Developing MitoPunch Mitochondrial Transfer to Enable Stable Transplantation of Human Mitochondrial DNA
Mitochondria, often considered simple cellular powerhouses that generate ATP by oxidative phosphorylation, are essential organelles found in all nucleated mammalian cells that play integral roles in apoptosis, Ca2+ signaling, reactive oxygen species generation, metabolism, and other critical cellular processes. Mitochondria are unique among organelles in that they contain multiple copies of their own genome (mtDNA) that is replicated and translated independently of the nuclear DNA (nDNA). Mitochondrial function is dependent upon the compatibility of the gene products of these two genomes and the coordination of their gene expression, and the current lack of methods to edit the mtDNA presents a major roadblock to dissecting the contribution of mtDNA sequence to cell fitness. There exist techniques to specify mtDNA-nDNA combinations in mammalian cells by delivery of isolated mitochondria into mtDNA depleted cells, however these approaches are technically challenging or restricted to a narrow range of manipulatable cell types. Novel methods that facilitate isolated mitochondrial transfer and generation of stable isolated mitochondrial recipient (SIMR) cells with known mtDNA- nDNA pairings are crucial to enable studies of mitonuclear communication, metabolism, and cell fate determination. To overcome these limitations we developed MitoPunch, a high- throughput, easily-assembled, pressure-driven mitochondrial transfer platform that can generate SIMR cell lines from mitochondria isolated from cell lines and donor tissue and a wide range of mtDNA depleted transformed or replication-limited cells. In this dissertation we present the method to construct and implement the MitoPunch apparatus, data demonstrating the generation of SIMR clones from mtDNA-depleted transformed and primary cell lines using MitoPunch, and a proof of principle pipeline for engineering induced pluripotent stem cells and differentiated cell types from SIMR clones produced using MitoPunch. The results found in this thesis suggest that MitoPunch enables for the first time the high throughput generation of stable mtDNA specified cell lines for use in detailed molecular studies of mtDNA-nDNA interactions.