Induced Pluripotent Stem Cell Approach to Glaucomatous Optic Neuropathy

描述(申请人提供)：青光眼是最常见的视神经病变，视网膜神经节细胞(RGC)进行性变性导致视力丧失。我们的长期目标是通过鉴定诱导多能干细胞(IPSCs)作为视网膜祖细胞的可再生来源来帮助防止青光眼视网膜节细胞的退化，以用于自体体外细胞治疗。此应用程序的目标是优化角膜缘的使用 IPSCs可产生功能性的、安全的、可用于临床的RGC。这项研究的中心假设是，RGC分化的分子机制在IPSC来源的视网膜前体细胞中是活跃的，并在特定的外部提示下被招募，以产生具有靶向性的RGC。我们的推理基于以下观察：(1)视网膜前体细胞可以从安全的非核酸方法产生的角膜缘IPSC分化而来；(2)IPSC来源的视网膜前体细胞对有利于RGC分化的线索有反应；(3)iPSC来源的RGC表现出靶向性。这项拟议研究的基本原理是，一旦确定了条件，我们就可以有效地产生RGC前体细胞，通过移植治疗RGC变性，并开发一个强大的模型系统来测试治疗视神经疾病的药物和遗传方法。根据我们的初步数据，我们提出了以下特定目标来检验这一假说：特定目标1：确定从IPSC产生视网膜前体细胞的条件，特定目标2：确定从IPSC来源的视网膜前体细胞产生视网膜节细胞的条件，以及特定目标3：确定IPSC来源的视网膜节细胞的靶向性和体内分化。视网膜电位将在我们实验室首创的非核酸方法产生的角膜缘IPSCs中进行检测。这种通过招募内源性多能性基因而不是引入外源基因来重新编程的方法，可能会导致插入突变，解决了基于IPSC的治疗的一个重大障碍。对照将包括由传统核酸方法提取的角膜缘IPSC，以比较两种不同的重新编程方法对视网膜和RGC电位获取的影响。IPSCs沿着神经谱系的诱导，其随后的分化为视网膜前体细胞，以及最终向RGC的分化将通过干扰特定的信号通路来概括发育机制而非细胞自主地完成。因此，我们提出的研究具有创新性，因为它提出了一种完全不同的、安全的方法，可以将体细胞重新编程为多能状态，并在不使用核酸或强制表达外源因子的情况下生成RGC。新出现的信息将是重要的，因为它不仅将解决目前使体外干细胞治疗方法不切实际的每个障碍，而且还将导致开发一个强大的模型系统，用于测试RGC发育的正常机制，并筛选药物和基因，以寻找解决青光眼视网膜变性的其他新方法。

DESCRIPTION (provided by applicant): Glaucoma is the most prevalent optic neuropathy where a progressive degeneration of retinal ganglion cells (RGCs) leads to vision loss. Our long-term goal is to help prevent the degeneration of glaucomatous RGCs by characterizing induced-pluripotent stem cells (iPSCs) as a renewable source of retinal progenitors for autologous ex vivo cell therapy. The objective of this application is to optimize the use of limbal iPSCs to generate RGCs that are functional, safe, and practical for clinical use. The central hypothesis of the proposed study is that the molecular mechanism underlying RGC differentiation is active in iPSC-derived retinal progenitors and recruited in response to specific extrinsic cues to generate RGCs with target specificity. Our reasoning is based on the following observations:(1) retinal progenitors can be derived from limbal iPSCs, generated through safe non-nucleic acid method (2) iPSC-derived retinal progenitors respond to cues conducive for RGC differentiation, and (3) iPSC-derived RGCs demonstrate target specificity. The rationale for the proposed research is that once conditions are identified, we can efficiently generate RGC precursors to treat RGC degeneration through transplantation, and develop a robust model system for testing drugs and genetic approaches for optic neuropathy. Based on our preliminary data the following specific aims are proposed to test the hypothesis: Specific Aim 1: To determine the conditions for generating retinal progenitors from iPSCs, Specific Aim 2: To determine conditions for the generation of RGCs from iPSC-derived retinal progenitors, and Specific Aim 3: To determine the target specificity and in vivo differentiation of iPSC-derived RGCs. The retinal potential will be examined in limbal iPSCs generated by non-nucleic acid means, pioneered in our lab. This approach of reprogramming by recruiting endogenous pluripotency genes instead of introducing exogenous genes, which can lead to insertional mutagenesis, addresses a significant barrier to iPSC-based therapy. Controls will include limbal iPSC derived by a conventional nucleic acid method to compare the effects of two different approaches of reprogramming on the acquisition of retinal and RGC potential. The induction of iPSCs along a neural lineage, their subsequent specification into retinal progenitors, and their final differentiation into RGCs will be accomplished non-cell autonomously by perturbing specific signaling pathways to recapitulate developmental mechanism. Therefore, our research proposed is innovative because it presents an entirely different and a safe approach for reprogramming somatic cells to a pluripotent state and generating RGCs without using nucleic acids or forced expression of exogenous factors. The emerging information will be significant because it will not only address each of the barriers that currently make the ex-vivo stem cell therapy approach impractical but also lead to the development of a robust model system for testing normal mechanisms of RGC development and for screening drugs and genes for additional new approaches for addressing glaucomatous retinal degeneration.