Study of mitochondria-depleted pluripotent stem cells in development and disease

线粒体耗尽的多能干细胞在发育和疾病中的研究

• 批准号: 10535808
• 负责人: Daniel Andrew Schmitz
• 金额: $ 3.77万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10535808
• 关键词: Adult; Affect; Autologous; Biological; Biological Assay; Biotechnology; Cardiac; Cardiac Myocytes; Cell Line; Cell Lineage; Cell physiology; Cells; Development; Disease; Disease model; Embryonic Development; Epiblast; Epigenetic Process; Gene Expression Profiling; Genetic Transcription; Genus Hippocampus; Health; Human; Human Development; In Vitro; Individual; MELAS Syndrome; Maintenance; Measures; Membrane Potentials; Metabolic; Metabolism; Methods; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Modeling; Modification; Mus; Mutation; Myopathy; Neurons; Neuropathy; Organelles; Oxygen Consumption; Patients; Persons; Phenotype; Pluripotent Stem Cells; Production; Research; Role; System; Teratoma; Therapeutic; Time; Tissues; cell replacement therapy; developmental disease; directed differentiation; disease phenotype; effective therapy; embryonic stem cell; extracellular; human disease; induced pluripotent stem cell; innovation; mitochondrial dysfunction; mitochondrial membrane; mitochondrial metabolism; mutant; pluripotency; regenerative therapy; relating to nervous system; self-renewal; stem; stem cells

PROJECT SUMMARY/ABSTRACT Mitochondria are critical for the healthy development of an individual. When dysfunctional, mitochondria give rise to a devastating group of developmental disorders that include several myopathies and neuropathies and affect an estimated 1:5,000 people. Many of these disorders are caused by mutations in mitochondrial DNA (mtDNA), and despite decades of research, many mtDNA diseases lack effective treatments. We have adapted a system of mitochondria-depletion to generate pluripotent stem cells (PSCs) that lack detectable levels of mitochondria (henceforth referred to as mito-depleted PSCs). We have found that mito-depleted PSCs survive in culture for several days and, moreover, by fusing healthy PSCs with mito-depleted PSCs, we were successful in restoring mitochondria to mito-depleted PSCs. These findings suggest mito-depleted PSCs provide a unique platform to uncover and study mechanisms by which mitochondria regulate pluripotency and early development. In addition, they may constitute an exciting biotechnology that can be harnessed in a potentially universal strategy for replacing mutant mitochondria with healthy mitochondria in mtDNA disease patient cells. This project aims to utilize the exciting and unstudied platform of mito-depleted PSCs to study mitochondrial roles in early development, as well as devise a new strategy to treat mtDNA disease. We will accomplish this by first determining the effects mito-depletion on mouse embryonic stem cells (mESCs). Using both targeted and unbiased methods, we will determine the overarching role of mitochondria in the pluripotent state and early embryonic development. Specifically, we will determine changes in the transcriptional, epigenetic, and metabolic states of mito-depleted mESCs. In addition, we will model the developmental ability of mito-depleted mESCs in vitro through pluripotent state changes and differentiation. We will also generate mtDNA-corrected hiPSCs reprogrammed from Mitochondrial Encephalopathy, Lactic acidosis, and Stroke-like episodes (MELAS) syndrome patients via mito-depletion and subsequent cytoplast fusion. We will determine reversal of metabolic and developmental phenotypes associated with MELAS through directed differentiation to neural and cardiac lineages, combined with metabolic Seahorse assays. Deciphering the biological mechanisms by which mitochondria regulate development and disease are of fundamental importance to advancing human health and developing innovative therapeutic strategies to treat mtDNA disease like MELAS syndrome.

项目摘要/摘要 线粒体对个体的健康发展至关重要。当功能失调时，线粒体就会产生 一组毁灭性的发育障碍，包括几种肌病和神经病，并影响 估计有1：5000人。其中许多疾病是由线粒体DNA(MtDNA)突变引起的， 尽管进行了数十年的研究，许多线粒体DNA疾病缺乏有效的治疗方法。我们已经调整了一个系统 线粒体耗竭，以产生缺乏可检测到的线粒体水平的多能干细胞(PSCs) (此后称为有丝分裂耗尽的PSC)。我们发现，丝裂原缺失的PSCs在培养中存活了 几天，而且，通过融合健康的PSCs和丝裂原耗尽的PSCs，我们成功地恢复了 线粒体到有丝分裂耗尽的PSCs。这些发现表明，丝裂原耗竭的PSC提供了一个独特的平台 揭示和研究线粒体调节多能性和早期发育的机制。此外, 它们可能构成一种令人兴奋的生物技术，可以被利用在潜在的普遍战略中 用线粒体DNA病患者细胞中的健康线粒体取代突变的线粒体。该项目旨在 利用线粒体缺失的PSCs这一令人兴奋且未被研究的平台研究线粒体在早期的作用 发展，以及设计一种治疗线粒体DNA疾病的新战略。我们将首先完成这项工作。 确定有丝分裂耗竭对小鼠胚胎干细胞的影响。同时使用目标和 不偏不倚的方法，我们将确定线粒体在多能性状态和早期的主要作用 胚胎发育。具体地说，我们将确定转录、表观遗传和代谢的变化 丝裂原耗竭的MESCs的状态。此外，我们还将模拟缺乏丝裂原的mESCs的发育能力。 体外通过多能状态的改变和分化。我们还将生成mtDNA校正的HiPSC 线粒体脑病、乳酸酸中毒和卒中样发作(MELAS)的重编程 综合征患者通过有丝分裂耗竭和随后的细胞质融合。我们将确定新陈代谢的逆转 与MELAS相关的发育表型通过定向分化为神经和心脏 血统，结合新陈代谢的海马氏分析。破译生物机制，通过它 线粒体调节发育和疾病对促进人类健康和 开发创新的治疗策略来治疗像MELAS综合征这样的线粒体DNA疾病。