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。

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.