不同体细胞来源人诱导多能干细胞（hiPSCs）向3D视网膜分化潜能及表观遗传机制

Establish differentiation technology of three-dimensional retina in vitro, which bring promising therapy to retinal degenerative disease. But, how to obtain high quality retina is urgently need to be solved. Through hiPSCs have the similar properties to ESCs, the differentiation potential take on unique propensity. However, whether somatic cell types can influence differentiation efficiency of hiPSCs to 3D retina and the mechanism remain unclear. Previous study have set one 3D retina differentiation system which can recapitulate spatiotemporally each of the main steps of retinal development observed in vivo, and generate multilayer and light response function retina. On that basis, the current project aim to compare the differentiation potential of blood, urine, Tenon’s capsule or skin derived hiPSCs to 3D retina accordingly. The main goals are to screen the high 3D retina differentiation potential hiPSCs, and investigate the mechanism of epigenetic memory further more. This project will provide cell source selection opinions for disease model based on stem cell or retina transplant therapy, and supply molecular information for reprogramming.

体外3D视网膜分化技术的建立，为退行性视网膜病变的治疗带来了希望。如何获得高质量的视网膜成为亟待解决的问题。研究表明hiPSCs具有胚胎干细胞（ESCs）样特性，但分化潜能有其独特倾向性。体细胞类型对hiPSCs向3D视网膜分化效率影响如何？hiPSCs的表观遗传记忆对其影响机理是什么？这种记忆是否可以消除？目前尚无定论。前期研究已经建立了一套可以模拟体内视网膜时空发育的分化系统，培育出具有板层结构及光反应功能的hiPSCs源的3D视网膜。在此基础上，本项目拟利用血液、尿液、Tenon’s囊或皮肤组织来源的hiPSCs，凭借该3D视网膜分化平台，比较不同体细胞来源hiPSC的3D视网膜分化潜能，拟获得高效3D视网膜分化潜能的hiPSCs系，并初步探讨hiPSCs向3D视网膜分化的表观遗传机制。该项目为基于干细胞的疾病模型研究或视网膜移植治疗提供细胞来源选择依据，同时为重编程提供分子信息。

视网膜退行性疾病具有不可逆性，该类疾病尚无有效治疗方法，视网膜移植带来新的希望。目前已利用hiPSCs成功获得3D视网膜组织，但是不同hiPSCs在分化效率方面存在很大差异，亟需筛选高效分化细胞株获得目的组织。基于此，本项目优化了3D视网膜分化系统，建立了单个EB分化体系，并系统性地比较了来自血液，尿液，Tenon’s囊和皮肤组织体细胞来源hiPSCs在视网膜早期分化过程的效率，合计约11株细胞系进行了视网膜分析比较。结果显示，血液来源hiPSCs在早期视网膜分化效率方面最高，尿液来源hiPSCs次之，Tenon’s囊和皮肤组织体细胞来源hiPSCs在早期视网膜分化效率方面相对较低。初步比较了血液和尿液hiPSCs的转录组差异，二者具有明显不同的转录组，存在约300多个差异基因。血液来源hiPSCs的Nog，Six3和Pax6基因表达水平高于尿液组，或许与视网膜早期分化有关，需进一步证明。然而，经过长期培养血液和尿液来源hiPSCs获得的3D视网膜组织都可以形成成熟的光感受器细胞。血液和尿液体细胞获得具有无创性，取材方面有优势，结合hiPSCs分化效率，为未来优化临床级别hiPSCs及视网膜组织提供基础依据。