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

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.

定型内胚层细胞谱系（definitive endodermal cell lineage）是高效生成成熟内胚层衍生物的必要前提，此类衍生物可分化为胰腺、肝脏等器官。本团队此前研究表明，间质定型内胚层细胞的诱导依赖于自分泌转化生长因子β（TGF-β）信号通路，而通过Repsox对TGF-β信号通路进行药物阻断会破坏内胚层特化过程。定型内胚层起源于原条（primitive streak），该过程高度依赖TGF-β信号通路。若采用Repsox阻断TGF-β通路，原条诱导后的细胞命运目前仍不明确。本文报道，诱导获得的原条细胞群中存在大量T/SOX2双阳性细胞，后续通过Repsox抑制TGF-β信号通路可促进神经外胚层细胞命运的定向分化，该结论经单细胞定量聚合酶链反应（single-cell qPCR）分析与免疫染色验证。上皮间质转化（epithelial-to-mesenchymal transition）是定型内胚层分化过程中固有的生物学过程，经Repsox处理后该过程同样遭到破坏。本研究结果可为神经前体细胞的制备提供全新的技术策略。