- Email: [email protected]
Direct Human Mitochondrial Transfer: A Novel Concept Based on the Endosymbiotic Theory
Direct Human Mitochondrial Transfer: A Novel Concept Based on the Endosymbiotic Theory T. Kitania, D. Kamib, T. Kawasakib, M. Nakataa, S. Matobaa, and S. Gojob,* a
Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan; Department of Regenerative Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
b
ABSTRACT Mitochondria play an essential role in eukaryotes, and mitochondrial dysfunction is implicated in several diseases. Therefore, intercellular mitochondrial transfer has been proposed as a mechanism for cell-based therapy. In addition, internalization of isolated mitochondria cells by simple coincubation was reported to improve mitochondrial function in the recipient cells. However, substantial evidence for internalization of isolated mitochondria is still lacking, and its precise mechanism remains elusive. We tested whether enriched mitochondria can be internalized into cultured human cells by simple coincubation using fluorescence microscopy and flow cytometry. Mitochondria were isolated from endometrial glandderived mesenchymal cells (EMCs) or EMCs stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito), and enriched by antimitochondrial antibody-conjugated microbeads. They were coincubated with isogeneic EMCs stably expressing green fluorescent protein (GFP). Live fluorescence imaging clearly showed that DsRed-labeled mitochondria accumulated in the cytoplasm of EMCs stably expressing GFP around the nucleus. Flow cytometry confirmed the presence of a distinct population of GFP and DsRed double-positive cells within the recipient cells. In addition, transfer efficiency depended on mitochondrial concentration, indicating that human cells may possess the inherent ability to internalize mitochondria. Therefore, this study supports the application of direct transfer of isogeneic mitochondria as a novel approach for the treatment of diseases associated with mitochondrial dysfunction.
M
ITOCHONDRIAL dysfunction is associated with a wide range of health problems, such as cancer, aging, and metabolic, cardiovascular, and neurodegenerative diseases [1,2]. Accordingly, the transfer of exogeneous mitochondria into human cells is currently envisioned as a mechanism in cell-based therapy [3,4]. Cellular uptake of exogeneous mitochondria, and the subsequent functional recovery of the recipient cells have been reported [5,6]. However, previous studies used mitochondrial dyes, which may leak from the donor mitochondria and result in artifactual transfer of exogeneous mitochondria. In the present study, we used genetically labeled mitochondria instead of dyes to determine whether exogeneous mitochondria may be internalized into isogeneic mesenchymal cells. Internalization was monitored by fluorescence microscopy and flow cytometry using mitochondria isolated from human uterine endometrial glandderived mesenchymal
cells (EMCs) stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito) and EMCs stably expressing green fluorescent protein (GFP). MATERIALS AND METHODS Cell Culture Human EMCs were kindly provided by Dr Umezawa [7]. EMCs stably expressing GFP or DsRed-mito were generated with a recombinant retrovirus carrying GFP or DsRed-mito driven by the pMX retroviral vector, as described previously [8]. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (Life *Address correspondence to Satoshi Gojo, MD, PhD, Department of Regenerative Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii cho, Kamigyo ku, Kyoto 602-8566, Japan. E-mail: [email protected]. ac.jp
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.11.133
Transplantation Proceedings, 46, 1233e1236 (2014)
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KITANI, KAMI, KAWASAKI ET AL
Technologies, Tokyo, Japan) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies).
were incubated in 6-well plates (1 105 cells/well) with various concentrations (2.5, 5, or 10 mg/mL) of enriched mitochondria for 24 hours at 37 C under 5% CO2.
