人胚胎干细胞向视网膜色素上皮分化的DNA/miRNA甲基化调控机制

Retinal pigmented epithelium (RPE) cells derived from human embryonic stem cells (hESCs) have been proposed as an attractive alternative source for transplantation in age-related macular degeneration. Improving differentiation procedure from hESCs to RPE cells has been put in the top studies for urgent attention . It is well established that epigenetic mechanisms play critical roles during hESCs differentiation, including methylation, miRNAs regulation, histone modification, etc. In our previous work, we studied the roles of miRNAs in directed differentiation of hESCs into RPE. A multitude of differentiatially expressed miRNAs were identified during differentiation. Among them miR-204, 211 and 302 were proven to participate in the regulation of Wnt/β-catenin and TGFβ/Nodal signaling pathways through targeting JUN, CTNNBIP1 and TGFBR2 genes. These results fully support our hypothesis that epigenetic mechanisms play important roles in the process of RPE induction from hESCs. In the current project, we plan to further explore the mechanisms of DNA methylation, together with microRNA regulation, aiming to achieve full scale understanding of the epigenentic regulation network underlying directed differentiation of hESCs into RPE. Out of the network, key epigenetic regulation factors, transcription factors and signaling molecules will be selected and combined to develop a more efficient RPE indcution regime from hESCs. The present study will undoubtedly deepen our understanding of molecular regulation mechanisms underlying directed differentiation of hESCs into RPE. The establishment of efficient RPE derivation protocol based on epigenetic regulation network will significantly promote the clinical application of RPE replacement therapy.

提高胚胎干细胞（ESCs）向视网膜色素上皮（RPE）细胞分化效率是目前干细胞治疗老年黄斑变性研究的焦点。甲基化修饰是表观遗传领域中决定胚胎干细胞（ESCs）分化的重要因素，通过调控DNA或miRNA启动子发挥作用。我们前期建立了稳定的人ESCs向RPE细胞的分化体系，并对与分化相关的miRNA和基因表达进行了初步研究。本项目将在此基础上，进一步研究人ESCs向RPE细胞分化过程中包括miRNA启动子在内的基因组甲基化变化；构建甲基化修饰对miRNA、信号通路的调控网络，验证前期发现的分化相关miRNA是否为甲基化调控网络的关键节点；并从甲基化程度、miRNA和信号通路多个层面，对挖掘的关键节点进行综合干预，应用于分化体系。从而从甲基化修饰出发，综合miRNA和下游信号通路因素阐明定向分化机制，为高效获得更为成熟的hESCs源性RPE细胞提供优化方案。

最近研究显示人胚胎干细胞（hESCs）诱导分化的视网膜色素上皮（RPE）为RPE损伤和功能丧失所致的致盲性眼病提供了新的治疗手段，探索高效诱导体系成为研究热点。本项目分化第5周出现色素斑，第7周观察到胞浆充满色素颗粒的六边形细胞；hESC-RPE RPE相关mRNA CRALBP, PEDF, MITF表达量高于hESC和胎儿RPE；Western blot结果显示MITF, RPE65蛋白表达量高于hESC。FCM结果显示纯化的色素化细胞ZO-1阳性率为(80.72±3.57)%，RPE65阳性率为(75.46±1.52)%；荧光显微镜观察发现色素化细胞表达Pax6, MITF, CRALBP, RPE65, ZO1蛋白；荧光微球吞噬实验证实hESC-RPE具有吞噬功能；本项目通过分析hESCs向RPE分化过程中mRNAs 和miRNAs变化规律及其相互调控作用,筛选出具备应用潜质的促分化miRNAs (miR-29b-3p, miR-130b-3p, miR-141-3p, miR-124-3p, miR-133b, miR-363-3p, miR-488-3p)，这些miRNAs靶向COL2A1, LMO4, PITX2, LMO4, FGF1, Pax3, POMC调控分化，提高hESCs向RPE细胞的分化效率，为获得大量较高纯度的hESCs源性RPE细胞提供新途径。