Characterizing Human RPE Cell Proliferation to Advance Endogenous Regeneration

项目摘要/摘要 数十年的研究表明，成人视网膜色素上皮(RPE)细胞具有强大的 体外增殖能力，表明RPE层具有自我修复的可能性。我们已经展示了 来自老年捐赠者或患有老年性黄斑变性(AMD)的捐赠者的成年RPE可以在 培养并产生接近本地的、更新的RPE单层。尽管如此，体内的RPE细胞不能再生 变性细胞患者的鹅卵石RPE层受损，例如干性AMD患者。这个 体内环境必须有效地防止鹅卵石RPE单层的修复，无论是通过缺乏 有丝分裂原或抑制分子，或两者的组合。这些研究的首要目标是实现 内源性RPE细胞安全、可控的增殖，使RPE层能够自我修复 AMD。这项建议的目标是描述积极和消极的环境因素。 控制成人RPE细胞的增殖。第一个具体目标是描述成人RPE的特征 利用创新的质谱学和生物信息学研究分裂细胞和未分裂细胞的细胞表面组 站台。这将首次对RPE质膜上的分子进行全面分析，并 揭示细胞表面受体和分泌的蛋白质，对影响细胞分裂的环境因素做出反应。 同时研究正常和AMD RPE将有助于更好地理解RPE细胞如何随着 疾病。第二个特定的目标是检查我们已经确定的与 用成人RPE细胞的增殖来确定哪些分子是关键的。为了有效地做到这一点，我们将 首先采用CRISPRi，然后使用额外的功能屏幕。第三个具体目标将检查外生性 因子可以激活静止的鹅卵石人RPE的增殖，包括Bruch‘s上的原位RPE 膜外植体，以及AMD患者的膜外植体。除了主要目标外，这项研究还将产生 关于可用于体内靶向RPE的RPE分子的新知识。拟议中的工作还具有 提高RPE细胞体外增殖的潜力，以实现更高效的细胞制造。最重要的是，这 该研究将为体内安全刺激RPE细胞增殖奠定基础。RPE的内源性激活 用细胞增殖来对抗AMD患者RPE细胞的丢失有可能避免手术和免疫抑制 参与RPE细胞移植，这将极大地造福老年AMD患者群体。

Project Summary / Abstract Decades of study have demonstrated that adult human retinal pigment epithelium (RPE) cells have strong proliferative capacity in vitro, which indicates that the RPE layer has the possibility of self-repair. We have shown that adult RPE from elderly donors or donors with age-related macular degeneration (AMD) can proliferate in culture and produce a near-native, renewed RPE monolayer. Despite this, RPE cells in vivo do not regenerate the damaged cobblestone RPE layer in patients with degenerating cells, such as those with dry AMD. The environment in vivo must effectively prevent repair of the cobblestone RPE monolayer, either through lack of mitogens or inhibitory molecules or a combination of both. The overarching goal of these studies is to achieve safe, controlled proliferation of endogenous RPE cells to enable self-repair of the RPE layer in patients with AMD. The objective of this proposal is to characterize the environmental factors that positively and negatively control the proliferation of adult human RPE cells. The first specific aim is to characterize the adult human RPE cell surfaceome on dividing and non-dividing cells using an innovative mass spectrometry and bioinformatic platform. This will provide the first comprehensive analysis of the molecules on the RPE plasma membrane and reveal cell surface receptors and secreted proteins that respond to environmental factors impacting cell division. Investigating both normal and AMD RPE will provide a greater understanding of how RPE cells change with disease. The second specific aim is to examine a transcriptional network we have identified that is associated with adult human RPE cell proliferation to determine which molecules are critical. To do this efficiently, we will first employ CRISPRi, then additional functional screens. The third specific aim will examine whether exogenous factors can activate proliferation of quiescent, cobblestone human RPE, including those in situ on Bruch’s membrane explants, and those from patients with AMD. In addition to the main objective, this study will generate new knowledge about RPE molecules that can be used to target RPE in vivo. The proposed work also has the potential to improve RPE cell proliferation ex vivo for more efficient cell manufacturing. Most importantly, this study will create a foundation for safely stimulating RPE cell proliferation in vivo. Endogenous activation of RPE cell proliferation to counteract RPE cell loss in AMD has the potential to avoid surgery and immunosuppression involved in RPE cell transplantation, which would greatly benefit the elderly AMD patient population.