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Background:. CD8+ Cytotoxic T lymphocytes play a key role in the pathogenesis of autoimmune diseases and clinical conditions such as graft versus host disease and graft rejection. Mesenchymal Stromal Cells (MSCs) are multipotent cells with tissue repair and immunomodulatory capabilities. Since they are able to suppress multiple pathogenic immune responses, MSCs have been proposed as a cellular therapy for the treatment of immune-mediated diseases. However, the mechanisms underlying their immunosuppressive properties are not yet fully understood. MSCs have the remarkable ability to sense tissue injury and inflammation and respond by donating their own mitochondria to neighboring cells. Whether mitochondrial transfer has any role in the repression of CD8+ responses is unknown. Methods and results:. We have utilized CD8+ T cells from Clone 4 TCR transgenic mice that differentiate into effector cells upon activation in vitro and in vivo to address this question. Allogeneic bone marrow derived MSCs, co-cultured with activated Clone 4 CD8+ T cells, decreased their expansion, the production of the effector cytokine IFNγ and their diabetogenic potential in vivo. Notably, we found that during this interaction leading to suppression, MSCs transferred mitochondria to CD8+ T cells as evidenced by FACS and confocal microscopy. Transfer of MSC mitochondria to Clone 4 CD8+ T cells also resulted in decreased expansion and production of IFNγ upon activation. These effects overlapped and were additive with those of prostaglandin E2 secreted by MSCs. Furthermore, preventing mitochondrial transfer in co-cultures diminished the ability of MSCs to inhibit IFNγ production. Finally, we demonstrated that both MSCs and MSC mitochondria downregulated T-bet and Eomes expression, key transcription factors for CTL differentiation, on activated CD8+ T cells. Conclusion:. In this report we showed that MSCs are able to interact with CD8+ T cells and transfer them their mitochondria. Mitochondrial transfer contributed to the global suppressive effect of MSCs on CD8+ T cell activation by downregulating T-bet and Eomes expression resulting in impaired IFNγ production of activated CD8+ T cells. [ABSTRACT FROM AUTHOR]

Abstract Background . CD8+ Cytotoxic T lymphocytes play a key role in the pathogenesis of autoimmune diseases and clinical conditions such as graft versus host disease and graft rejection. Mesenchymal Stromal Cells (MSCs) are multipotent cells with tissue repair and immunomodulatory capabilities. Since they are able to suppress multiple pathogenic immune responses, MSCs have been proposed as a cellular therapy for the treatment of immune-mediated diseases. However, the mechanisms underlying their immunosuppressive properties are not yet fully understood. MSCs have the remarkable ability to sense tissue injury and inflammation and respond by donating their own mitochondria to neighboring cells. Whether mitochondrial transfer has any role in the repression of CD8+ responses is unknown. Methods and results . We have utilized CD8+ T cells from Clone 4 TCR transgenic mice that differentiate into effector cells upon activation in vitro and in vivo to address this question. Allogeneic bone marrow derived MSCs, co-cultured with activated Clone 4 CD8+ T cells, decreased their expansion, the production of the effector cytokine IFNγ and their diabetogenic potential in vivo. Notably, we found that during this interaction leading to suppression, MSCs transferred mitochondria to CD8+ T cells as evidenced by FACS and confocal microscopy. Transfer of MSC mitochondria to Clone 4 CD8+ T cells also resulted in decreased expansion and production of IFNγ upon activation. These effects overlapped and were additive with those of prostaglandin E2 secreted by MSCs. Furthermore, preventing mitochondrial transfer in co-cultures diminished the ability of MSCs to inhibit IFNγ production. Finally, we demonstrated that both MSCs and MSC mitochondria downregulated T-bet and Eomes expression, key transcription factors for CTL differentiation, on activated CD8+ T cells. Conclusion . In this report we showed that MSCs are able to interact with CD8+ T cells and transfer them their mitochondria. Mitochondrial transfer contributed to the global suppressive effect of MSCs on CD8+ T cell activation by downregulating T-bet and Eomes expression resulting in impaired IFNγ production of activated CD8+ T cells.

