Stem Cell Reviews and Reports ( IF 4.5 ) Pub Date : 2021-02-23 , DOI: 10.1007/s12015-021-10136-8
Vijay V Vishnu 1 , Bh Muralikrishna 1 , Archana Verma 1 , Sanjeev Chavan Nayak 1 , Divya Tej Sowpati 1 , Vegesna Radha 1 , P Chandra Shekar 1
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C3G (RAPGEF1), engaged in multiple signaling pathways, is essential for the early development of the mouse. In this study, we have examined its role in mouse embryonic stem cell self-renewal and differentiation. C3G null cells generated by CRISPR mediated knock-in of a targeting vector exhibited enhanced clonogenicity and long-term self-renewal. They did not differentiate in response to LIF withdrawal when compared to the wild type ES cells and were defective for lineage commitment upon teratoma formation in vivo. Gene expression analysis of C3G KO cells showed misregulated expression of a large number of genes compared with WT cells. They express higher levels of self-renewal factors like KLF4 and ESRRB and show high STAT3 activity, and very low ERK activity compared to WT cells. Reintroduction of C3G expression in a KO line partially reverted expression of ESRRB, and KLF4, and ERK activity similar to that seen in WT cells. The expression of self-renewal factors was persistent for a longer time, and induction of lineage-specific markers was not seen when C3G KO cells were induced to form embryoid bodies. C3G KO cells showed poor adhesion and significantly reduced levels of pFAK, pPaxillin, and Integrin-β1, in addition to downregulation of the cluster of genes involved in cell adhesion, compared to WT cells. Our results show that C3G is essential for the regulation of STAT3, ERK, and adhesion signaling, to maintain pluripotency of mouse embryonic stem cells and enable their lineage commitment for differentiation.
Graphical abstract
中文翻译：
C3G 调节 STAT3、ERK、粘附信号，对于胚胎干细胞的分化至关重要
C3G (RAPGEF1) 参与多种信号通路，对小鼠的早期发育至关重要。在这项研究中，我们研究了它在小鼠胚胎干细胞自我更新和分化中的作用。由 CRISPR 介导的靶向载体敲入产生的 C3G 无效细胞表现出增强的克隆形成性和长期自我更新。与野生型 ES 细胞相比，它们对 LIF 退出的反应没有分化，并且在体内畸胎瘤形成时谱系定型有缺陷。与 WT 细胞相比，C3G KO 细胞的基因表达分析显示大量基因的错误表达。与 WT 细胞相比，它们表达更高水平的自我更新因子，如 KLF4 和 ESRRB，并显示出高 STAT3 活性和非常低的 ERK 活性。在 KO 系中重新引入 C3G 表达部分恢复了 ESRRB 和 KLF4 的表达，以及与在 WT 细胞中看到的相似的 ERK 活性。自我更新因子的表达持续时间较长，在诱导C3G KO细胞形成胚状体时未观察到谱系特异性标志物的诱导。与 WT 细胞相比，C3G KO 细胞显示出较差的粘附性，并且 pFAK、pPaxillin 和 Integrin-β1 的水平显着降低，此外还下调了参与细胞粘附的基因簇。我们的研究结果表明，C3G 对于调节 STAT3、ERK 和粘附信号传导、维持小鼠胚胎干细胞的多能性并使其谱系分化成为必需。自我更新因子的表达持续时间较长，在诱导C3G KO细胞形成胚状体时未观察到谱系特异性标志物的诱导。与 WT 细胞相比，C3G KO 细胞显示出较差的粘附性，并且 pFAK、pPaxillin 和 Integrin-β1 的水平显着降低，此外还下调了参与细胞粘附的基因簇。我们的研究结果表明，C3G 对于调节 STAT3、ERK 和粘附信号传导、维持小鼠胚胎干细胞的多能性并使其谱系分化成为必需。自我更新因子的表达持续时间较长，在诱导C3G KO细胞形成胚状体时未观察到谱系特异性标志物的诱导。与 WT 细胞相比，C3G KO 细胞显示出较差的粘附性，并且 pFAK、pPaxillin 和 Integrin-β1 的水平显着降低，此外还下调了参与细胞粘附的基因簇。我们的研究结果表明，C3G 对于调节 STAT3、ERK 和粘附信号传导、维持小鼠胚胎干细胞的多能性并使其谱系分化成为必需。与 WT 细胞相比，除了下调参与细胞粘附的基因簇。我们的研究结果表明，C3G 对于调节 STAT3、ERK 和粘附信号传导、维持小鼠胚胎干细胞的多能性并使其谱系分化成为必需。与 WT 细胞相比，除了下调参与细胞粘附的基因簇。我们的研究结果表明，C3G 对于调节 STAT3、ERK 和粘附信号传导、维持小鼠胚胎干细胞的多能性并使其谱系分化成为必需。
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