Characterizing Human RPE Cell Proliferation to Advance Endogenous Regeneration

表征人类 RPE 细胞增殖以促进内源性再生

• 批准号: 10534177
• 负责人: SALLY TEMPLE
• 金额: $ 56.84万
• 依托单位: REGENERATIVE RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10534177
• 关键词: Acute; Address; Adult; Age related macular degeneration; Bioinformatics; Blindness; Bruch' s basal membrane structure; CRISPR interference; Cadaver; Cell Surface Receptors; Cell Survival; Cell Transplantation; Cell division; Cell membrane; Cell secretion; Cell surface; Cells; Clinical Trials; Data; Disease; Elderly; Environment; Environmental Risk Factor; Epithelial Cell Proliferation; Exogenous Factors; Extracellular Domain; Eye; FGF2 gene; Female; Foundations; Genes; Genetic Transcription; Glycoproteins; Goals; Growth Factor; Human; Immunosuppression; In Situ; In Vitro; Interphase Cell; KITLG gene; Knowledge; Ligands; Mass Spectrum Analysis; Methods; Mitogens; Natural regeneration; Nonexudative age-related macular degeneration; Operative Surgical Procedures; Pathway Analysis; Pathway interactions; Patients; Photoreceptors; Proliferating; Protein Secretion; Proteins; Proteomics; Retina; Retinal Degeneration; Source; Structure of retinal pigment epithelium; System; Testing; Translating; Translations; Work; bioinformatics tool; epithelial repair; fibroblast growth factor 18; genetic manipulation; improved; in vivo; induced pluripotent stem cell; innovation; interest; male; manufacture; monolayer; novel strategies; patient population; prevent; programs; protein expression; receptor; repaired; retinal stimulation; small hairpin RNA; small molecule; stem cells; therapy development; transcriptome sequencing

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.

项目摘要 /摘要 数十年的研究表明，成年人类视网膜色素上皮（RPE）细胞具有很强的 体外的增殖能力，这表明RPE层具有自我修复的可能性。我们已经显示了 来自老年人或与年龄相关的黄斑变性（AMD）的捐助者或捐助者的成年RPE可以在 培养并产生近乎本地的RENEW RPE单层。尽管如此，体内的RPE细胞不会再生 在患有退化细胞的患者（例如干燥AMD的患者）中，鹅卵石RPE层受损。 体内环境必须有效地防止鹅卵石RPE单层维修，要么通过缺乏 有丝分子或抑制分子或两者的组合。这些研究的总体目标是实现 内源性RPE细胞的安全，受控的增殖，以使RPE层的自我修复 AMD。该提议的目的是表征积极和负面的环境因素 控制成年人类RPE细胞的增殖。第一个具体目的是表征成人人类RPE 使用创新的质谱法和生物信息学上的分裂和非分裂细胞上的细胞表面组 平台。这将对RPE质膜上的分子进行首次综合分析和 揭示细胞表面受体和分泌的蛋白质，这些蛋白质应对影响细胞分裂的环境因素。 研究正常和AMD RPE将对RPE细胞如何随着 疾病。第二个具体目的是检查我们已经确定的转录网络 成人人RPE细胞增殖以确定哪些分子至关重要。为了有效地做到这一点，我们将 第一员工CRISPRI，然后是其他功能屏幕。第三个特定目的将检查外源是否 因素可以激活静止，鹅卵石人类RPE的增殖，包括Bruch的原位 膜外植体和来自AMD患者的膜外植体。除了主要目标外，本研究还将产生 有关RPE分子的新知识，可用于靶向体内RPE。拟议的工作也有 可以改善RPE细胞增殖的外体，以进行更有效的细胞制造。最重要的是，这 研究将为体内安全刺激RPE细胞的增殖创造基础。 rpe的内源激活 在AMD中抵消RPE细胞损失的细胞增殖有可能避免手术和免疫抑制 参与RPE细胞移植，这将极大地使AMD患者群体受益。