Limits...
Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations.

Cho YM, Kim JH, Kim M, Park SJ, Koh SH, Ahn HS, Kang GH, Lee JB, Park KS, Lee HK - PLoS ONE (2012)

Bottom Line: Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea.

ABSTRACT
It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which 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.

Show MeSH

Related in: MedlinePlus

Genetic analysis of rapidly proliferating cells survived after coculture.(A) The cells survived after coculture showed genetic identities with 143B ρ0 cell nuclei. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker. (B) Since ρ0 cells did not have any detectable mtDNA, the mtDNA of the cells survived after coculture came solely from the MSCs. The hypervariable region sequences of the cells were identical with the MSCs. The arrows indicate sequence variations compared to the Cambridge reference sequence. (C) PCR-RFLP for 10394 Dde I and 10397 Alu I of mtDNA. The recuperated cells in the coculture experiment with R6G treatment to a cybrid harboring mtDNA with +10394 Dde I and +10397 Alu I were revealed to have mtDNAs with both −10394 Dde I and −10397 Alu I, both of which were identical to the MSCs.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3295770&req=5

pone-0032778-g003: Genetic analysis of rapidly proliferating cells survived after coculture.(A) The cells survived after coculture showed genetic identities with 143B ρ0 cell nuclei. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker. (B) Since ρ0 cells did not have any detectable mtDNA, the mtDNA of the cells survived after coculture came solely from the MSCs. The hypervariable region sequences of the cells were identical with the MSCs. The arrows indicate sequence variations compared to the Cambridge reference sequence. (C) PCR-RFLP for 10394 Dde I and 10397 Alu I of mtDNA. The recuperated cells in the coculture experiment with R6G treatment to a cybrid harboring mtDNA with +10394 Dde I and +10397 Alu I were revealed to have mtDNAs with both −10394 Dde I and −10397 Alu I, both of which were identical to the MSCs.

Mentions: Normally, none of 143B ρ0, R6G-treated 143B ρ+, or MSC could survive in uridine− BrdU+ media; therefore, the exponentially growing cells in uridine− BrdU+ media must be the cells with normal mitochondrial function but with no thymidine kinase activity—characters that could be obtained through the exchange of genetic materials between cocultured cells. Nuclear DNA fingerprints showed that the cells that survived in uridine− BrdU+ media in both coculture conditions had the same genetic identities as the 143B cells (Fig. 3 A). More detailed data on DNA fingerprinting are shown in Table S1. On the other hand, the mtDNA hypervariable region sequences of those cells were completely identical to those of the MSCs from both coculture experiments of 143B ρ0 with MSCs (Fig. 3 B and Table S2) and R6G-treated 143B ρ+ cells with MSCs (data not shown). To further address whether mtDNAs from the MSCs replaced the mtDNAs of R6G-treated cells, we repeated the coculture experiment with R6G treatment, with a cybrid harboring 143B nuclear genome and mtDNA with +10394 Dde I and +10397 Alu I sites. The cells that survived in uridine− BrdU+ media were found to have mtDNAs with both −10394 Dde I and −10397 Alu I sites, which were identical to those of the MSCs (Fig. 3 C). Taken together, the cells that survived in uridine− BrdU+ media were trans-mitochondrial hybrid 143B cells (TM143B).


Mesenchymal stem cells transfer mitochondria to the cells with virtually no mitochondrial function but not with pathogenic mtDNA mutations.

Cho YM, Kim JH, Kim M, Park SJ, Koh SH, Ahn HS, Kang GH, Lee JB, Park KS, Lee HK - PLoS ONE (2012)

