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

项目总结/摘要 线粒体对个体的健康发育至关重要。当线粒体功能失调时， 一组毁灭性的发育障碍，包括几种肌病和神经病， 估计有1：5,000的人。这些疾病中的许多是由线粒体DNA（mtDNA）突变引起的， 尽管经过几十年的研究，许多mtDNA疾病缺乏有效的治疗方法。我们采用了一个系统 以产生缺乏可检测水平的线粒体的多能干细胞（PSC） （此后称为线粒体耗尽的PSC）。我们已经发现，线粒体缺失的PSC在培养物中存活， 此外，通过将健康的PSC与线粒体缺失的PSC融合，我们成功地恢复了 线粒体转化为线粒体缺失的PSC。这些发现表明，线粒体缺失的PSC提供了一个独特的平台， 发现和研究线粒体调节多能性和早期发育的机制。此外，本发明还提供了一种方法， 它们可能构成一种令人兴奋的生物技术，可以用于一种潜在的普遍战略， 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 利用令人兴奋的和未研究的平台，线粒体耗竭的PSC研究线粒体的作用， 开发，以及设计一种新的策略来治疗mtDNA疾病。我们将在第一时间完成这项工作 确定线粒体耗竭对小鼠胚胎干细胞（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， 线粒体耗竭的mESCs的状态。此外，我们将模拟线粒体缺失的mESCs的发育能力， 体外通过多能状态的变化和分化。我们还将产生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 综合征患者通过线粒体耗竭和随后的胞质融合。我们将确定代谢逆转 和通过定向分化为神经和心脏的与MELAS相关的发育表型 谱系，结合代谢海马测定。解读生物学机制， 线粒体调节发育和疾病对于促进人类健康具有根本重要性， 开发创新的治疗策略来治疗线粒体DNA疾病，如MELAS综合征。

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.