Mitochondrial Isolation and Transfer
Flow Cytometry
Mitochondria were isolated from EMCs or EMCs-DsRed-mito using the magnetic beads isolation kit (Miltenyi Biotec, Tokyo, Japan) with a modified protocol for further enrichment. In brief, harvested cells were ruptured by 20 strokes of a 27-gauge needle. The homogenate was centrifuged (400 g; 5 minutes) twice to remove unbroken cells. After magnetic labeling with antimitochondria outer membrane receptor 22 microbeads (Miltenyi Biotec), the suspension was loaded onto a MACS Column (Miltenyi Biotec) placed in the magnetic field of a MACS Separator (Miltenyi Biotec). After removing the column from the magnetic field, the retained mitochondria were flushed out and collected. The concentration of isolated mitochondria was expressed relative to protein concentration using a Bio-Rad protein assay kit (Bio-Rad, Richmond, Calif, United States). The surface charge (zeta potential: electrostatic potential generated by the accumulation of ions at the surface of colloidal particles) of isolated mitochondria was determined using a Zetasizer Nano ZS (Malvern Instruments, Malvern, United Kingdom) [9]. Mitochondria (100 mg) were fixed with 2% paraformaldehyde (TAAB Laboratory Equipment Ltd, Aldermaston, United Kingdom) and 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, United States) in 0.1 mol/L cacodylate buffer (pH 7.4) (Electron Microscopy Sciences) at 4 C overnight. After fixation, the samples were analyzed by transmission electron microscopy. For mitochondrial transfer, EMCs
Cells were dispersed with 0.25% trypsineethylenediaminetetraacetic acid and subjected to flow cytometry. The cell population was evaluated using 488- and 561-nm laser excitation lines to detect GFP and Ds-Red, respectively. Fluorescence data were collected using the Cell Sorter SH800 (Sony, Tokyo, Japan), and the flow cytometry data were analyzed using FlowJo software (TreeStar, San Carlos, Calif, United States).
RESULTS Isolated Mitochondria from EMCs-DsRed-mito
Mitochondria were isolated from EMCs-DsRed-mito (Figs 1A, B). Flow cytometry revealed that isolated mitochondria may be efficiently enriched (Fig 1C). Transmission electron microscopy confirmed a morphologically maintained ultrastructure of cristae and intact outer membranes with microbeads after enrichment (Fig 1D). Zetasizer measurement showed that isolated mitochondria had a negatively charged surface (mean ¼ 18.7 10.1 [standard deviation] mV), indicating that mitochondria could have viable functionality (Fig 1E).
Fig 1. Mitochondria were isolated and enriched from human uterine endometrial gland-derived mesenchymal cells (EMCs) or EMCs stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito). (A) Representative image of EMCs-DsRedmito by fluorescence microscopy. Scale bar, 100 mm. (B) Representative images of mitochondria isolated from EMCs-DsRed-mito: phase contrast (left), fluorescence (middle), and merged images (right). Scale bar, 20 mm. (C) Flow cytometry of isolated mitochondria. Black and red curves represent mitochondria from EMCs and EMCs-DsRed-mito, respectively. (D) Transmission electron microscopy of isolated mitochondria. Black arrows indicate microbeads attached to the mitochondrial outer membrane. Scale bar, 500 nm. (E) Zeta potential distribution of isolated mitochondria. (AV, average zeta potential.)
DIRECT HUMAN MITOCHONDRIAL TRANSFER
Detection of Internalized Mitochondria
Live fluorescence imaging clearly showed that isogeneic DsRed-labeled mitochondria were internalized into human EMCs expressing GFP after 24 hours of incubation (Fig 2A). In addition, flow cytometry of the recipient EMCs expressing GFP confirmed the presence of a distinct population of GFP and DsRed double-positive cells (Fig 2B). Moreover, the percentages of GFP and DsRed double-positive cells increased with the delivered mitochondrial concentration (2.5, 5, or 10 mg/mL) by 0.39%, 0.81%, and 1.66%, respectively. DISCUSSION
This study shows that exogeneous human mitochondria are internalized into isogeneic cells during in vitro experiments. Previous studies used dyes to stain mitochondria, which is subject to misleading artifacts because of possible leakage of the dye [5,6]. To avoid false-positive results, we genetically labeled the donor mitochondria and recipient cells. Transmission electron microscopy confirmed that the isolated and enriched mitochondria preserved a morphologically intact outer membrane. Furthermore, their surface was negatively charged, indicating that the mitochondrial membrane potential was maintained after isolation. Fluorescence microscopy revealed that DsRed-labeled mitochondria were internalized into isogeneic EMCs expressing GFP by simple
1235
coincubation. Furthermore, EMCs expressing GFP carrying DsRed-labeled mitochondria were found to have a single nucleus, which excludes the possibility of cell fusion between EMCs expressing GFP and EMCs containing DsRed-labeled mitochondria. Flow cytometry confirmed the presence of a distinct population of GFP and DsRed double-positive cells. These results are consistent with those of cellular uptake of exogeneous human mitochondria by simple coincubation in vitro. Masuzawa et al recently reported that direct injection of autologous mitochondria into the heart considerably improved post-infarct cardiac functions in a rabbit myocardial infarction model [10]. They also reported that only a limited number of engrafted mitochondria were internalized into cells. Similar to their result, we found that cells containing exogeneous mitochondria were relatively rare. Our experiments indicate that the percentage of mitochondrial internalization increases in a dose-dependent manner in vitro. The more sophisticated delivery of mitochondria might repair ischemic injury more efficiently. To the best of our knowledge, only cancer cell lines have been reported to be capable of internalizing exogeneous mitochondria [5,6,10e14]. In this study, we show that noncancerous human cells also have the ability to internalize exogeneous mitochondria. The present study supports applications for mitochondrial transfer as a novel therapy for diseases associated with mitochondrial dysfunction.
