Lhx2调控上皮-间质转换的表观遗传学机制研究及其在神经外胚层细胞编程与重编程中的作用

The transition in between epithelial and mesenchymal cell types accompanies the cell fate determining processes when pluripotent stem cells are programmed to three germ layers or somatic cells are reprogrammed back into pluripotency. However, it is still unclear whether epithelial-mesenchymal transition (EMT) or mesenchymal-epithelial transition (MET) is required for cell fate conversion during programming or reprogramming. Moreover, the determining factors for EMT or MET still needs to be defined. According to our preliminary data, transcription factor Lhx2 strongly induces EMT in human embryonic stem cells and this EMT is a prerequisite for proper differentiation of human neuroectoderm. In this application, we will first investigate the role of Lhx2 in determining the epithelial and mesenchymal fates as well as its role in neuroectoderm programming and reprogramming. By comparison of normal human embryonic stem cells (“E” type) and Lhx2 overexpressed human embryonic stem cells (“M” type), epigenetic landscapes related to “E” and “M” fates will be drawn by studying DNA and histone modifications. By comparison of Lhx2 binding sites and specific epigenetic modifications related to “E” and “M”, molecular mechanisms underlying Lhx2 regulated EMT will be studied extensively. We will also try to investigate the function of this Lhx2-epigenetic modification-EMT pathway in guiding human embryonic stem cells to neuroectoderm or reprogramming of human fibroblasts to neuroectoderm.

在多能干细胞向三胚层编程分化以及成体细胞向诱导多能干细胞重编程的过程中均伴随有明显的上皮细胞（“E”）和间质细胞（“M”）类型间的相互转换。这种“E”-“M”转换或“M”-“E”转换是否是实现编程和重编程过程所必需，其转换的关键决定因素与分子机制如何尚有待明确。我们的前期研究发现，转录因子Lhx2能诱导人胚胎干细胞发生“E”-“M”转换，而且这种转换是人胚胎干细胞发育为神经外胚层细胞的先决条件。在本申请中，我们将首先确立Lhx2在“E”和“M”细胞类型决定及神经外胚层细胞“编程”与“重编程”中的作用；进而通过比较人胚胎干细胞“E”和过表达Lhx2人胚胎干细胞“M”两者间的表观遗传图谱，提炼“E”和“M”的特异表观标记，解析出Lhx2通过表观遗传学实现“E”-“M”转换的调控网络；并研究这一调控网络在人胚胎干细胞定向分化为神经外胚层细胞以及人成纤维细胞直接转分化为神经外胚层细胞中的关键作用。

在多能干细胞向三胚层编程分化以及成体细胞向诱导多能干细胞重编程的过程中均伴随有明显的上皮细胞（“E”）和间质细胞（“M”）类型间的相互转换。这种“E”-“M”转换或“M”-“E”转换是否是实现编程和重编程过程所必需，其转换的关键决定因素与分子机制如何尚有待明确。我们成功的建立了将人多能干细胞定向分化为神经外胚层细胞的编程技术，同时也建立了将人成纤维细胞重编程为神经外胚层细胞的技术。通过慢病毒介导的Lhx2敲降或Lhx2基因敲除，我们发现Lhx2是多能干细胞转变为神经外胚层细胞的必要条件。转录组学分析发现，Lhx2敲降后“E”-“M”转换的相关基因出现明显下调，提示Lhx2和EMT直接相关，而且EMT是神经外胚层细胞分化所必需经历的阶段。过表达Lhx2引起人多能干细胞发生EMT，表现为细胞形态的改变和愈合能力的增强。这些结果提示，EMT是人胚胎干细胞发育为神经外胚层细胞的必需过程，而Lhx2在EMT中起关键作用。我们正通过Lhx2的Chip-seq分析其靶基因，并通过干预和EMT相关靶基因，研究EMT在谱系发育中的作用。