Induced pluripotent stem cell approach to optic nerve regeneration

摘要 青光眼是最常见的视神经病变，其中视网膜神经节细胞的进行性变性 （RGC）导致视力丧失。我们的长期目标是通过以下方法帮助预防青光眼RGC的变性： 本发明的目的是将多能干细胞表征为用于自体离体细胞疗法的RGC的可再生来源。的目标 该更新申请是为了解决下一个与人多能的潜在临床应用相关的问题 细胞来源的RGC：这些细胞是否可以精心制作可引导的轴突，这些轴突可以导航出宿主视网膜， 寻求真正的目标，这对逆转视力丧失至关重要。据我们所知，这个问题对于实际的离体 干细胞方法治疗昏迷性变性，仍然没有答案。拟议研究的中心假设是 人类诱导多能干细胞衍生的RGC（hiPSC-RGC）精心制作了可引导的轴突，由 mTOR途径，一种内在调节剂轴突发生和再生。我们的推理是基于我们的观察， hiPSC-RGC是（1）稳定、功能性和安全的（2）表达导向受体并对近端（内） 视网膜）和远端（视网膜外）引导线索，和（3）具有活性mTOR通路，调节发育和 轴突发生我们的基本原理是，hiPSC-RGC重演轴突生长机制的能力， 指导将证明它们是一种可行的试剂，可以在青光眼中功能性地替代变性的RGC。以下 目的1：确定hiPSC-RGC对轴突导向的能力 目的2：确定hiPSC-RGC用于mTOR依赖性轴突发生的能力， 目的3：确定新生儿和成人视网膜中mTOR依赖性hiPSC-RGC轴突发生。 hiPSC-RGC用于轴突发生和轴突引导的潜力将在共培养范例中使用以下方法进行检查： 微流控系统在受控条件下。已知途径和转录的免疫细胞化学分析 谱分析将识别候选调节因子。mTOR背景下hiPSC-RGC的再生能力 通路将在我们实验室建立的轴突切断模型的微流体模型中进行检查。转录谱在 轴突切断前、轴突切断和轴突切断后阶段将鉴定再生基因调控网络。最后， hiPSC-RGC的再生能力和mTOR途径的影响将在新生儿视网膜中进行体内检查， 其中环境有利于轴突生长，并且在青光眼动物模型中处于退化的成人环境中。 我们的研究方案是创新的，因为它将决定从头产生的神经元是否可以在功能上 取代那些长距离连接的细胞，如RGC，并填补我们对人类RGC知识的空白 发育和轴突路径发现，视神经再生的障碍。新出现的信息将是重要的 因为它不仅解决了目前离体干细胞治疗中最重要的障碍， 这种方法不切实际，但也导致开发用于测试正常/病理的鲁棒模型系统 RGC的发展机制，并筛选药物和基因的额外的新的治疗方法， 青光眼性视网膜变性。

ABSTRACT 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 pluripotent stem cells as a renewable source of RGCs for autologous ex vivo cell therapy. The objective of this renewal application is to address the next question relevant to the potential clinical application of human pluripotent cell-derived RGCs: whether or not these cells can elaborate guidable axons that can navigate out of the host retina and seek bonafide targets, essential for reversing vision loss. To our knowledge this question, essential for practical ex-vivo stem cell approach to glaucomatous degeneration, remains unanswered. The central hypothesis of the proposed study is that human induced pluripotent stem cells derived RGCs (hiPSC-RGCs) elaborate guidable axons, regulated by the mTOR pathway, an intrinsic regulator axonogenesis and regeneration. Our reasoning is based on our observations that hiPSC-RGCs are (1) stable, functional, and safe (2) express guidance receptors and respond to both proximal (intra- retinal) and distal (extra-retinal) guidance cues, and (3) have active mTOR pathway, regulating development and neuritogenesis. Our rationale is that the ability of hiPSC-RGCs to recapitulate the mechanism of axon growth and guidance will posit them as a viable reagent to functionally replace degenerated RGCs in glaucoma. The following specific aims are proposed to test the hypothesis: Aim 1: To determine the competence of hiPSC-RGCs for axon guidance and target specificity, Aim 2: To determine the competence of hiPSC-RGCs for mTOR-dependent axonogenesis and regeneration in vitro, and Aim 3: To determine mTOR-dependent hiPSC-RGC axonogenesis in neonatal and adult retina. The potential of hiPSC-RGCs for axonogenesis and axon guidance will be examined in co-culture paradigm using the microfluidic system in controlled conditions. Immunocytochemical analysis of known pathways and transcriptional profiling would identify candidate regulatory factors. The regenerative ability of hiPSC-RGCs in the context of mTOR pathway will be examined in a microfluidic model of the axotomy model, established in our lab. Transcription profile at pre-axotomy, axotomy, and post-axotomy stages would identify regenerative gene regulatory network. Finally, regenerative capacity of hiPSC-RGCs and the influence of the mTOR pathway will be examined in vivo in neonatal retina, where environment is conducive for axon growth and in a degenerative adult environment in animal model of glaucoma. Our research proposal is innovative because it will determine whether the de novo generated neurons can functionally replace those that make long distance connections such as RGCs and bridge a gap in our knowledge about human RGC development and axon path finding, a barrier to optic nerve regeneration. The emerging information will be significant because it will not only address each of the most significant 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/pathological mechanisms of RGC development and for screening drugs and genes for additional new therapeutic approaches for glaucomatous retinal degeneration.