诱导多能干细胞来源的神经干细胞在猴脑中的安全性和功能研究

Although induced pluripotent cells (iPS) from patient somatic cells provide the possibility to get enough donor cells for understanding disease mechanisms in vitro and for use in drug screens and the clinic stem cell therapy, it is unclear the safety of primate iPS-derived differentiated cells as donor cells in vivo. Additionally, it is obscure whether these differentiated cells can form functional cells in host tissues.In the previous studies, we used three different strategies (retrovirus infection, sendai virus infection and episomal vector transfection) to produce iPS cells from skin fibroblast of cynomolgus monkeys, and then induced these iPS cells differentiation into neural stem cells (NSCs). We called these NSCs as riPS-NSCs (retrovirus), siPS-NSCs (sendai virus) and eiPS-NSCs (episomal vector), respectively. Furthermore, we established the culture condition by which NSCs can be maintained in the neuroepithelium stage over long-term culture and support single NSC growing into a stable cell line. The objective in the study is to evaluate the safety and functionality of riPS-NSCs, siPS-NSCs and eiPS-NSCs and figure out their differences among three types of NSCs after grafted into monkey brain. To avoid the contamination of few iPS cells, single cell from three types of iPS-NSCs is cultured into a stable cell line by our established condition, respectively. Rosette-like NSCs lines are identified by the gene expression profiles, staining and differentiation potentials, and neuroepithelium stage NSCs are chosen to test their safety and functionality in the study. Two different experiment assays would be carried out. First experiment is to inject neuroepithelium stage riPS-NSCs, siPS-NSCs and eiPS-NSCs into cynomolgus monkey embryos' brains, respectively. At the one-month, one-year and three-year-old postnatal, these monkeys are euthanasia and safety and functionality are evaluated by the analysis of cell fate in the brain. In the words, test whether there is any tumor growth, or grafted cells integrate into host tissue by migration and differentiate into neurons which can form the communication with host neurons. To avoid the interference of different genetic background with our data, riPS-NSCs, siPS-NSCs and eiPS-NSCs are labeled by three different fluorescein color proteins, respectively. For example, riPS-NSCs by GFP (green), siPS-NSCs by mCherry (red) and eiPS-NSCs by BFP (blue). The mixed cells are injected into the same embryos' brain and competing assay is used to figure out what the difference among them.To further evaluate the safety and functionality of iPS-NSCs, adult brains injured by mechanical methods are used to receive the injection of these labeled iPS-NSCs. Safety and functionality are also figured out by the analysis of cell fate of transplanted cells in vivo. The study would provide the important data about safety and functionality of iPS cells and promote its application in the future clinic.

病人体细胞的重编程为干细胞替代治疗、疾病机制研究和药物筛选提供了大量的细胞来源。但是灵长类iPS（包括人和猴）在体内的安全性以及能否产生功能的分化细胞并不清楚。本项目建立在我们的前期研究基础上，将三种不同策略产生的多株猴iPS细胞系分化的神经干细胞（NSCs）,通过单细胞克隆去除少量iPS细胞污染，并挑选处于早期发育阶段的NSCs，即神经上皮细胞，作为细胞模型，以与人脑结构、功能和发育相似的猴作为动物模型，开展iPS-NSCs在脑内的长期安全性和功能性的研究。通过分别将iPS-NSCs移植到发育同步的胎猴脑以及损伤的成年猴大脑皮层中，分析细胞在体内的命运，即能否整合到脑部参与早期胎猴脑部的发育，能否和受体神经元建立功能环路，以及能否替代成年受损的神经元，回答三种不同方法产生的iPS细胞的安全性和功能性是否存在显著差异。通过本项目的研究将为灵长类iPS细胞的安全性和有效性提供重要的数据。

本项目围绕目标，在多能干细胞（PSCs）分离培养、PSCs分化和体细胞直接转分化产生的功能神经上皮干细胞、特定神经细胞以及细胞移植方面取得了多项重要的突破，建立了一系列神经干细胞和特定神经细胞培养、分化的系统，证明了重编程产生的神经干细胞的安全性，完成了整个课题的全部预期目标。并根据学科的发展情况，增加多项研究内容。项目取得了一系列重要的成果：1）建立体内发育多能性的猴子多能干细胞，并首次在世界上证明猴子多能干细胞能产生嵌合体，其成果产生了重要的影响力，在该领域引起了极大关注；2）首次证明传统培养的Primed猴ESCs、诱导多能干细胞（iPSCs）以及核移植来源的ESCs（nt-ESCs）当注射到桑椹胚后，抑制其凋亡后能产生嵌合猴，表现出安全性；3）建立了一套可规模化的三维悬浮培养体系。采用该体系，成功实现了人和猴PSCs的长期稳定扩增，为细胞的大量生产提供了重要的基础；4）建立了一套PSCs分化为神经上皮干细胞、神经上皮干细胞的稳定扩增以及利用神经上皮干细胞开展神经疾病与发育研究的创新性体系；5）成功将人PSCs高效分化为功能的、可移植的人大脑皮层投射神经元，为再生出特定成熟神经元提供了基础；6）建立了灵长类成纤维细胞直接转分化为功能端脑神经上皮干细胞的体系，为个性化医疗和精准治疗提供了重要的研究基础；7）证明了神经干细胞在大脑里的存活和分化与细胞移植区域以及移植前细胞的特性有关。项目共发表6篇SCI论文，分别发表于Cell Stem Cell、Biomaterials（2篇）、Stem cell Reports、Scientific Reports和Current Stem Cell Research & Therapy，共计影响因子达到55。发表国内核心期刊２篇，1篇SCI论文正在审稿。授权1项专利，申请获得1项PCT专利，申请1项中国专利和1项美国专利。培养2名博士研究生和６名硕士研究生。