Abstract
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.
Highlights
Mutations in the multi-copy mitochondrial genome can impair the biosynthesis of ATP, metabolites, fatty acids, reactive oxygen species, and iron sulfur clusters[1,2,3,4]
The presence of undisrupted donor cells was minimized by introducing additional centrifugation spins in the mitochondrial isolation procedure and by passing the mitochondrial isolate through a 3 μm filter before reaching the recipient cells, which is an indirect benefit of the MitoPunch transfer pipeline
MtDNA diseases do correlate with reduced mtDNA copy numbers in cells, no ρ0 cells exist in vivo with the exception of red blood c ells34,52–55. ρ0 tumor cells in experimental mouse systems acquire exogenous mtDNA from host cells, which stimulates tumor progression and a ggression[36,56,57]
Summary
Mutations in the multi-copy mitochondrial genome (mtDNA) can impair the biosynthesis of ATP, metabolites, fatty acids, reactive oxygen species, and iron sulfur clusters[1,2,3,4]. (e) Seahorse Extracellular Flux analysis to quantify oxygen consumption rate of bulk culture generated from 143BTK− ρ0 cells transferred HEK293T or MELAS mitochondria. Current mitochondrial transfer approaches for somatic cells include MitoCeption24, microinjection[25], cell fusion[26], co-culturing[27,28], isolated mitochondrial co-incubation29, magnetomitotransfer[30], and large cargo delivery p latforms[31] These techniques have in common the provision of mitochondria containing exogenous mtDNA into mtDNA-deficient (ρ0) recipient cells, often followed by selection in uridine-deficient culture medium. Ρ0 cells are typically generated using DNA intercalating drugs, such as ethidium bromide, or DNA polymerase chain terminators, such as 2′,3′- dideoxycytidine, to remove recipient cell mtDNA33,34 These drugs can cause off target nDNA mutations and are not effective in removing all endogenous mtDNA from all cell types. We evaluated whether introduced CAP-R mtDNA into ρ+ recipient cybrid cells was retained or transient and lost when the recipient cell nDNA matched and co-evolved, or was mismatched, with the cybrid cell mtDNA strain, and the resultant effect on respiratory function
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