Abstract Background Although mesenchymal stem cells (MSCs) have been effective in tendinopathy, the mechanisms by which MSCs promote tendon healing have not been fully elucidated. In this study, we tested the hypothesis that MSCs transfer mitochondria to injured tenocytes in vitro and in vivo to protect against Achilles tendinopathy (AT). Methods Bone marrow MSCs and H2O2-injured tenocytes were co-cultured, and mitochondrial transfer was visualized by MitoTracker dye staining. Mitochondrial function, including mitochondrial membrane potential, oxygen consumption rate, and adenosine triphosphate content, was quantified in sorted tenocytes. Tenocyte proliferation, apoptosis, oxidative stress, and inflammation were analyzed. Furthermore, a collagenase type I-induced rat AT model was used to detect mitochondrial transfer in tissues and evaluate Achilles tendon healing. Results MSCs successfully donated healthy mitochondria to in vitro and in vivo damaged tenocytes. Interestingly, mitochondrial transfer was almost completely blocked by co-treatment with cytochalasin B. Transfer of MSC-derived mitochondria decreased apoptosis, promoted proliferation, and restored mitochondrial function in H2O2-induced tenocytes. A decrease in reactive oxygen species and pro-inflammatory cytokine levels (interleukin-6 and -1β) was observed. In vivo, mitochondrial transfer from MSCs improved the expression of tendon-specific markers (scleraxis, tenascin C, and tenomodulin) and decreased the infiltration of inflammatory cells into the tendon. In addition, the fibers of the tendon tissue were neatly arranged and the structure of the tendon was remodeled. Inhibition of mitochondrial transfer by cytochalasin B abrogated the therapeutic efficacy of MSCs in tenocytes and tendon tissues. Conclusions MSCs rescued distressed tenocytes from apoptosis by transferring mitochondria. This provides evidence that mitochondrial transfer is one mechanism by which MSCs exert their therapeutic effects on damaged tenocytes.

Abstract Background Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer. Methods We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors. Results Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore. Conclusions RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria.

Abstract Background Mesenchymal stem/stromal cells (MSCs) are multipotent cells with strong tissue repair and immunomodulatory properties. Due to their ability to repress pathogenic immune responses, and in particular T cell responses, they show therapeutic potential for the treatment of autoimmune diseases, organ rejection and graft versus host disease. MSCs have the remarkable ability to export their own mitochondria to neighboring cells in response to injury and inflammation. However, whether mitochondrial transfer occurs and has any role in the repression of CD4+ Th1 responses is unknown. Methods and results In this report we have utilized CD4+ T cells from HNT TCR transgenic mice that develop Th1-like responses upon antigenic stimulation in vitro and in vivo. Allogeneic bone marrow-derived MSCs reduced the diabetogenic potential of HNT CD4+ T cells in vivo in a transgenic mouse model of disease. In co-culture experiments, we have shown that MSCs were able to reduce HNT CD4+ T cell expansion, expression of key effector markers and production of the effector cytokine IFNγ after activation. This was associated with the ability of CD4+ T cells to acquire mitochondria from MSCs as evidenced by FACS and confocal microscopy. Remarkably, transfer of isolated MSC mitochondria to CD4+ T cells resulted in decreased T cell proliferation and IFNγ production. These effects were additive with those of prostaglandin E2 secreted by MSCs. Finally, we demonstrated that both co-culture with MSCs and transfer of isolated MSC mitochondria prevent the upregulation of T-bet, the master Th1 transcription factor, on activated CD4+ T cells. Conclusion The present study demonstrates that transfer of MSC mitochondria to activated CD4+ T cells results in the suppression of Th1 responses in part by downregulating T-bet expression. Furthermore, our studies suggest that MSC mitochondrial transfer might represent a general mechanism of MSC-dependent immunosuppression.

