It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. was also supported by transcriptome analysis. Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition. However, we could not find any evidence of mitochondrial transfer to the cells harboring human pathogenic mtDNA mutations (A3243G mutation or 4,977 bp deletion). Thus, the mitochondrial transfer is limited to the condition of a near total absence of mitochondrial function. Elucidation of the mechanism of mitochondrial transfer will help us create a potential cell therapy-based mitochondrial restoration or mitochondrial gene therapy for human diseases caused by mitochondrial dysfunction. Introduction In the 1940s, Lederberg and Tatum discovered the conjugation phenomenon, which is a process of a unidirectional transfer of genetic information essential for survival on a minimal medium through the formation of a physical bridge between a donor bacterium and a recipient bacterium . Similar to the earlier findings in prokaryotes, it was recently reported that human mesenchymal stem cells (MSCs) can transfer mitochondria (microorganelles containing their own genetic information) to the cells with nonfunctional mitochondria incapable of aerobic respiration due to defective or deleted mitochondrial DNA (mtDNA) . However, the mechanism of intercellular mitochondrial transfer is still elusive. It was postulated that the MSC actively transfers mitochondria to the cells with severely compromised mitochondrial function through a tunneling, tube-like structure . In addition, it is conceivable that the cells with defective mitochondrial function could uptake the vesicles containing mitochondria that WZ3146 supplier LIMK2 budded off from the donor cells. However, isolated mitochondria or platelets, which contain mitochondria, could not restore mitochondrial function of the cells with nonfunctional mitochondria . Furthermore, it is uncertain whether the restoration of mitochondrial function is mediated by the transfer of mtDNA alone, which is similar to the conjugation phenomenon, or by the transfer of intact mitochondrial particles. In this study, firstly, WZ3146 supplier we investigated whether the reported intercellular mitochondrial transfer  could be replicated in different types of cells or under different experimental conditions. In this regard, we used mtDNA-less 0 cells derived from human osteosarcoma 143B cells lacking thymidine kinase activity by long-term treatment with ethidium bromide (EtBr) . Secondly, to address whether the restoration of mitochondrial function is mediated through a transfer of mtDNA alone or intact mitochondrial particles, we used rhodamine-6G (R6G), which causes abrupt and irreversible damage to the mitochondrial function without the removal of mtDNA , , , . Finally, to explore the possibility of therapeutic implications, we examined whether the mitochondrial transfer could occur in the cells harboring mtDNA mutations relevant to human diseases, such as A to G substitution at np 3243 in tRNAleu(UUR) (OMIM #540000 or #520000) and 4,977 bp large deletion (OMIM #530000). Materials and Methods Coculture experiments The 143B + cell and the 143B 0 cell, which was established from the 143B + cell through long-term treatment with EtBr, were generous gifts from Professor Yau-Huei Wei from National Yang-Ming University in Taiwan. Since the 143B 0 cell is characterized by the absence of a functional respiratory chain, no thymidine kinase activity, and dependence on uridine and pyruvate , we grew the cells in Dulbecco’s Modified WZ3146 supplier Eagle’s Media (DMEM, Invitrogen, Carlsbad, CA) supplemented with 100 g/ml BrdU (Sigma, St. Louis, MO), 50 g/ml uridine (Sigma), and 10% FBS. Southern blot analysis and PCR amplification of mtDNA target sequences confirmed the absence of any residual mtDNA (data not shown). Trans-mitochondrial cytoplasmic hybrids (or cybrids) harboring either the A3243G or 4,977 bp deletion mutation with varying degrees of mutational load (heteroplasmy rate) that shared an identical nuclear background with 143B, were also cultured in the same media. The cybrids with A3243G mutation were derived by fusion of 143B 0 cells with platelets from a patient harboring A3243G mutation, according to the standard protocol . The cybrids with 4,977 bp deletion mutation were also gifts from Professor Wei. The schemes of the coculture procedures are shown WZ3146 supplier in Fig. 1. We cocultured human bone marrow derived MSCs at 1.5105 cells with those cells listed above (i.e., 143B 0, cybrids with A3243G, and cybrids with 4,977 bp deletion) at 3105 cells in a 100 mm culture dish with DMEM supplemented with 10% FBS and 50 g/ml uridine for 24 h. At this stage, we removed BrdU from the culture medium; we did not add pyruvate, since DMEM contains a sufficient amount of pyruvate (110 g/ml). Then, we changed the culture medium with DMEM supplemented by 10% FBS, but without.