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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.

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Scheme of coculture experiments.Mitochondrial dysfunction was induced by long-term treatment of ethidium bromide (EtBr), which is known to remove mtDNA while sparing nuclear DNA, or by short-term treatment of rhodamine 6G (R6G), which tightly binds to the mitochondrial inner membrane and destroys the mitochondrial respiratory function. Gel image shows that the mtDNA was still present until five days after R6G treatment (inset). We cocultured MSCs with 143B-derived cells with severely compromised mitochondrial function (Δmt) in uridine− BrdU− media for five days (Stage I) and in uridine− BrdU+ media thereafter (Stage II). Cybrid cells harboring either the A3243G or 4,977 bp deletion mutation were also cultured in a similar way, with slight modification. For details, refer to the Materials and Methods section. The gray-colored box refers to the condition, which was expected to be fatal for the cells.
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pone-0032778-g001: Scheme of coculture experiments.Mitochondrial dysfunction was induced by long-term treatment of ethidium bromide (EtBr), which is known to remove mtDNA while sparing nuclear DNA, or by short-term treatment of rhodamine 6G (R6G), which tightly binds to the mitochondrial inner membrane and destroys the mitochondrial respiratory function. Gel image shows that the mtDNA was still present until five days after R6G treatment (inset). We cocultured MSCs with 143B-derived cells with severely compromised mitochondrial function (Δmt) in uridine− BrdU− media for five days (Stage I) and in uridine− BrdU+ media thereafter (Stage II). Cybrid cells harboring either the A3243G or 4,977 bp deletion mutation were also cultured in a similar way, with slight modification. For details, refer to the Materials and Methods section. The gray-colored box refers to the condition, which was expected to be fatal for the cells.

Mentions: The schemes of the coculture procedures are shown in Fig. 1. We cocultured human bone marrow derived MSCs at 1.5×105 cells with those cells listed above (i.e., 143B ρ0, cybrids with A3243G, and cybrids with 4,977 bp deletion) at 3×105 cells in a 100 mm culture dish with DMEM supplemented with 10% FBS and 50 µg/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 µg/ml). Then, we changed the culture medium with DMEM supplemented by 10% FBS, but without uridine (Stage I). After five days of coculture in the DMEM without uridine, we added 100 µg/ml BrdU to the same culture condition and continued cultivation for several weeks (Stage II). These experiments were repeated by adding cytochalasin B (5 µg/ml) (Sigma), an inhibitor of chemotaxis and cytoskeletal assembly.


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)

Scheme of coculture experiments.Mitochondrial dysfunction was induced by long-term treatment of ethidium bromide (EtBr), which is known to remove mtDNA while sparing nuclear DNA, or by short-term treatment of rhodamine 6G (R6G), which tightly binds to the mitochondrial inner membrane and destroys the mitochondrial respiratory function. Gel image shows that the mtDNA was still present until five days after R6G treatment (inset). We cocultured MSCs with 143B-derived cells with severely compromised mitochondrial function (Δmt) in uridine− BrdU− media for five days (Stage I) and in uridine− BrdU+ media thereafter (Stage II). Cybrid cells harboring either the A3243G or 4,977 bp deletion mutation were also cultured in a similar way, with slight modification. For details, refer to the Materials and Methods section. The gray-colored box refers to the condition, which was expected to be fatal for the cells.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3295770&req=5

pone-0032778-g001: Scheme of coculture experiments.Mitochondrial dysfunction was induced by long-term treatment of ethidium bromide (EtBr), which is known to remove mtDNA while sparing nuclear DNA, or by short-term treatment of rhodamine 6G (R6G), which tightly binds to the mitochondrial inner membrane and destroys the mitochondrial respiratory function. Gel image shows that the mtDNA was still present until five days after R6G treatment (inset). We cocultured MSCs with 143B-derived cells with severely compromised mitochondrial function (Δmt) in uridine− BrdU− media for five days (Stage I) and in uridine− BrdU+ media thereafter (Stage II). Cybrid cells harboring either the A3243G or 4,977 bp deletion mutation were also cultured in a similar way, with slight modification. For details, refer to the Materials and Methods section. The gray-colored box refers to the condition, which was expected to be fatal for the cells.
Mentions: The schemes of the coculture procedures are shown in Fig. 1. We cocultured human bone marrow derived MSCs at 1.5×105 cells with those cells listed above (i.e., 143B ρ0, cybrids with A3243G, and cybrids with 4,977 bp deletion) at 3×105 cells in a 100 mm culture dish with DMEM supplemented with 10% FBS and 50 µg/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 µg/ml). Then, we changed the culture medium with DMEM supplemented by 10% FBS, but without uridine (Stage I). After five days of coculture in the DMEM without uridine, we added 100 µg/ml BrdU to the same culture condition and continued cultivation for several weeks (Stage II). These experiments were repeated by adding cytochalasin B (5 µg/ml) (Sigma), an inhibitor of chemotaxis and cytoskeletal assembly.

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