Background: Although mesenchymal stem cells (MSCs) have been effective in tendinopathy, the mechanisms by which MSCs promote tendon healing have not been fully elucidated. In this study, we tested the hypothesis that MSCs transfer mitochondria to injured tenocytes in vitro and in vivo to protect against Achilles tendinopathy (AT). Methods: Bone marrow MSCs and H2O2-injured tenocytes were co-cultured, and mitochondrial transfer was visualized by MitoTracker dye staining. Mitochondrial function, including mitochondrial membrane potential, oxygen consumption rate, and adenosine triphosphate content, was quantified in sorted tenocytes. Tenocyte proliferation, apoptosis, oxidative stress, and inflammation were analyzed. Furthermore, a collagenase type I-induced rat AT model was used to detect mitochondrial transfer in tissues and evaluate Achilles tendon healing. Results: MSCs successfully donated healthy mitochondria to in vitro and in vivo damaged tenocytes. Interestingly, mitochondrial transfer was almost completely blocked by co-treatment with cytochalasin B. Transfer of MSC-derived mitochondria decreased apoptosis, promoted proliferation, and restored mitochondrial function in H2O2-induced tenocytes. A decrease in reactive oxygen species and pro-inflammatory cytokine levels (interleukin-6 and -1β) was observed. In vivo, mitochondrial transfer from MSCs improved the expression of tendon-specific markers (scleraxis, tenascin C, and tenomodulin) and decreased the infiltration of inflammatory cells into the tendon. In addition, the fibers of the tendon tissue were neatly arranged and the structure of the tendon was remodeled. Inhibition of mitochondrial transfer by cytochalasin B abrogated the therapeutic efficacy of MSCs in tenocytes and tendon tissues. Conclusions: MSCs rescued distressed tenocytes from apoptosis by transferring mitochondria. This provides evidence that mitochondrial transfer is one mechanism by which MSCs exert their therapeutic effects on damaged tenocytes. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial dysfunction caused by mutations in mitochondrial DNA (mtDNA) or nuclear DNA, which codes for mitochondrial components, are known to be associated with various genetic and congenital disorders. These mitochondrial disorders not only impair energy production but also affect mitochondrial functions and have no effective treatment. Mesenchymal stem cells (MSCs) are known to migrate to damaged sites and carry out mitochondrial transfer. MSCs grown using conventional culture methods exhibit heterogeneous cellular characteristics. In contrast, highly purified MSCs, namely the rapidly expanding clones (RECs) isolated by single-cell sorting, display uniform MSCs functionality. Therefore, we examined the differences between RECs and MSCs to assess the efficacy of mitochondrial transfer. Methods: We established mitochondria-deficient cell lines (ρ0 A549 and ρ0 HeLa cell lines) using ethidium bromide. Mitochondrial transfer from RECs/MSCs to ρ0 cells was confirmed by PCR and flow cytometry analysis. We examined several mitochondrial functions including ATP, reactive oxygen species, mitochondrial membrane potential, and oxygen consumption rate (OCR). The route of mitochondrial transfer was identified using inhibition assays for microtubules/tunneling nanotubes, gap junctions, or microvesicles using transwell assay and molecular inhibitors. Results: Co-culture of ρ0 cells with MSCs or RECs led to restoration of the mtDNA content. RECs transferred more mitochondria to ρ0 cells compared to that by MSCs. The recovery of mitochondrial function, including ATP, OCR, mitochondrial membrane potential, and mitochondrial swelling in ρ0 cells co-cultured with RECs was superior than that in cells co-cultured with MSCs. Inhibition assays for each pathway revealed that RECs were sensitive to endocytosis inhibitor, dynasore. Conclusions: RECs might serve as a potential therapeutic strategy for diseases linked to mitochondrial dysfunction by donating healthy mitochondria. [ABSTRACT FROM AUTHOR]

