Pressure-Driven Mitochondrial Transfer Pipeline Generates Mammalian Cells of Desired Genetic Combinations and Fates

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. [Display omitted] •We report a “proof-of-principle” mitochondrial transfer pipeline by MitoPunch•MitoPunch generates cells with unique mtDNA-nDNA pairs, regardless of cell source•Replacement mtDNA in non-immortal cells remains stable with cell fate conversions•Enables studies of mtDNA-nDNA interactions with reprogramming and differentiation Patananan and colleagues demonstrate a pipeline for transferring isolated mitochondria into mtDNA-deficient recipient cells. mtDNA-depleted fibroblasts permanently retain acquired non-native mtDNA through cell fate transitions. Initially, mitochondrial recipients show mtDNA-deficient cell transcriptome and metabolome profiles, with improvement to control profiles by reprogramming to pluripotency and subsequent differentiation.