The permanent transfer of specific mtDNA sequences into mammalian cells could generate improved models of mtDNA disease and support future cell-based therapies. Previous studies documented multiple biochemical changes in recipient cells shortly after mtDNA transfer, but the long-term retention and function of transferred mtDNA remains unknown. Here, we evaluate mtDNA retention in new host cells using 'MitoPunch', a device that transfers isolated mitochondria into mouse and human cells. We show that newly introduced mtDNA is stably retained in mtDNA-deficient (ρ0) recipient cells following uridine-free selection, although exogenous mtDNA is lost from metabolically impaired, mtDNA-intact (ρ+) cells. We then introduced a second selective pressure by transferring chloramphenicol-resistant mitochondria into chloramphenicol-sensitive, metabolically impaired ρ+ mouse cybrid cells. Following double selection, recipient cells with mismatched nuclear (nDNA) and mitochondrial (mtDNA) genomes retained transferred mtDNA, which replaced the endogenous mutant mtDNA and improved cell respiration. However, recipient cells with matched mtDNA-nDNA failed to retain transferred mtDNA and sustained impaired respiration. Our results suggest that exogenous mtDNA retention in metabolically impaired ρ+ recipients depends on the degree of recipient mtDNA-nDNA co-evolution. Uncovering factors that stabilize exogenous mtDNA integration will improve our understanding of in vivo mitochondrial transfer and the interplay between mitochondrial and nuclear genomes.

Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.