Metabolism of AMD iPSC-derived RPE

AMD iPSC 衍生的 RPE 的代谢

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要年龄相关性黄斑变性 (AMD) 是导致成年人视力障碍和失明的主要原因 65 岁，预计到 2040 年将影响全球约 2.88 亿人。 AMD 患者和表型相似的患者产生的多能干细胞 (iPSC) 衍生的 RPE 单基因疾病已被证明与培养中 AMD 疾病表型的要素相似，包括亚 RPE 沉积物的形成，重新组装玻璃膜疣、失调的补体和线粒体我们的团队和其他人测量了代谢利用、糖酵解、线粒体功能和各种 iPSC RPE 模型系统中的脂质代谢，而体外 RPE 模型显示出巨大的前景。在发现疾病机制和治疗靶点的同时，人们也越来越认识到潜在的局限性，包括模型系统的再现性和对本地条件的保真度。对最近 iPSC RPE 研究的综合回顾表明，最常用的传统培养基是营养成分和代谢物含量高度多样化，可能会显着改变 RPE 代谢。使用多种类型的电镀基质可能会导致营养环境的变化。对基线营养环境及其对 RPE 代谢影响的了解达成共识，使得该提案的目标是描述代谢和代谢的特征。 AMD iPSC RPE 细胞在三种不同且常用的传统方法中的疾病相关表型特征培养基和生理培养基的成分非常接近人类血液两种AMD iPSC RPE。本研究将使用 RPE 品系及其经过 CRISPR 校正的同基因对照来区分。一个 NIH/NYSCF AREDS2 受试者 iPSC 系，具有多个已知的高风险等位基因，根据性别和性别进行选择从具有早发性黄斑玻璃疣（EOMD）剪接的个体生成的复合型匹配的 RPE 系。 CFH 基因突变导致这种严重的 AMD 亚型，我们的初步数据表明 EOMD iPSC RPE 显示 AMD 疾病相关特征，包括补体失调、亚 RPE 沉积 iPSC RPE 的形成和代谢改变将在 twp 基质（Matrigel®、玻连蛋白）上培养，并且维持在四种培养基制剂中（基于 MEM-α、基于 DMEM/F-12、X-VIVO 10TM 和 PlasmaxTM）。项目旨在确定培养微环境对 AMD 和 EOMD iPSC RPE 代谢的影响该项目的成果将是对疾病表型的新的、更全面的了解。传统和生理介质如何影响正常人的代谢特征和表型特征这一新的认识将有助于解释代谢物研究。模型系统并有助于为未来研究设计更多的生理细胞培养基。