Fig 2. Isogeneic DsRed-labeled mitochondria were internalized into human EMCs expressing GFP. (A) Representative live fluorescence images of DsRed-labeled mitochondria internalized into EMCs expressing GFP. Internalized DsRed-labeled mitochondria (far left), recipient EMCs expressing GFP (middle left), phase contrast image (middle right), and merged images (far right). White arrows indicate internalized DsRed-labeled mitochondria. Scale bars, 100 mm. (B) Flow cytometry analysis of EMCs expressing GFP after 24 hours of coincubation with 0 (control), 2.5, 5.0, or 10 mg/mL DsRed-labeled mitochondria. (Control, no mitochondria delivery; Mito, 2.5e10, 2.5e10 mg/mL of mitochondria delivery.)
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REFERENCES [1] Lesnefsky EJ, Moghaddas S, Tandler B, et al. Mitochondrial dysfunction in cardiac disease: ischemiadreperfusion, aging, and heart failure. J Mol Cell Cardiol 2001;33:1065. [2] Nunnari J, Suomalainen A. Mitochondria: in sickness and in health. Cell 2012;148:1145. [3] Islam MN, Das SR, Emin MT, et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nature Medicine 2012;18:759. [4] Spees JL, Olson SD, Whitney MJ, et al. Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A 2006;103:1283. [5] Clark MA, Shay JW. Mitochondrial transformation of mammalian cells. Nature 1982;295:605. [6] Katrangi E, D’Souza G, Boddapati SV, et al. Xenogenic transfer of isolated murine mitochondria into human Rho0 cells can improve respiratory function. Rejuvenation Res 2007;10:561. [7] Ikegami Y, Miyoshi S, Nishiyama N, et al. Serum-independent cardiomyogenic transdifferentiation in human endometriumderived mesenchymal cells. Artificial Organs 2010;34:280. [8] Ishii R, Kami D, Toyoda M, et al. Placenta to cartilage: direct conversion of human placenta to chondrocytes with transformation by defined factors. Mol Biol Cell 2012;23:3511.
KITANI, KAMI, KAWASAKI ET AL [9] Jo J, Aoki I, Tabata Y. Design of iron oxide nanoparticles with different sizes and surface charges for simple and efficient labeling of mesenchymal stem cells. J Control Release 2010;142: 465. [10] Masuzawa A, Black KM, Pacak CA, et al. Transplantation of autologously-derived mitochondria protects the heart from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2013;304:H966. [11] Ber R, Stauver MG, Shay JW. Use of isolated mitochondria to transfer chloramphenicol resistance in hamster cells. Israel J Med Sci 1984;20:244. [12] Chang JC, Liu KH, Li YC, et al. Functional recovery of human cells harbouring the mitochondrial DNA mutation Merrf A8344g via peptide-mediated mitochondrial delivery. Neurosignals 2012;21:160. [13] Elliott RL, Jiang XP, Head JF. Mitochondria organelle transplantation: introduction of normal epithelial mitochondria into human cancer cells inhibits proliferation and increases drug sensitivity. Breast Cancer Res Treatment 2012; 136:347. [14] McCully JD, Cowan DB, Pacak CA, et al. Injection of isolated mitochondria during early reperfusion for cardioprotection. Am J Physiol Heart Circ Physiol 2009;296:H94.
Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan; Department of Regenerative Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan
b
ABSTRACT Mitochondria play an essential role in eukaryotes, and mitochondrial dysfunction is implicated in several diseases. Therefore, intercellular mitochondrial transfer has been proposed as a mechanism for cell-based therapy. In addition, internalization of isolated mitochondria cells by simple coincubation was reported to improve mitochondrial function in the recipient cells. However, substantial evidence for internalization of isolated mitochondria is still lacking, and its precise mechanism remains elusive. We tested whether enriched mitochondria can be internalized into cultured human cells by simple coincubation using fluorescence microscopy and flow cytometry. Mitochondria were isolated from endometrial glandderived mesenchymal cells (EMCs) or EMCs stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito), and enriched by antimitochondrial antibody-conjugated microbeads. They were coincubated with isogeneic EMCs stably expressing green fluorescent protein (GFP). Live fluorescence imaging clearly showed that DsRed-labeled mitochondria accumulated in the cytoplasm of EMCs stably expressing GFP around the nucleus. Flow cytometry confirmed the presence of a distinct population of GFP and DsRed double-positive cells within the recipient cells. In addition, transfer efficiency depended on mitochondrial concentration, indicating that human cells may possess the inherent ability to internalize mitochondria. Therefore, this study supports the application of direct transfer of isogeneic mitochondria as a novel approach for the treatment of diseases associated with mitochondrial dysfunction.
M
ITOCHONDRIAL dysfunction is associated with a wide range of health problems, such as cancer, aging, and metabolic, cardiovascular, and neurodegenerative diseases [1,2]. Accordingly, the transfer of exogeneous mitochondria into human cells is currently envisioned as a mechanism in cell-based therapy [3,4]. Cellular uptake of exogeneous mitochondria, and the subsequent functional recovery of the recipient cells have been reported [5,6]. However, previous studies used mitochondrial dyes, which may leak from the donor mitochondria and result in artifactual transfer of exogeneous mitochondria. In the present study, we used genetically labeled mitochondria instead of dyes to determine whether exogeneous mitochondria may be internalized into isogeneic mesenchymal cells. Internalization was monitored by fluorescence microscopy and flow cytometry using mitochondria isolated from human uterine endometrial glandderived mesenchymal
cells (EMCs) stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito) and EMCs stably expressing green fluorescent protein (GFP). MATERIALS AND METHODS Cell Culture Human EMCs were kindly provided by Dr Umezawa [7]. EMCs stably expressing GFP or DsRed-mito were generated with a recombinant retrovirus carrying GFP or DsRed-mito driven by the pMX retroviral vector, as described previously [8]. Both cell lines were maintained in Dulbecco’s modified Eagle’s medium (Life *Address correspondence to Satoshi Gojo, MD, PhD, Department of Regenerative Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii cho, Kamigyo ku, Kyoto 602-8566, Japan. E-mail: [email protected]. ac.jp
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/14/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.11.133
Transplantation Proceedings, 46, 1233e1236 (2014)
1233
1234
KITANI, KAMI, KAWASAKI ET AL
Technologies, Tokyo, Japan) supplemented with 10% fetal bovine serum (Life Technologies) and 1% penicillin/streptomycin (Life Technologies).
were incubated in 6-well plates (1 105 cells/well) with various concentrations (2.5, 5, or 10 mg/mL) of enriched mitochondria for 24 hours at 37 C under 5% CO2.
