Characterization of human RPE subpopulations at the single cell level

项目摘要/摘要 视网膜色素上皮(RPE)通过多种活动在支持视力方面起着至关重要的作用 保持视网膜健康。老年性黄斑变性(AMD)早期会出现RPE细胞的丢失，并伴有 随之而来的是失明。AMD是老年人失明的主要原因，其发病率预计将 随着美国人口老龄化而增加，伴随着巨大的相关个人和经济成本。AMD可以分为 分为两类：‘干性’AMD约占病例的90%，‘湿性’AMD占其余约10%。目前没有 干性AMD有有效的治疗方法，但干细胞疗法有望取代RPE细胞。 失落于干性AMD的发病机制。为此，我们已经确定了一种成人RPE干细胞(RPESC)作为潜在的 RPE细胞更换的来源。我们之前的工作利用AMD的动物模型展示了这种能力 移植的RPESC来源的RPE(RPESC-RPE)以挽救视力。我们发现一个先祖阶段 RPESC-RPE成熟化在挽救视力方面比完全分化的成熟后代更有效。在这 建议，我们的目标是首先调查存在于原生人类RPE层的RPE亚群，包括 RPESC，使用单细胞转录(AIM 1)和细胞表面筛选(AIM2)。在目标3中，我们评估 作为视觉基础的细胞和分子通路受益于移植的RPE视觉。在预赛中 使用有效评估每个RPE亚群的体外整合试验的实验，我们发现 RPE前体阶段的整合比分化的RPE后代更有效。我们计划在#年确认这些。 体外结果和描述动物模型中移植后个体亚群的整合。 此外，识别有效RPE亚群的转录本描述了一组 与视觉救援有关。我们使用生物信息学的方法来识别这些基因，然后提出 基因敲除和过表达实验，以评估其在体外和体内的功能。完成这些工作 研究将显著增加我们在细胞、分子和功能水平上对RPE的理解。

Project Summary / Abstract The retinal pigment epithelium (RPE) plays a crucial role in supporting vision through a variety of activities that maintain retinal health. A loss of RPE cells occurs early in age related macular degeneration (AMD) with a concomitant loss of vision. AMD is the leading cause of blindness in the elderly and its incidence is expected to increase as the US population ages, with great associated personal and economic costs. AMD can be divided into two categories: `dry' AMD constitutes ~90% of cases and `wet' AMD the remaining ~10%. Currently no effective treatment is available for dry AMD, but stem cell therapy holds great promise to replace the RPE cells lost in dry AMD pathogenesis. To this end, we have identified an adult RPE stem cell (RPESC) as a potential source for RPE cell replacement. Our previous work utilizing an animal model of AMD demonstrated the ability of transplanted RPESC-derived RPE (RPESC-RPE) to rescue vision. We found that a progenitor stage of RPESC-RPE maturation is more effective than fully differentiated, mature progeny at vision rescue. In this proposal, we aim to first survey the RPE subpopulations present in the native human RPE layer, including the RPESC, using single cell transcriptomics (Aim 1) and a cell surface screen (Aim2). In Aim 3, we assess the cellular and molecular pathways that underlie vision benefit by transplanted RPE vision. In preliminary experiments using an in vitro integration assay that efficiently evaluates each RPE subpopulation, we found the RPE progenitor stage integrates more effectively than differentiated RPE progeny. We plan to confirm these in vitro results and describe integration of individual subpopulations after transplantation in the animal model. Furthermore, the transcripts that identify the effective RPE subpopulation describe the set of genes that are associated with vision rescue. We use a bioinformatics approach to identify these genes and then propose knock-down and overexpression experiments to assess their function in vitro and in vivo. Completion of these studies will significantly increase our understanding of the RPE at cellular, molecular and functional levels.