definitive endoderm cells depends on autocrine TGF-β signaling and that pharmacological blockage of TGF-β signaling by Repsox disrupts endoderm specification. The definitive endoderm arises from a primitive streak, which depends largely on TGF-β signaling. If the TGF-β pathway is blocked by Repsox, cell fate after the primitive streak induction is so-far unknown. We report here, that an induced primitive streak cell-population contained many T/SOX2 co-expressing cells, and subsequent inhibition of TGF-β signaling by Repsox promoted neuroectodermal cell fate, which was characterized using single-cell qPCR analysis and immunostaining. The process of epithelial-to-mesenchymal transition, which is inherent to the process of definitive endoderm differentiation, was also disrupted upon Repsox treatment. Our findings may provide a new approach to produce neural progenitors.

Highlights

Differentiation of human pluripotent stem cells into definitive endoderm (DE) is the critical first step for generating visceral organs, such as liver, pancreas, gut, and lungs [1]
The cells stained for immunofluorescence (IF) analysis showed a loss of the DE marker SOX17 and of the epithelial-to-mesenchymal transition (EMT)-related protein N-cadherin (CDH2) in the presence of Repsox; the epithelial marker E-cadherin (CDH1) was maintained consistently
We showed that increasing ectoderm marker levels were associated with decreasing endoderm marker levels upon the addition of Repsox after primitive streak (PS) induction, as SOX1 and SOX2 expression and SOX17 expression were mutually exclusive, and no EMT was observed as measured by the ratio of CDH1 and CDH2 expression

Summary

Introduction

Differentiation of human pluripotent stem cells (hPSCs) into definitive endoderm (DE) is the critical first step for generating visceral organs, such as liver, pancreas, gut, and lungs [1]. Most protocols for efficient production of DE cells employ exogenous Wnt and recombinant activin A to induce a primitive streak (PS) intermediate within 24 h, followed by continued TGF-β/ activin/nodal signaling for the subsequent 2–5 days. By systematically optimizing the differentiation protocol, Loh et al were able to differentiate hPSCs into > 98% pure SOX17-expressing DE cells within 48 h [2, 3]. In vertebrate embryos and during hPSC differentiation, activation of TGF-β/activin/nodal signaling by activin A is imperative for DE specification [4]. Epiblast cells undergo an epithelial-to-mesenchymal transition (EMT) at the primitive streak.

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