Highly-purified rapidly expanding clones, RECs, are superior for functional-mitochondrial transfer

BackgroundMitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer.MethodsWe established mitochondria-deficient cell lines (rho(0) A549 and rho(0) HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to rho(0) cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors.ResultsCo-culture of rho(0) cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to rho(0) cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in rho(0) cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore.ConclusionsRECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.

Automated summary

By examining the differences between RECs and MSCs, the efficacy of mitochondrial transfer was assessed. It was found that RECs were more effective in transferring mitochondria to rho(0) cells compared to MSCs and resulted in superior recovery of mitochondrial function. Therefore, RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction.