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Induced pluripotent stem cell approach to optic nerve regeneration

诱导多能干细胞方法促进视神经再生

基本信息

批准号：
10411954
负责人：
Iqbal Ahmad
金额：
$ 36.98万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-12-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10411954
关键词：
Address Adult Affect African American population American Animal Model Autologous Axon Axotomy Biological Models Blindness Brain Cell Therapy Cell Transplantation Cells Chemicals Coculture Techniques Competence Cues Development Disease Distal Drug Screening Early Diagnosis Environment FRAP1 gene Ganglion Cell Layer Gene Expression Profile Gene Expression Profiling Generations Genes Glaucoma Goals Human In Vitro Knowledge Lead Light Methods Microfluidics Mind Modeling Molecular Target Mus Natural regeneration Neonatal Neurons Optic Chiasm Outcome Pathologic Pathway interactions Physiologic Intraocular Pressure Pluripotent Stem Cells Primary Open Angle Glaucoma Process Program Development Rattus Reagent Regenerative capacity Research Research Proposals Retina Retinal Degeneration Retinal Ganglion Cells Rodent Signal Transduction Somatic Cell Source Specificity System Testing Transplantation axon growth axon guidance base clinical application clinically significant disease-in-a-dish effective therapy experimental study gain of function gene regulatory network immunosuppressed in vitro Model in vivo induced pluripotent stem cell innovation loss of function molecular targeted therapies novel therapeutic intervention optic nerve disorder optic nerve regeneration pluripotency prevent receptor recruit regenerative response retinal ganglion cell degeneration retinal ganglion cell regeneration retinal progenitor cell stem cell therapy stem cells synaptogenesis

项目摘要

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.

摘要青光眼是最常见的视神经病变，视网膜神经节细胞进行性变性。 (RGCs)会导致视力丧失。我们的长期目标是帮助防止青光眼视网膜节细胞退化将多能干细胞表征为用于自体体外细胞治疗的RGC的可再生来源。的目标是这一更新申请是为了解决与人类多能性潜在临床应用相关的下一个问题。细胞来源的RGC：这些细胞是否能够形成可引导的轴突，这些轴突可以导航到宿主视网膜和寻找真正的目标，这对扭转视力损失至关重要。据我们所知，这个问题对于实际的体外实验是必不可少的干细胞治疗青光眼变性的方法仍然没有答案。这项拟议研究的中心假设是人类诱导多能干细胞来源的视网膜节细胞(hiPSC-RGCs)形成可引导的轴突，受 MTOR通路--轴突发生和再生的内在调节因子。我们的推理是基于我们观察到的 HIPSC-RGCs是(1)稳定、功能和安全的(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，以及(3)具有活跃的mTOR途径，调节发育和神经发生。我们的理论基础是，HiPSC-RGCs概括轴突生长和指南将证明它们是一种可行的试剂，可以在功能上取代青光眼中退化的视网膜节细胞。以下是提出了检验假设的具体目标：目标1：确定HiPSC-RGCs对轴突引导的能力和靶特异性，目标2：确定HiPSC-RGCs对mTOR依赖的轴突发生和目的3：检测新生和成人视网膜中mTOR依赖的HiPSC-RGC轴突发生。 HiPSC-RGCs在轴突发生和轴突引导方面的潜力将在共培养范式中进行研究，使用受控条件下的微流控系统。已知途径和转录的免疫细胞化学分析概况分析将确定候选的监管因素。MTOR环境下HIPSC-RGCs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测。转录配置文件位于轴突切断前、切断轴突和切断轴突后三个阶段将识别再生基因调控网络。最后， HIPSC-RGCs的再生能力和mTOR通路的影响将在新生视网膜中进行活体检测。在青光眼动物模型中，环境有利于轴突生长和退化的成人环境。我们的研究方案是创新的，因为它将确定从头生成的神经元是否可以在功能上取代那些建立远距离联系的人，如RGC，并弥合我们对人类RGC知识的差距发育和轴突路径寻找，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使体外干细胞疗法方法不切实际，但也导致了用于测试正常/病理的健壮模型系统的开发 RGC的发育机制及筛选药物和基因以寻找新的治疗方法青光眼视网膜变性。