生物反应器重编程来源细胞对角膜内皮和视网膜色素上皮损伤修复的作用

Corneal endothelial cells (CE) and retinal pigment epithelial cells (RPE) are key cells for cornea and retina. iPSCs can be used for the treatment of CE and RPE lesions, but the safety of iPSCs has been difficult to resolve. Based on the problem, the project carries out the study to safely produce iPSCs, CE and RPE derived from reprogramming and their transplantations for the repair of CE and RPE disorders. The project makes adipose derived stem cells (ADSCs) into p-c-iPSCs by using bioreactors, recombinant reprogramming proteins and small molecular compounds. SDIA co-cultures are used to promote the differentiation of iPSCs into CE and RPE. The integrated use of microgravity and perfused bioreactors for direct reprogramming makes ADSCs into CE and RPE and their transplantations are operated. The tests of gene differences, multiple signal pathways, electric potential differences of CE and ERG are carried out. The experimental and theoretical systems of non-DNA direct reprogramming and differentiating CE and RPE with function are established. This project is innovative in the use of bioreactors for c-p-iPSCs induction, direct reprogramming and cell sheet constructs, MAX and CHC for obtaining high activity of CE and RPE, and 18F-FDG-PET live tracking and positioning of transplanted cells in the acute stage. The project can provide new ways and theoretical basis for the treatment of the lesions of CE and RPE, which are important for the recovery of blind diseases from cornea and retina.

角膜内皮细胞(CE)和视网膜色素上皮细胞(RPE)是角膜和视网膜的重要细胞，iPSCs可用于治疗CE和RPE病变，但iPSCs的安全性一直难以解决。本项目基于此开展安全的iPSCs诱导、重编程获得CE和RPE并进行移植。利用生物反应器、重编程蛋白和小分子，诱导ADSCs为p-c-iPSCs；SDIA共培养使iPSCs分化为CE和RPE；一体化微重力和灌注培养直接重编程使ADSCs成为CE和RPE；CE和RPE移植。进行基因差异、多信号通路、CE电位差、ERG等检测，建立非DNA重编程定向分化为功能CE和RPE的实验和理论体系。本项目在生物反应器参与的c-p-iPSCs诱导、直接重编程和细胞片构建，MAX和CHC获取高活性的CE和RPE，18F-FDG-PET活体示踪进行急性期移植细胞定位等方面具有创新性。本项目为治疗影响角膜与视网膜致盲疾病的重要细胞CE和RPE病变提供新的手段和理论依据。

1. 主要背景及研究内容：本项目拟探索安全的干细胞重编程、分化及移植用于具有同样六边形单层结构的角膜内皮 (CE)及视网膜色素上皮(RPE)的规律及分子机制。（1）非DNA的p-c-iPSCs诱导及其CE细胞和RPE细胞分化。 （2）生物反应器在非DNA重编程诱导分化的作用。（3）p-c-iPSCs及非DNA直接重编程来源的CE和RPE移植修复CE和RPE损伤。2. 重要结果及数据：（1）微重力生物反应器可以促进干细胞重编程。（2）通过多细胞微球模具技术产生的角膜内皮细胞球在注射移植方面有着更多的优势。（3）促进人iPSCs单细胞生长半换液自条件培养液有利于安全的干细胞重编程定向分化为功能CE和RPE的实验体系建立。（4）非基因处理的球形细胞培养促进靶细胞干性。PTD-Oct4/Klf4/Sox2 +VPA促进细胞球形聚集克隆生长。（5）应用灌注生物培养器系统及球形培养，可以提供仿生微环境，促进CE增殖扩增，以及CE细胞片构建。与细胞蛋白激酶活性、cAMP 反应元素结合和AMP活化蛋白激酶活性通路有关。（6）ADSCs存在PAC1受体，PAC1受体特异激动剂Maxadilan对ADSCs有促增殖和抗凋亡作用。促进ADSCs 的神经分化与去分化后的再分化。（7）Matrigel和Activin A（MA）处理促进了人角膜视网膜色素上皮细胞（RPE）间的接触与连接和抗凋亡。通过外加小分子循序定向诱导单散hiPSCs分化为RPE，分化产生的hiPSCs-RPE能阳性表达RPE特异性相关标志蛋白和基因，hiPSCs-RPE纯度：74.4% (RPE-65), 85.8% (Mitf), 97.4% (CRALBP)和93.8% (EMMPRIN)。ERG结果显示hiPSCs-RPE细胞球移植可部分恢复碘酸钠致RPE缺损色素兔的ERG波形，并能检测到b波信号 (*P<0.05）。且石蜡切片显示体内移植的hiPSCs-RPE细胞球（带PKH26标记）能较好的在体内存活, 并成局部片状生长。3. 科学意义：CE 和RPE是角膜和视网膜关键细胞。iPSCs可用于治疗CE和RPE病变，但iPSCs的安全性一直难以解决。本项目进行安全的iPSCs诱导和分化为CE 和RPE，具有重要的意义。本项目为治疗影响角膜与视网膜致盲疾病的重要细胞CE和RPE病变提供新的手段和理论依据。