Characterization of human RPE subpopulations at the single cell level

项目总结/摘要 视网膜色素上皮（RPE）通过各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。RPE细胞的损失发生在年龄相关性黄斑变性（AMD）的早期， 伴随的视力丧失。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的增加，伴随着巨大的个人和经济成本。AMD可以分为 分为两类：“干性”AMD占病例的约90%，“湿性”AMD占剩余的约10%。目前没有 干细胞疗法是治疗干性AMD的有效方法，但干细胞疗法有望取代RPE细胞 在干性AMD发病机制中丢失。为此，我们已经确定了成人RPE干细胞（RPESC）作为一种潜在的干细胞。 用于RPE细胞替换的源。我们以前的工作利用AMD的动物模型证明了这种能力 移植的RPESC衍生的RPE（RPESC-RPE）来拯救视力。我们发现， RPESC-RPE成熟在视力拯救方面比完全分化的成熟后代更有效。在这 根据这项提议，我们的目标是首先调查存在于天然人RPE层中的RPE亚群，包括 RPESC，使用单细胞转录组学（Aim 1）和细胞表面筛选（Aim 2）。在目标3中， 细胞和分子通路的基础视力受益于移植的RPE视力。初步 使用有效评估每个RPE亚群的体外整合测定的实验，我们发现 RPE祖细胞阶段比分化的RPE后代更有效地整合。我们计划在 体外结果并描述了在动物模型中移植后个体亚群的整合。 此外，鉴定有效RPE亚群的转录本描述了与RPE亚群相关的基因集。 与视力拯救有关。我们使用生物信息学的方法来识别这些基因，然后提出 敲低和过表达实验以评估它们在体外和体内的功能。完成这些 这些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.