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.

项目总结/摘要 几十年的研究已经证明，成人视网膜色素上皮（RPE）细胞具有强的 体外增殖能力，表明RPE层具有自我修复的可能性。我们已经表明 来自老年供体或患有年龄相关性黄斑变性（AMD）的供体的成人RPE可以在 培养并产生接近天然的更新的RPE单层。尽管如此，体内的RPE细胞不会再生 视网膜色素上皮层的损害与退化细胞的患者，如干性AMD。的 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。这些研究的总体目标是实现 安全、可控的内源性RPE细胞增殖，使RPE层能够自我修复， AMD.该提案的目的是描述积极和消极的环境因素， 控制成人RPE细胞的增殖。第一个具体目标是表征成人RPE 使用创新的质谱和生物信息学技术， 平台这将提供对RPE质膜上分子的首次全面分析， 揭示了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作出反应。 研究正常和AMD RPE将更好地了解RPE细胞如何随着年龄的增长而变化。 疾病第二个具体的目标是检查我们已经确定的转录网络， 与成人RPE细胞增殖，以确定哪些分子是关键的。为了有效地做到这一点，我们将 首先使用CRISPRi，然后使用其他功能性屏幕。第三个具体目标将审查是否外源 因子可以激活静止的鹅卵石状人RPE的增殖，包括那些在Bruch's 膜外植体和来自AMD患者的那些。除了主要目标，这项研究将产生 关于可用于体内靶向RPE的RPE分子的新知识。拟议的工作也有 改善RPE细胞离体增殖以更有效地制造细胞的潜力。最重要的是这 这项研究将为在体内安全刺激RPE细胞增殖奠定基础。RPE的内源性激活 细胞增殖以抵消AMD中RPE细胞损失具有避免手术和免疫抑制的潜力 参与RPE细胞移植，这将极大地有益于老年AMD患者群体。