Abstract The most frequent biochemical defect of inherited mitochondrial disease is isolated complex I deficiency. There is no cure for this disorder, and treatment is mainly supportive. In this study, we investigated the effects of human mesenchymal stem cells (MSCs) on skin fibroblast derived from three individuals with complex I deficiency carrying different pathogenic variants in mitochondrial DNA-encoded subunits (MT-ND3, MT-ND6). Complex I-deficient fibroblasts were transiently co-cultured with bone marrow-derived MSCs. Mitochondrial transfer was analysed by fluorescence labelling and validated by Sanger sequencing. Levels of reactive oxygen species (ROS) were measured using MitoSOX Red. Moreover, mitochondrial respiration was analysed by Seahorse XFe96 Extracellular Flux Analyzer. Levels of antioxidant proteins were investigated via immunoblotting. Co-culturing of complex I-deficient fibroblast with MSCs lowered cellular ROS levels. The effect on ROS production was more sustained compared to treatment of patient fibroblasts with culture medium derived from MSC cultures. Investigation of cellular antioxidant defence systems revealed an upregulation of SOD2 (superoxide dismutase 2, mitochondrial) and HO-1 (heme oxygenase 1) in patient-derived cell lines. This adaptive response was normalised upon MSC treatment. Moreover, Seahorse experiments revealed a significant improvement of mitochondrial respiration, indicating a mitigation of the oxidative phosphorylation defect. Experiments with repetitive MSC co-culture at two consecutive time points enhanced this effect. Our study indicates that MSC-based treatment approaches might constitute an interesting option for patients with mitochondrial DNA-encoded mitochondrial diseases. We suggest that this strategy may prove more promising for defects caused by mitochondrial DNA variants compared to nuclear-encoded defects.

Background: Mesenchymal stem/stromal cells (MSCs) are multipotent cells with strong tissue repair and immunomodulatory properties. Due to their ability to repress pathogenic immune responses, and in particular T cell responses, they show therapeutic potential for the treatment of autoimmune diseases, organ rejection and graft versus host disease. MSCs have the remarkable ability to export their own mitochondria to neighboring cells in response to injury and inflammation. However, whether mitochondrial transfer occurs and has any role in the repression of CD4+ Th1 responses is unknown. Methods and results: In this report we have utilized CD4+ T cells from HNT TCR transgenic mice that develop Th1-like responses upon antigenic stimulation in vitro and in vivo. Allogeneic bone marrow-derived MSCs reduced the diabetogenic potential of HNT CD4+ T cells in vivo in a transgenic mouse model of disease. In co-culture experiments, we have shown that MSCs were able to reduce HNT CD4+ T cell expansion, expression of key effector markers and production of the effector cytokine IFNγ after activation. This was associated with the ability of CD4+ T cells to acquire mitochondria from MSCs as evidenced by FACS and confocal microscopy. Remarkably, transfer of isolated MSC mitochondria to CD4+ T cells resulted in decreased T cell proliferation and IFNγ production. These effects were additive with those of prostaglandin E2 secreted by MSCs. Finally, we demonstrated that both co-culture with MSCs and transfer of isolated MSC mitochondria prevent the upregulation of T-bet, the master Th1 transcription factor, on activated CD4+ T cells. Conclusion: The present study demonstrates that transfer of MSC mitochondria to activated CD4+ T cells results in the suppression of Th1 responses in part by downregulating T-bet expression. Furthermore, our studies suggest that MSC mitochondrial transfer might represent a general mechanism of MSC-dependent immunosuppression. [ABSTRACT FROM AUTHOR]

