Generation of somatic mitochondrial DNA-replaced cells for mitochondrial dysfunction treatment

Abstract

Mitochondrial diseases currently have no cure regardless of whether the cause is a nuclear or mitochondrial genome mutation. Mitochondrial dysfunction notably affects a wide range of disorders in aged individuals, including neurodegenerative diseases, cancers, and even senescence. Here, we present a procedure to generate mitochondrial DNA-replaced somatic cells with a combination of a temporal reduction in endogenous mitochondrial DNA and coincubation with exogeneous isolated mitochondria. Heteroplasmy in mitochondrial disease patient-derived fibroblasts in which the mutant genotype was dominant over the wild-type genotype was reversed. Mitochondrial disease patient-derived fibroblasts regained respiratory function and showed lifespan extension. Mitochondrial membranous components were utilized as a vehicle to deliver the genetic materials into endogenous mitochondria-like horizontal genetic transfer in prokaryotes. Mitochondrial DNA-replaced cells could be a resource for transplantation to treat maternal inherited mitochondrial diseases.

Mitochondrial Transplantation: Is It a Feasible Therapy to Prevent the Cardiorenal Side Effects of Cisplatin?

“…Mitochondria isolation requires transferring the tissue in homogenizing buffers that closely depend on the needs of cells or tissues. [116,124,125]. Homogenizers need to provide sterilized and effective homogeni- MSCs primarily take up platelet derived mitochondria via dynamin dependent, clathrin mediated endocytosis [121], but MSCs have also been shown to donate mitochondria to neural stem cells (NSCs) through actin based intercellular structures and the mitochondrial motor protein Rho-GTPase 1 (Miro1) during cisplatin induced damage in vitro [101].…”

“…Mitochondria isolation requires transferring the tissue in homogenizing buffers that closely depend on the needs of cells or tissues. [116,124,125]. Homogenizers need to provide sterilized and effective homogenization, allowing the consistent and uniform dissociation of the tissue.…”

“…Tissue homogenates, such as those obtained from muscle, the liver or the heart, are usually subjected to brief digestion, generally with subtilisin A, and the mixture can be passed through several disposable mesh filters or differential centrifugation [87,91,111,123]. Cells are usually ruptured on ice with strokes of 27 gauge and then differentially centrifugated [116,124,125]. Mitochondria cannot survive much time after extraction, and although many improvements in extraction protocols have reduced the time of extraction, some characteristics, such as temperature, respiration buffer, and counting methods, need to be taken into account to improve mitochondrial survival [91,93,123].…”

“…Therefore, the source of mitochondria and previous genetic analysis of mtDNA will have to be carried out before transplantation [140]. Concerning this, Maeda et al [124] established a novel technology to generate somatic mitochondrial DNA replaced cells (MirCs), combined with a temporal reduction in endogenous mtDNA and coincubation with exogenous isolated mitochondria. The results showed that mitochondrial disease patient derived fibroblasts regained respiratory function and showed lifespan extension, suggesting a possible application to treat maternal inherited mitochondrial diseases and improving the possibilities of using mitochondria from the same individual (autologous).…”

“…It would be necessary to investigate the adverse effects of DAMPs present in isolated heterogeneous or allergenic mitochondria when several injections could be needed [108]. Solutions of isolated mitochondria could contain some damaged mitochondrial membrane fragments and genomes that may act as DAMPs, inducing innate immune activation in the cytosol and highlighting the care of technical procedures during isolation [124]. The leakage of mtDNA from the mitochondrial matrix is a ligand of Toll-like receptor 9 (TLR-9), which induces immune responses and cell injury through the stimulation of NF-κB [125,130].…”