多能干细胞在DNA损伤应激反应中的异质性对其命运选择的影响和调控

DNA damage response and cell cycle arrest can induce diversified fate in pluripotent stem cells. In our preliminary study, we found that the activation of DNA damage checkpoint and upregulation of P53 in S-phase-delayed embryonic stem cells (ESCs), resulted in the tendency of pluripotency maintenance, and at the mean time also triggered apoptotic response in other ESCs. Yet how the S phase arrest and checkpoint activation control pluripotency network and how the two apparently contradictory cell fates could be resulted are not clear. Here we employ a combination of molecular biology and quantitative live single cell imaging methods, to capture the heterogeneity in the DNA damage response in individual pluripotent stem cells and study how this could lead to the diversification of fate choice. We plan to employ a combination of bio-sensors and fluorescent reporters, to quantitatively measure the activity and dynamics of the key checkpoint players, chromatin maturation kinetics, as well as the pluripotency and apoptosis markers in single cell. With the multi-dimensional, quantitative, and time-resolved data, we plan to build up a system model to uncover novel kinetics and linkage between the heterogeneity of these factors and the stem cell fate choices. With this new approach, we hope to understand the pluripotent stem cell fate decision in a systematic, quantitative and precise angle to explain those events which were previously considered as stochastic and unpredictable. Moreover, this could direct us to design novel method to induce the stem cell toward unified cell fate which would be essential for stem cell based regenerative therapy.

细胞周期停滞和DNA损伤应激反应可诱导多能干细胞命运的改变。初期实验中我们发现S期停滞的胚胎干细胞会开启DNA损伤检查点并上调P53，结果导致了干细胞多能性的维持，与此同时这些通路也可直接促进干细胞凋亡。但S期停滞和检查点开启如何调控多能性网络，以及如何诱发截然不同的干细胞命运表型，这些机制尚不清楚。本项目将结合分子生物学和单细胞定量追踪成像的方法，捕捉多能干细胞在DNA损伤应激时的异质性，及其如何导致干细胞产生多样化的命运。通过一系列生物传感器和荧光报告因子，定量实时测量关键的检查点通路，染色质成熟动态以及多能性和细胞凋亡控制因子在单细胞中的活性水平和动态变化，由此建立系统模型以描述和解释以上因素在细胞之间的异质性与细胞命运选择多样性之间的关系。运用这个新颖的的平台我们希望精确定量的了解多能干细胞命运选择的控制规律，诱导干细胞走向单一的、我们期待的命运， 由此效益于再生医学与干细胞治疗。

干细胞自我更新和分化过程中，经常受到内外源DNA损伤的干扰,这会对胚胎发育造成不同层面的影响。胚胎干细胞如何应对DNA损伤不仅决定了胚胎干细胞自身基因组的稳定性，更将直接影响其子代细胞的分化命运，并影响胚胎发育。为研究胚胎干细胞中DNA损伤对干细胞自我更新和分化等命运选择的影响，我们在多能干细胞及不同谱系分化体系中，构建了一系列DNA损伤应激通路的荧光报告因子和生物传感器，来进行活细胞追踪，利用实时定量成像的方法分析干细胞细胞周期改变、DNA损伤应激等伴随干细胞分化的变化规律，以及与DNA损伤应激激活对于干细胞自我更新和分化的影响。围绕着这些研究内容，我们发现DNA损伤检查点不同程度的激活，可以通过ERK与P53互作诱导干细胞在自我更新和不同谱系分化的命运中进行选择。同时在全能性重编程、退出和进入的过程中，DNA损伤应激也发挥不同的作用。转录因子KLF5可以同时调控细胞周期和原始态多能性相关的逆转座子从而协同细胞命运。. 本项目目前共发表论文6篇，其中以通讯作者（含共同）身份发表论文4篇，发表在Cell Rep.（最后通讯），Stem Cell Int.(通讯作者)， Frontiers in Cell and Dev Biol.(通讯作者)，Stem Cell Res.(共同通讯)杂志上。同时以承担人为共同作者的其他文章发表在Stem Cell Rep., Gastroenterology杂志上。本项目的研究发现提示了干细胞周期和逆转座子调控在早期干细胞和胚胎发育中的重要影响，不仅将在细胞生物学、发育或再生生物学等基础研究领域引起新的兴趣，也将影响生殖医学和人类胚胎发育等相关的临床研究和应用。