小分子化合物组合诱导人成纤维细胞向视网膜色素上皮细胞转分化的研究

Retinal pigment epithelium (RPE) cells play a key role on retinal function. Loss of RPE is one of the main causes in retinal degeneration. Currently, there is no radical treatment for this disease. A number of studies have shown that human embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) could be induced into RPE cells. Stem cell-derived RPE transplantation could restore vision in clinical trials of human retinal degenerative disease. However the clinical application has been greatly limited by the ethical issue of ESC and the genetic manipulation involved of iPSC. Recently, pluripotent stem cells and functional neurons can be generated from mouse somatic cells using a cocktail of seven small molecules. Based on the previous dedication, we plan to transdifferentiate functional RPE cells from human fibroblasts by small molecule compounds, and culture in serum-free medium, then transplant them into the subretinal space of the RCS rats, a conventional model for the disease. We will develope xeno-free and defined functional RPE cells differentiation method from adult cells by small molecules.Functional RPE cells will be transplantated into RCS rats by subretinal transplantation.This study will provide key basis for clinical treatment of human retinal degenerative disease.

视网膜色素上皮（RPE）细胞对神经视网膜的功能维持至关重要。RPE的变性是导致视网膜变性类疾病发生的关键原因，而目前，临床上尚无此类疾病有效的治疗方法。大量研究已表明人胚胎干细胞（ESC）和诱导多能干细胞（iPSC）均可向RPE细胞分化。但ESC涉及的伦理问题和异体排斥反应以及iPSC外源性基因的安全性问题极大地限制了多能干细胞来源RPE的临床转化应用。最近的研究显示，小分子化合物可将鼠成纤维细胞重编程为多能干细胞以及向神经细胞转分化。本项目基于以往的工作基础，拟应用小分子化合物组合诱导人成纤维细胞向功能性RPE细胞的转分化，将基于无血清无异源性成分诱导培养的RPE细胞通过视网膜下腔移植治疗RCS大鼠模型并进行视觉功能评价。本项目将建立从人成体细胞向功能性RPE细胞转分化的小分子策略，建立无血清无异源性成分向RPE细胞诱导分化的体系，为视网膜变性相关疾病的临床细胞治疗提供关键科学依据。

视网膜色素上皮（RPE）细胞移植最关键的问题是供体细胞的来源。小分子化合物调控细胞命运转变是干细胞与再生医学领域的研究热点。本项目通过筛选发现，小分子化合物616452，CHIR99021，LDN193189，A83-01， RA，SMER28, RG108, Parnate，5-Aza等能够有效诱导体外培养的成纤维细胞直接转分化RPE细胞；RPE细胞的生物学特征与视网膜来源的RPE生物学特性相似，包括细胞增殖、自我更新、基因表达谱、体外和体内条件下功能；将小分子化合物组合诱导来源的功能性RPE细胞，通过视网膜下腔移植治疗RCS大鼠模型。视觉功能评价表明转分化来源RPE细胞在体内能发挥功能。在以上研究基础之上，进一步发现小分子化合物组合在将成纤维细胞诱导RPE转分化过程中经历一个中间态过程。我们初步证明整个中间态过程为神经干细胞。我们的研究不但开启了细胞命运调控的新方向，而且为基于RPE的细胞治疗提供了新的种子细胞来源，为视网膜色素类疾病治疗提供了全新方案。