Mitochondria Dynamically Transplant into Cells in Vitro and in Mice and Rescue Aerobic Respiration of Mitochondrial DNA-Depleted Motor Neuron NSC-34

Abstract

It has been reported that transplantation of pheochromocytoma P12 and hepatoma cells' mitochondria improve the locomotive activity and prevent disease progress in experimental Parkinson's disease rats. To prepare for mitochondrial transplantation study in human neurodegenerative diseases, we select human fibroblasts as mitochondrial donor because that fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). We isolate human primary fibroblasts and develop a mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34 ρ˚ cells). Fibroblast and NSC-34 cell's mitochondria are co-cultured with NSC-34 ρ˚ cells. Mitochondrial transplantation is observed by fluorescent microscopy. Gene expression is determined by polymerase chain reaction (PCR) and real time PCR (qPCR). Also, mitochondria are injected to mice bearing mammary adenocarcinoma 4T1 cells. We find results as following: 1) There are abundant mitochondria in fibroblasts (337 ± 80 mitochondria per fibroblast). 42.4% of viable mitochondria are obtained by using differential centrifugation. The isolated mitochondria actively transplant into NSC-34 ρ˚ cells after co-culture. 2) Fibroblasts transfer mitochondria to human mammary adenocarcinoma MCF-7 cells. 3) There is no expression of HLA-I antigen in fibroblast's mitochondria indicating they can be used for allogeneic mitochondrial transplantation without HLA antigen match. 4) PCR and qPCR show that NSC-34 ρ˚ cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate expression of glycolysis-associated genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA). 5) Transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates gene expression of HK2, SLC2A1 and LDHA. 6) Normal mammary epithelial mitochondria

Essentiality and dynamic expression of the human tRNA pool during viral infection

“…We further investigated the expression level of mitochondrial tRNAs. Given that the expression level of mitochondrial tRNAs is a cumulative sum of contributions from many mitochondria within each cell, we normalize it by the estimated mitochondrial genome copy number per cell, which is about 300 particles in human skin fibroblasts (Jiang et al, 2020). Per mitochondrial genome copies, the expression of mitochondrial tRNA is comparable to the lowest among the cytosolic tRNAs (Figure S1A).…”

Extracellular Vesicles from Mesenchymal Stromal Cells (imEVs) Improve Cold Preservation of Isolated Mitochondria

“…were detected by PCR [14]. PCR was performed in a total 25 µl volume including 2 µl of PCR Master mix (Thermofisher Scientific, Waltham, MA, USA), 0.5 µl of 100 µM forward and reverse primers and 1 µl of heat-denatured imEVs samples.…”

Mitochondrial Organelle Transplantation Is a Potential Therapeutic for Mitochondria Dysfunction in Severe Acute Respiratory Syndrome (SARS) Coronavirus Diseases

“…MOT is an innovative technology to transfer the isolated healthy mitochondria to injured or diseased cells. We published that the mitochondria isolated from cells [45]. Early our research showed that human normal mammary epithelial mitochondria transplanted into human breast cancer cell line MCF-7, T47D and MDA-MB-231, and suppressed glycolytic metabolism and glucose uptake of human breast cancer cells and reduced lactate production [46].…”