Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, USA.
Department of Human Oncology, University of Wisconsin-Madison, Madison, WI, USA.
Cell Rep. 2019 May 7;27(6):1726-1741.e5. doi: 10.1016/j.celrep.2019.04.056.
Elucidating the mechanism of reprogramming is confounded by heterogeneity due to the low efficiency and differential kinetics of obtaining induced pluripotent stem cells (iPSCs) from somatic cells. Therefore, we increased the efficiency with a combination of epigenomic modifiers and signaling molecules and profiled the transcriptomes of individual reprogramming cells. Contrary to the established temporal order, somatic gene inactivation and upregulation of cell cycle, epithelial, and early pluripotency genes can be triggered independently such that any combination of these events can occur in single cells. Sustained co-expression of Epcam, Nanog, and Sox2 with other genes is required to progress toward iPSCs. Ehf, Phlda2, and translation initiation factor Eif4a1 play functional roles in robust iPSC generation. Using regulatory network analysis, we identify a critical role for signaling inhibition by 2i in repressing somatic expression and synergy between the epigenomic modifiers ascorbic acid and a Dot1L inhibitor for pluripotency gene activation.
阐明重编程的机制受到体细胞获得诱导多能干细胞（iPSCs）效率低和动力学差异的影响而变得复杂。因此，我们通过组合使用表观遗传修饰剂和信号分子来提高效率，并对单个重编程细胞的转录组进行了分析。与既定的时间顺序相反，体细胞基因失活和细胞周期、上皮和早期多能性基因的上调可以独立触发，因此这些事件的任何组合都可以在单个细胞中发生。Epcam、Nanog 和 Sox2 与其他基因的持续共表达对于向 iPSCs 前进是必需的。Ehf、Phlda2 和翻译起始因子 Eif4a1 在稳健的 iPSC 生成中发挥功能作用。通过调控网络分析，我们发现 2i 通过抑制体细胞表达和表观遗传修饰剂与 Dot1L 抑制剂协同作用来激活多能性基因，在抑制信号中起着关键作用。
