Toward enhancing organization and defining synaptic connectivity of transplanted human pluripotent stem cell-derived photoreceptor grafts

项目摘要 导致光感受器（PR）细胞死亡的外视网膜变性疾病（RDD）是视网膜变性的主要原因。 视力障碍是世界范围内最常见的疾病，但在许多此类患者中，挽救或恢复视力的选择有限。 人类多能干细胞（hPSC）衍生的PR移植越来越多地被研究作为治疗方法 视网膜内的神经再生最近已被确定为一个领域， 国家眼科研究所（NEI）的战略重点。一些临床前研究表明， 在动物模型中使用推注输送的PR移植物进行视力恢复，以及研究安全性的临床试验 在患有严重视网膜变性的患者中， 目前正在进行中。尽管最近取得了这些成功，但该领域仍面临着几个关键的障碍， 对于大多数RDD患者可以实现临床PR替代治疗。视网膜下团注治疗的当前策略 分离的PR的递送不能准确地重建外视网膜的复杂组织，并且它们 通常伴随着混乱，不可预测的剂量，以及在治疗后立即出现的总体细胞计数低 注射由于回流到玻璃体腔。此外，目前还不清楚视觉反应是否通常 归因于移植供体PR实际上是由于解剖整合和功能性突触形成 在宿主退化的视网膜内事实上，PR移植后突触形成的效率， 从头突触发生和视觉功能测量之间的关系尚未被测试， 约会 在这里，我们寻求使用最先进的生物材料和PR支架沿着严格的突触追踪 在严重的感光细胞变性的大鼠模型中，这些方法可以解决这些挑战。目标1： 将使用一种新型的微图案化，可生物降解的支架靶向hPSC-PR移植，以评估 体内推注递送和支架递送的PR的保留、存活和成熟。在目标2中，我们 定义hPSC-PR在变性视网膜外植体和活宿主变性视网膜外植体中的突触连接性。 组织与创新的单突触逆行追踪分析。威斯康星大学麦迪逊分校 理想的科学和知识环境，成功地完成这些目标， 合作研究社区跨越眼科学，生物医学工程领域， 再生医学这里详细介绍的研究建议和研究金培训计划旨在解决 目前在公关更换领域的障碍，同时也提供必要的技能，以解决 下一代的挑战面临着蓬勃发展的领域的视网膜再生。

PROJECT SUMMARY Outer retinal degenerative diseases (RDDs) resulting in photoreceptor (PR) cell death are a leading cause of visual impairment worldwide, but options for rescuing or restoring vision in many of these patients are limited. Human pluripotent stem cell (hPSC)-derived PR transplantation is increasingly being studied as a therapeutic strategy for these patients, and neural regeneration within the retina has recently been identified as an area of strategic focus by the National Eye Institute (NEI). Several preclinical studies have shown some degree of visual restoration with bolus-delivered PR transplants in animal models, and clinical trials studying the safety and efficacy of bolus-delivered fetal-derived retinal precursors in patients with severe retinal degeneration are currently underway. Despite these recent successes, the field still faces several critical roadblocks before clinical PR replacement therapy can be realized for most RDD patients. Current strategies for bolus subretinal delivery of dissociated PRs fail to accurately reconstitute the complex organization of the outer retina, and they are often accompanied by disorganization, unpredictable dosing, and overall low cell counts immediately after injection due to reflux into the vitreous cavity. Further, it remains unclear whether visual responses commonly attributed to transplanted donor PRs are actually due to anatomic integration and functional synapse formation within the host degenerate retina. Indeed, the efficiency of synapse formation following PR transplantation, and the relationship between de novo synaptogenesis and measurements of visual function has not been tested to date. Here, we seek to use state-of-the-art biomaterials and PR scaffolds along with rigorous synaptic tracing methodologies to address these challenges in a rat model of severe photoreceptor degeneration. In Aim 1, we will use a novel micro-patterned, biodegradable scaffold for targeted hPSC-PR transplantation to assess the retention, survival, and maturation of bolus-delivered and scaffold-delivered PRs in vivo. In Aim 2, we will define the synaptic connectivity of hPSC-PRs in degenerate retinal explants and live host degenerate retinal tissue with an innovative monosynaptic retrograde tracing assay. The University of Wisconsin-Madison fosters the ideal scientific and intellectual environment for successful completion of these aims with strong, collaborative research communities spanning the fields of ophthalmology, biomedical engineering, and regenerative medicine. The research proposal and fellowship training plans detailed here seek to address current roadblocks within the field of PR replacement while also providing the necessary skillset to address the next generation of challenges facing the burgeoning field of retinal regeneration.