Mitochondrial Isolation and Transfer
Flow Cytometry
Mitochondria were isolated from EMCs or EMCs-DsRed-mito using the magnetic beads isolation kit (Miltenyi Biotec, Tokyo, Japan) with a modified protocol for further enrichment. In brief, harvested cells were ruptured by 20 strokes of a 27-gauge needle. The homogenate was centrifuged (400 g; 5 minutes) twice to remove unbroken cells. After magnetic labeling with antimitochondria outer membrane receptor 22 microbeads (Miltenyi Biotec), the suspension was loaded onto a MACS Column (Miltenyi Biotec) placed in the magnetic field of a MACS Separator (Miltenyi Biotec). After removing the column from the magnetic field, the retained mitochondria were flushed out and collected. The concentration of isolated mitochondria was expressed relative to protein concentration using a Bio-Rad protein assay kit (Bio-Rad, Richmond, Calif, United States). The surface charge (zeta potential: electrostatic potential generated by the accumulation of ions at the surface of colloidal particles) of isolated mitochondria was determined using a Zetasizer Nano ZS (Malvern Instruments, Malvern, United Kingdom) [9]. Mitochondria (100 mg) were fixed with 2% paraformaldehyde (TAAB Laboratory Equipment Ltd, Aldermaston, United Kingdom) and 2% glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, United States) in 0.1 mol/L cacodylate buffer (pH 7.4) (Electron Microscopy Sciences) at 4 C overnight. After fixation, the samples were analyzed by transmission electron microscopy. For mitochondrial transfer, EMCs
Cells were dispersed with 0.25% trypsineethylenediaminetetraacetic acid and subjected to flow cytometry. The cell population was evaluated using 488- and 561-nm laser excitation lines to detect GFP and Ds-Red, respectively. Fluorescence data were collected using the Cell Sorter SH800 (Sony, Tokyo, Japan), and the flow cytometry data were analyzed using FlowJo software (TreeStar, San Carlos, Calif, United States).
RESULTS Isolated Mitochondria from EMCs-DsRed-mito
Mitochondria were isolated from EMCs-DsRed-mito (Figs 1A, B). Flow cytometry revealed that isolated mitochondria may be efficiently enriched (Fig 1C). Transmission electron microscopy confirmed a morphologically maintained ultrastructure of cristae and intact outer membranes with microbeads after enrichment (Fig 1D). Zetasizer measurement showed that isolated mitochondria had a negatively charged surface (mean ¼ 18.7 10.1 [standard deviation] mV), indicating that mitochondria could have viable functionality (Fig 1E).
Fig 1. Mitochondria were isolated and enriched from human uterine endometrial gland-derived mesenchymal cells (EMCs) or EMCs stably expressing mitochondrial-targeted red fluorescent protein (EMCs-DsRed-mito). (A) Representative image of EMCs-DsRedmito by fluorescence microscopy. Scale bar, 100 mm. (B) Representative images of mitochondria isolated from EMCs-DsRed-mito: phase contrast (left), fluorescence (middle), and merged images (right). Scale bar, 20 mm. (C) Flow cytometry of isolated mitochondria. Black and red curves represent mitochondria from EMCs and EMCs-DsRed-mito, respectively. (D) Transmission electron microscopy of isolated mitochondria. Black arrows indicate microbeads attached to the mitochondrial outer membrane. Scale bar, 500 nm. (E) Zeta potential distribution of isolated mitochondria. (AV, average zeta potential.)
DIRECT HUMAN MITOCHONDRIAL TRANSFER
Detection of Internalized Mitochondria
Live fluorescence imaging clearly showed that isogeneic DsRed-labeled mitochondria were internalized into human EMCs expressing GFP after 24 hours of incubation (Fig 2A). In addition, flow cytometry of the recipient EMCs expressing GFP confirmed the presence of a distinct population of GFP and DsRed double-positive cells (Fig 2B). Moreover, the percentages of GFP and DsRed double-positive cells increased with the delivered mitochondrial concentration (2.5, 5, or 10 mg/mL) by 0.39%, 0.81%, and 1.66%, respectively. DISCUSSION
This study shows that exogeneous human mitochondria are internalized into isogeneic cells during in vitro experiments. Previous studies used dyes to stain mitochondria, which is subject to misleading artifacts because of possible leakage of the dye [5,6]. To avoid false-positive results, we genetically labeled the donor mitochondria and recipient cells. Transmission electron microscopy confirmed that the isolated and enriched mitochondria preserved a morphologically intact outer membrane. Furthermore, their surface was negatively charged, indicating that the mitochondrial membrane potential was maintained after isolation. Fluorescence microscopy revealed that DsRed-labeled mitochondria were internalized into isogeneic EMCs expressing GFP by simple
1235
coincubation. Furthermore, EMCs expressing GFP carrying DsRed-labeled mitochondria were found to have a single nucleus, which excludes the possibility of cell fusion between EMCs expressing GFP and EMCs containing DsRed-labeled mitochondria. Flow cytometry confirmed the presence of a distinct population of GFP and DsRed double-positive cells. These results are consistent with those of cellular uptake of exogeneous human mitochondria by simple coincubation in vitro. Masuzawa et al recently reported that direct injection of autologous mitochondria into the heart considerably improved post-infarct cardiac functions in a rabbit myocardial infarction model [10]. They also reported that only a limited number of engrafted mitochondria were internalized into cells. Similar to their result, we found that cells containing exogeneous mitochondria were relatively rare. Our experiments indicate that the percentage of mitochondrial internalization increases in a dose-dependent manner in vitro. The more sophisticated delivery of mitochondria might repair ischemic injury more efficiently. To the best of our knowledge, only cancer cell lines have been reported to be capable of internalizing exogeneous mitochondria [5,6,10e14]. In this study, we show that noncancerous human cells also have the ability to internalize exogeneous mitochondria. The present study supports applications for mitochondrial transfer as a novel therapy for diseases associated with mitochondrial dysfunction.
