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.

项目概要/摘要 线粒体对于个体的健康发育至关重要。当功能失调时，线粒体会产生 一组毁灭性的发育障碍，包括多种肌病和神经病，并影响 估计为 1:5,000 人。许多这些疾病是由线粒体 DNA (mtDNA) 突变引起的， 尽管经过数十年的研究，许多线粒体DNA疾病仍缺乏有效的治疗方法。我们已经调整了一个系统 线粒体消耗产生缺乏可检测水平线粒体的多能干细胞（PSC） （以下称为线粒体耗尽的 PSC）。我们发现线粒体耗尽的 PSC 在培养物中可以存活很长时间 几天后，通过将健康的 PSC 与线粒体耗尽的 PSC 融合，我们成功地恢复了 线粒体到线粒体耗尽的 PSC。这些发现表明线粒体耗尽的 PSC 提供了一个独特的平台 揭示并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，可用于潜在的通用战略 在 mtDNA 疾病患者细胞中用健康线粒体替换突变线粒体。该项目旨在 利用线粒体耗尽 PSC 的令人兴奋且未经研究的平台来研究线粒体在早期的作用 开发，并制定治疗 mtDNA 疾病的新策略。我们将首先实现这一点 确定线粒体缺失对小鼠胚胎干细胞 (mESC) 的影响。同时使用有针对性的和 公正的方法，我们将确定线粒体在多能状态和早期的总体作用 胚胎发育。具体来说，我们将确定转录、表观遗传和代谢的变化 线粒体耗尽的 mESC 的状态。此外，我们将模拟线粒体耗尽的 mESC 的发育能力 通过多能状态变化和分化在体外。我们还将生成 mtDNA 校正的 hiPSC 从线粒体脑病、乳酸酸中毒和中风样发作 (MELAS) 中重新编程 综合征患者通过有丝分裂耗竭和随后的细胞质融合。我们将确定代谢的逆转 通过定向分化为神经和心脏，与 MELAS 相关的发育表型 谱系，结合代谢海马测定。破译其生物学机制 线粒体调节发育和疾病对于促进人类健康至关重要 开发创新的治疗策略来治疗 MELAS 综合征等 mtDNA 疾病。