Metabolism of AMD iPSC-derived RPE

项目摘要/摘要 老年性黄斑变性(AMD)是老年人视力受损和失明的主要原因之一。 预计到2040年，全球将有约2.88亿人受到影响。最近，诱导 AMD患者和表型相似者多能干细胞(IPSC)来源的RPE 单基因疾病已被证明在培养中与AMD疾病表型相似， 包括形成类似于玻璃体的亚RPE沉积、调节失调的补体和线粒体。 功能障碍。我们的团队和其他人测量了代谢物的使用、糖酵解、线粒体功能和 不同IPSC RPE模型体系中的脂质代谢。虽然体外RPE模型显示出重大的前景 在疾病机制和治疗靶点的发现中，人们也越来越意识到 潜在的限制，包括模型系统的重现性和对本地条件的保真度。一个 对最近IPSC RPE研究的综合审查表明，最常用的传统文化媒体是 营养和代谢物含量高度多样化，这可能会显著改变RPE的代谢。此外， 所使用的多种类型的电镀底物可能会导致营养环境的变化。缺乏 就基线营养环境及其对RPE新陈代谢影响的知识达成共识 两个发现之间的比较具有挑战性。这项提案的目标是描述新陈代谢和 三种不同和常用的传统AMD IPSC RPE细胞的疾病相关表型特征 与人类血液的成分非常接近的介质和生理介质。两台AMD iPSC RPE 品系及其CRISPR校正的等基因对照将在本研究中使用。RPE将区别于 一个NIH/NYSCF AREDS2受试者IPSC系，具有多个已知的高危等位基因，按性别和 来自早发性黄斑玻璃体病(EOMD)患者的复杂类型匹配的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EOMD IPSC RPE显示与AMD疾病相关的特征，包括补体失调、亚RPE沉积 形成，并改变新陈代谢。IPSC RPE将在两种底物(Matrigel®，Vitronectin)上培养，以及 维持在四种培养基制剂中(基于α、基于DMEM/F-12、X-vivo 10TM和Platmax TM)。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 传统和生理介质如何影响正常人的代谢谱和表型特征 和病变的RPE细胞。这种新的理解将有助于对代谢物研究的解释。 模型系统，并有助于为将来的研究提供更多生理细胞培养液的设计信息。

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.