Fig 2. Isogeneic DsRed-labeled mitochondria were internalized into human EMCs expressing GFP. (A) Representative live fluorescence images of DsRed-labeled mitochondria internalized into EMCs expressing GFP. Internalized DsRed-labeled mitochondria (far left), recipient EMCs expressing GFP (middle left), phase contrast image (middle right), and merged images (far right). White arrows indicate internalized DsRed-labeled mitochondria. Scale bars, 100 mm. (B) Flow cytometry analysis of EMCs expressing GFP after 24 hours of coincubation with 0 (control), 2.5, 5.0, or 10 mg/mL DsRed-labeled mitochondria. (Control, no mitochondria delivery; Mito, 2.5e10, 2.5e10 mg/mL of mitochondria delivery.)
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REFERENCES [1] Lesnefsky EJ, Moghaddas S, Tandler B, et al. Mitochondrial dysfunction in cardiac disease: ischemiadreperfusion, aging, and heart failure. J Mol Cell Cardiol 2001;33:1065. [2] Nunnari J, Suomalainen A. Mitochondria: in sickness and in health. Cell 2012;148:1145. [3] Islam MN, Das SR, Emin MT, et al. Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury. Nature Medicine 2012;18:759. [4] Spees JL, Olson SD, Whitney MJ, et al. Mitochondrial transfer between cells can rescue aerobic respiration. Proc Natl Acad Sci U S A 2006;103:1283. [5] Clark MA, Shay JW. Mitochondrial transformation of mammalian cells. Nature 1982;295:605. [6] Katrangi E, D’Souza G, Boddapati SV, et al. Xenogenic transfer of isolated murine mitochondria into human Rho0 cells can improve respiratory function. Rejuvenation Res 2007;10:561. [7] Ikegami Y, Miyoshi S, Nishiyama N, et al. Serum-independent cardiomyogenic transdifferentiation in human endometriumderived mesenchymal cells. Artificial Organs 2010;34:280. [8] Ishii R, Kami D, Toyoda M, et al. Placenta to cartilage: direct conversion of human placenta to chondrocytes with transformation by defined factors. Mol Biol Cell 2012;23:3511.
KITANI, KAMI, KAWASAKI ET AL [9] Jo J, Aoki I, Tabata Y. Design of iron oxide nanoparticles with different sizes and surface charges for simple and efficient labeling of mesenchymal stem cells. J Control Release 2010;142: 465. [10] Masuzawa A, Black KM, Pacak CA, et al. Transplantation of autologously-derived mitochondria protects the heart from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 2013;304:H966. [11] Ber R, Stauver MG, Shay JW. Use of isolated mitochondria to transfer chloramphenicol resistance in hamster cells. Israel J Med Sci 1984;20:244. [12] Chang JC, Liu KH, Li YC, et al. Functional recovery of human cells harbouring the mitochondrial DNA mutation Merrf A8344g via peptide-mediated mitochondrial delivery. Neurosignals 2012;21:160. [13] Elliott RL, Jiang XP, Head JF. Mitochondria organelle transplantation: introduction of normal epithelial mitochondria into human cancer cells inhibits proliferation and increases drug sensitivity. Breast Cancer Res Treatment 2012; 136:347. [14] McCully JD, Cowan DB, Pacak CA, et al. Injection of isolated mitochondria during early reperfusion for cardioprotection. Am J Physiol Heart Circ Physiol 2009;296:H94.