Genetic analysis of rapidly proliferating cells survived after coculture.(A) The cells survived after coculture showed genetic identities with 143B ρ0 cell nuclei. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker. (B) Since ρ0 cells did not have any detectable mtDNA, the mtDNA of the cells survived after coculture came solely from the MSCs. The hypervariable region sequences of the cells were identical with the MSCs. The arrows indicate sequence variations compared to the Cambridge reference sequence. (C) PCR-RFLP for 10394 Dde I and 10397 Alu I of mtDNA. The recuperated cells in the coculture experiment with R6G treatment to a cybrid harboring mtDNA with +10394 Dde I and +10397 Alu I were revealed to have mtDNAs with both −10394 Dde I and −10397 Alu I, both of which were identical to the MSCs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3295770&req=5

pone-0032778-g003: Genetic analysis of rapidly proliferating cells survived after coculture.(A) The cells survived after coculture showed genetic identities with 143B ρ0 cell nuclei. The boxed numbers and corresponding peaks represent locations of polymorphisms for each short tandem-repeat marker. (B) Since ρ0 cells did not have any detectable mtDNA, the mtDNA of the cells survived after coculture came solely from the MSCs. The hypervariable region sequences of the cells were identical with the MSCs. The arrows indicate sequence variations compared to the Cambridge reference sequence. (C) PCR-RFLP for 10394 Dde I and 10397 Alu I of mtDNA. The recuperated cells in the coculture experiment with R6G treatment to a cybrid harboring mtDNA with +10394 Dde I and +10397 Alu I were revealed to have mtDNAs with both −10394 Dde I and −10397 Alu I, both of which were identical to the MSCs.
Mentions: Normally, none of 143B ρ0, R6G-treated 143B ρ+, or MSC could survive in uridine− BrdU+ media; therefore, the exponentially growing cells in uridine− BrdU+ media must be the cells with normal mitochondrial function but with no thymidine kinase activity—characters that could be obtained through the exchange of genetic materials between cocultured cells. Nuclear DNA fingerprints showed that the cells that survived in uridine− BrdU+ media in both coculture conditions had the same genetic identities as the 143B cells (Fig. 3 A). More detailed data on DNA fingerprinting are shown in Table S1. On the other hand, the mtDNA hypervariable region sequences of those cells were completely identical to those of the MSCs from both coculture experiments of 143B ρ0 with MSCs (Fig. 3 B and Table S2) and R6G-treated 143B ρ+ cells with MSCs (data not shown). To further address whether mtDNAs from the MSCs replaced the mtDNAs of R6G-treated cells, we repeated the coculture experiment with R6G treatment, with a cybrid harboring 143B nuclear genome and mtDNA with +10394 Dde I and +10397 Alu I sites. The cells that survived in uridine− BrdU+ media were found to have mtDNAs with both −10394 Dde I and −10397 Alu I sites, which were identical to those of the MSCs (Fig. 3 C). Taken together, the cells that survived in uridine− BrdU+ media were trans-mitochondrial hybrid 143B cells (TM143B).

Bottom Line: Cytochalasin B, an inhibitor of chemotaxis and cytoskeletal assembly, blocked mitochondrial transfer phenomenon in the above condition.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Seoul National University College of Medicine, Seoul, South Korea.

ABSTRACT
It has been reported that human mesenchymal stem cells (MSCs) can transfer mitochondria to the cells with severely compromised mitochondrial function. We tested whether the reported intercellular mitochondrial transfer could be replicated in different types of cells or under different experimental conditions, and tried to elucidate possible mechanism. Using biochemical selection methods, we found exponentially growing cells in restrictive media (uridine(-) and bromodeoxyuridine [BrdU](+)) during the coculture of MSCs (uridine-independent and BrdU-sensitive) and 143B-derived cells with severe mitochondrial dysfunction induced by either long-term ethidium bromide treatment or short-term rhodamine 6G (R6G) treatment (uridine-dependent but BrdU-resistant). The exponentially growing cells had nuclear DNA fingerprint patterns identical to 143B, and a sequence of mitochondrial DNA (mtDNA) identical to the MSCs. Since R6G causes rapid and irreversible damage to mitochondria without the removal of mtDNA, the mitochondrial function appears to be restored through a direct transfer of mitochondria rather than mtDNA alone. Conditioned media, which were prepared by treating mtDNA-less 143B ρ(0) cells under uridine-free condition, induced increased chemotaxis in MSC, which 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.

Show MeSH
Related in: MedlinePlus