Abstract Background Non-alcoholic fatty liver disease (NAFLD) has become a global epidemic disease. Its incidence is associated with type 2 diabetes mellitus (T2DM). Presently, there is no approved pharmacological agents specially developed for NAFLD. One promising disease-modifying strategy is the transplantation of stem cells to promote metabolic regulation and repair of injury. Method In this study, a T2DM model was established through 28-week high-fat diet (HFD) feeding resulting in T2DM-associated NAFLD, followed by the injection of bone marrow mesenchymal stem cells (BMSCs). The morphology, function, and transfer of hepatocyte mitochondria were evaluated in both vivo and in vitro. Results BMSC implantation resulted in the considerable recovery of increasing weight, HFD-induced steatosis, liver function, and disordered glucose and lipid metabolism. The treatment with BMSC transplantation was accompanied by reduced fat accumulation. Moreover, mitochondrial transfer was observed in both vivo and vitro studies. And the mitochondria-recipient steatotic cells exhibited significantly enhanced OXPHOS activity, ATP production, and mitochondrial membrane potential, and reduced reactive oxygen species levels, which were not achieved by the blocking of mitochondrial transfer. Conclusion Mitochondrial transfer from BMSCs is a feasible process to combat NAFLD via rescuing dysfunction mitochondria, and has a promising therapeutic effect on metabolism-related diseases.

The most frequent biochemical defect of inherited mitochondrial disease is isolated complex I deficiency. There is no cure for this disorder, and treatment is mainly supportive. In this study, we investigated the effects of human mesenchymal stem cells (MSCs) on skin fibroblast derived from three individuals with complex I deficiency carrying different pathogenic variants in mitochondrial DNA-encoded subunits (MT-ND3, MT-ND6). Complex I-deficient fibroblasts were transiently co-cultured with bone marrow-derived MSCs. Mitochondrial transfer was analysed by fluorescence labelling and validated by Sanger sequencing. Levels of reactive oxygen species (ROS) were measured using MitoSOX Red. Moreover, mitochondrial respiration was analysed by Seahorse XFe96 Extracellular Flux Analyzer. Levels of antioxidant proteins were investigated via immunoblotting. Co-culturing of complex I-deficient fibroblast with MSCs lowered cellular ROS levels. The effect on ROS production was more sustained compared to treatment of patient fibroblasts with culture medium derived from MSC cultures. Investigation of cellular antioxidant defence systems revealed an upregulation of SOD2 (superoxide dismutase 2, mitochondrial) and HO-1 (heme oxygenase 1) in patient-derived cell lines. This adaptive response was normalised upon MSC treatment. Moreover, Seahorse experiments revealed a significant improvement of mitochondrial respiration, indicating a mitigation of the oxidative phosphorylation defect. Experiments with repetitive MSC co-culture at two consecutive time points enhanced this effect. Our study indicates that MSC-based treatment approaches might constitute an interesting option for patients with mitochondrial DNA-encoded mitochondrial diseases. We suggest that this strategy may prove more promising for defects caused by mitochondrial DNA variants compared to nuclear-encoded defects. [ABSTRACT FROM AUTHOR]

Abstract Mesenchymal stem cells (MSCs) are multipotent cells that show self-renewal, multi-directional differentiation, and paracrine and immune regulation. As a result of these properties, the MSCs have great clinical application prospects, especially in the regeneration of injured tissues, functional reconstruction, and cell therapy. However, the transplanted MSCs are prone to ageing and apoptosis and have a difficult to control direction differentiation. Therefore, it is necessary to effectively regulate the functions of the MSCs to promote their desired effects. In recent years, it has been found that mitochondria, the main organelles responsible for energy metabolism and adenosine triphosphate production in cells, play a key role in regulating different functions of the MSCs through various mechanisms. Thus, mitochondria could act as effective targets for regulating and promoting the functions of the MSCs. In this review, we discuss the research status and current understanding of the role and mechanism of mitochondrial energy metabolism, morphology, transfer modes, and dynamics on MSC functions.

