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

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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.