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

项目总结 视网膜外变性疾病(RDDS)导致光感受器(PR)细胞死亡是导致 全球范围内的视力受损，但拯救或恢复其中许多患者的视力的选择有限。 人类多能干细胞(HPSC)来源的PR移植越来越多地被研究为一种治疗方法 这些患者的治疗策略，视网膜内的神经再生最近被确定为 国家眼科研究所(NEI)的战略重点。一些临床前研究表明，在某种程度上 团注PR移植修复视力的动物模型及安全性临床试验 团注胎源性视网膜前体细胞对严重视网膜变性患者的疗效是 目前正在进行中。尽管最近取得了这些成功，但在此之前，该领域仍面临着几个关键障碍 大多数RDD患者可以实现临床PR替代治疗。目前视网膜下推注的治疗策略 游离PR的传递不能准确地重建外部视网膜的复杂组织，并且它们 通常伴随着组织混乱、不可预测的剂量和紧随其后的细胞总数低 由于玻璃体腔内回流而注射。此外，尚不清楚视觉反应是否通常 归因于移植的供体PR实际上是由于解剖整合和功能性突触的形成 在宿主退化的视网膜内。事实上，PR移植后突触形成的效率，以及 新生突触发生和视功能测量之间的关系还没有被测试到 约会。 在这里，我们寻求使用最先进的生物材料和PR支架以及严格的突触追踪 在严重光感受器变性的大鼠模型中解决这些挑战的方法。在目标1中，我们 将使用一种新型的微图案、可生物降解的支架进行靶向hPSC-PR移植 团剂传递和支架传递的PR在体内的保留、存活和成熟。在目标2中，我们将 在变性视网膜和活体宿主变性视网膜中确定hPSC-PR的突触连接性 组织与创新的单突触逆行示踪分析。威斯康星大学麦迪逊分校培养 为成功完成这些目标提供了理想的科学和智力环境， 合作研究社区，涵盖眼科、生物医学工程和 再生医学。此处详述的研究提案和研究金培训计划旨在解决 目前公关更换领域内的障碍，同时还提供必要的技能集来解决 新兴的视网膜再生领域面临的下一代挑战。

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.