Background: Non-alcoholic fatty liver disease (NAFLD) has become a global epidemic disease. Its incidence is associated with type 2 diabetes mellitus (T2DM). Presently, there is no approved pharmacological agents specially developed for NAFLD. One promising disease-modifying strategy is the transplantation of stem cells to promote metabolic regulation and repair of injury. Method: In this study, a T2DM model was established through 28-week high-fat diet (HFD) feeding resulting in T2DM-associated NAFLD, followed by the injection of bone marrow mesenchymal stem cells (BMSCs). The morphology, function, and transfer of hepatocyte mitochondria were evaluated in both vivo and in vitro. Results: BMSC implantation resulted in the considerable recovery of increasing weight, HFD-induced steatosis, liver function, and disordered glucose and lipid metabolism. The treatment with BMSC transplantation was accompanied by reduced fat accumulation. Moreover, mitochondrial transfer was observed in both vivo and vitro studies. And the mitochondria-recipient steatotic cells exhibited significantly enhanced OXPHOS activity, ATP production, and mitochondrial membrane potential, and reduced reactive oxygen species levels, which were not achieved by the blocking of mitochondrial transfer. Conclusion: Mitochondrial transfer from BMSCs is a feasible process to combat NAFLD via rescuing dysfunction mitochondria, and has a promising therapeutic effect on metabolism-related diseases. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) are multipotent cells that show self-renewal, multi-directional differentiation, and paracrine and immune regulation. As a result of these properties, the MSCs have great clinical application prospects, especially in the regeneration of injured tissues, functional reconstruction, and cell therapy. However, the transplanted MSCs are prone to ageing and apoptosis and have a difficult to control direction differentiation. Therefore, it is necessary to effectively regulate the functions of the MSCs to promote their desired effects. In recent years, it has been found that mitochondria, the main organelles responsible for energy metabolism and adenosine triphosphate production in cells, play a key role in regulating different functions of the MSCs through various mechanisms. Thus, mitochondria could act as effective targets for regulating and promoting the functions of the MSCs. In this review, we discuss the research status and current understanding of the role and mechanism of mitochondrial energy metabolism, morphology, transfer modes, and dynamics on MSC functions. [ABSTRACT FROM AUTHOR]

Abstract Background Recent studies have demonstrated mesenchymal stem cells (MSCs) as effective mitochondrial donors with therapeutic success in multiple experimental models of human disease. MSCs obtained from different tissue sources such as bone marrow (BM), adipose (AD), dental pulp (DP), and Wharton’s jelly (WJ) are routinely used in clinical trials with no known study of their mitochondrial donor capacity. Here, we show for the first time that MSCs derived from different tissue sources have different mitochondrial donor properties and that this is correlated with their intrinsic respiratory states. Methods MitoTracker®-labeled MSCs were co-cultured with Cell Trace–labeled U87-MG cells or rat cardiomyocytes. Mitochondrial transfer abilities of MSCs were assessed by using flow cytometry analysis and fluorescence imaging. Mitochondrial reactive oxygen species (mtROS) levels were analyzed by using MitoSOX red–based staining, and mitochondrial respiration parameters were analyzed by using a Seahorse XF Analyzer. Results AD-MSCs and BM-MSCs displayed higher mitochondrial transfer than DP-MSCs and WJ-MSCs. Counterintuitively, DP-MSCs and WJ-MSCs were more effective in suppressing mtROS levels in stressed recipient cells than AD-MSCs or BM-MSCs. Interestingly, the oxygen consumption rates and intrinsic mitochondrial respiration parameters like ATP levels, basal and maximal respiration, and mitochondrial DNA copy number in donor MSCs showed a highly significant inverse correlation with their mitochondrial donation. Conclusions We find that there are intrinsic differences in the mitochondrial respiration, donation capacity, and therapeutic efficacy among MSCs of different tissue origin. MSCs with high mitochondrial respiration capacities are associated with lower mitochondrial transfer but more effective suppression of mtROS in stressed recipient cells. This is most compatible with a model where recipient cells optimally regulate mitochondrial transfer such that they take more mitochondria from MSCs with lower mitochondrial function. Furthermore, it appears to be advantageous to use MSCs such as DP-MSCs or WJ-MSCs with higher mitochondrial respiratory abilities that achieved better therapeutic effect with lower mitochondrial transfer in our study. This opens up a new direction in stem cell therapeutics.