Metabolism of AMD iPSC-derived RPE

AMD iPSC 衍生的 RPE 的代谢

• 批准号: 10576558
• 负责人: Jennifer Rayming Chao
• 金额: $ 43.54万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-30 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10576558
• 关键词: Adult; Affect; Age related macular degeneration; Awareness; Biological Models; Blindness; Blood; Cell Culture Techniques; Cell Line; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Consensus; Consumption; Culture Media; Data; Deposition; Development; Differentiation and Growth; Disease; Drusen; Elements; Energy Metabolism; Environment; Experimental Designs; Future; Gender; Genes; Genus Hippocampus; Glycolysis; Goals; Human; In Vitro; Individual; Knowledge; Laboratories; Maintenance; Measures; Mendelian disorder; Metabolic; Metabolic Diseases; Metabolic Pathway; Metabolic dysfunction; Metabolism; Mitochondria; Modeling; Morphology; Mutation; Nutrient; Outcome; Oxygen saturation measurement; Pathogenesis; Patients; Persons; Phenotype; Physiological; Play; Preparation; RNA Splicing; Regulation; Reporting; Reproducibility; Resources; Retinal Diseases; Role; Structure; Structure of retinal pigment epithelium; System; Tight Junctions; United States National Institutes of Health; Variant; Visual impairment; Vitronectin; base; cancer cell; cell growth; design; dichlorodifluoromethane; disease phenotype; early onset; extracellular; high risk; human old age (65+); induced pluripotent stem cell; lipid metabolism; macular drusen; matrigel; metabolic phenotype; metabolic profile; metabolomics; mitochondrial dysfunction; retinal progenitor cell; risk variant; stem cells; therapeutic target

Project Summary/Abstract Age-related macular degeneration (AMD) is a leading cause of visual impairment and blindness in adults over the age of 65 and is expected to affect ~288 million people worldwide by the year 2040. Recently, induced pluripotent stem cells (iPSC)-derived RPE generated from AMD patients and those with phenotypically similar monogenic diseases have been shown to approximate elements of AMD disease phenotype in culture, including the formation of sub-RPE deposits resembling drusen, dysregulated complement, and mitochondrial dysfunction. Our groups and others have measured metabolite usage, glycolysis, mitochondrial function, and lipid metabolism in a variety of iPSC RPE model systems. While in vitro RPE models show significant promise in the discovery of disease mechanisms and therapeutic targets, there is also increasing awareness of potential limitations, including reproducibility across model systems and fidelity to native conditions. A comprehensive review of recent iPSC RPE studies shows that the most used traditional culture media are highly diverse in nutrient and metabolite content which may significantly alter RPE metabolism. Moreover, multiple types of plating substrates used could contribute to the variability in nutrient environments. A lack of consensus on baseline nutrient environments and knowledge of their impact on RPE metabolism makes comparisons between findings challenging. The goal of this proposal is to characterize the metabolic and disease-relevant phenotypic profiles of AMD iPSC RPE cells in three distinct and commonly used traditional media and physiological medium closely approximating the composition of human blood. Two AMD iPSC RPE lines and their CRISPR-corrected isogenic controls will be used in this study. RPE will be differentiated from one NIH/NYSCF AREDS2 subject iPSC line with multiple known high-risk alleles, selected to gender and complotype-match RPE lines generated from an individual with early onset macular drusen (EOMD). A splicing mutation in the CFH gene results in this severe subtype of AMD, and our preliminary data show that EOMD iPSC RPE display AMD disease-relevant features, including complement dysregulation, sub-RPE deposit formation, and altered metabolism. iPSC RPE will be cultured on twp substrates (Matrigel®, vitronectin), and maintained in four media preparations (MEM-α based, DMEM/F-12 based, X-VIVO 10TM and PlasmaxTM). This project aims to determine the impact of culture microenvironment on AMD and EOMD iPSC RPE metabolism and disease phenotype. The outcome of this project will be a new and more comprehensive understanding of how traditional and physiologic media influence the metabolic profile and phenotypic characteristics of normal and diseased RPE cells. This new understanding will aid in the interpretation of metabolite studies across model systems and help to inform the design of more physiologic cell culture media for future studies.

项目总结/摘要 视网膜相关性黄斑变性（AMD）是导致视力损害和失明的主要原因， 65岁，预计到2040年将影响全球约2.88亿人。最近，诱导 多能干细胞（iPSC）衍生的RPE产生自AMD患者和具有表型相似的表型的那些患者。 单基因疾病已经显示出在培养物中接近AMD疾病表型的要素， 包括类似玻璃疣的亚RPE沉积物的形成、失调的补体和线粒体 功能障碍我们的团队和其他人已经测量了代谢物的使用，糖酵解，线粒体功能， 在各种iPSC RPE模型系统中的脂质代谢。虽然体外RPE模型显示出显著的前景， 在发现疾病机制和治疗靶点的过程中， 潜在的局限性，包括跨模型系统的再现性和对天然条件的保真度。一 对最近iPSC RPE研究的全面回顾表明，最常用的传统培养基是 营养和代谢物含量高度多样化，可显著改变RPE代谢。此外，委员会认为， 所用的多种类型的电镀基质可能导致营养环境的变化。缺乏 关于基线营养环境及其对RPE代谢影响的知识的共识使得 调查结果之间的比较具有挑战性。该提案的目标是表征代谢和 AMD iPSC RPE细胞的疾病相关表型特征在三种不同和常用的传统 介质和生理介质非常接近人体血液的组成。两个AMD iPSC RPE 品系及其CRISPR校正的同基因对照将用于本研究。RPE将区别于 一个NIH/NYSCF AREDS 2受试者iPSC系，具有多个已知的高风险等位基因，根据性别选择， 从患有早发性黄斑玻璃膜疣（EOMD）的个体产生的补体型匹配RPE线。拼接 CFH基因突变导致了这种严重的AMD亚型，我们的初步数据显示， iPSC RPE显示AMD疾病相关特征，包括补体失调、亚RPE存款 形成和代谢改变。iPSC RPE将在两种底物（Matrigel®，玻连蛋白）上培养， 在四种培养基制备物（基于MEM-α、基于DMEM/F-12、X-VIVO 10 TM和Plasmax TM）中维持。这 该项目旨在确定培养微环境对AMD和EOMD iPSC RPE代谢的影响 和疾病表型。该项目的成果将是对以下方面的新的和更全面的了解： 传统和生理媒介如何影响正常人的代谢谱和表型特征 和患病的RPE细胞。这一新的理解将有助于解释代谢物研究， 模型系统，并有助于为未来研究提供更多生理细胞培养基的设计。