线粒体转移

Cancer cells in the tumour microenvironment use various mechanisms to evade the immune system, particularly T cell attack1. For example, metabolic reprogramming in the tumour microenvironment and mitochondrial dysfunction in tumour-infiltrating lymphocytes (TILs) impair antitumour immune responses2, 3–4. However, detailed mechanisms of such processes remain unclear. Here we analyse clinical specimens and identify mitochondrial DNA (mtDNA) mutations in TILs that are shared with cancer cells. Moreover, mitochondria with mtDNA mutations from cancer cells are able to transfer to TILs. Typically, mitochondria in TILs readily undergo mitophagy through reactive oxygen species. However, mitochondria transferred from cancer cells do not undergo mitophagy, which we find is due to mitophagy-inhibitory molecules. These molecules attach to mitochondria and together are transferred to TILs, which results in homoplasmic replacement. T cells that acquire mtDNA mutations from cancer cells exhibit metabolic abnormalities and senescence, with defects in effector functions and memory formation. This in turn leads to impaired antitumour immunity both in vitro and in vivo. Accordingly, the presence of an mtDNA mutation in tumour tissue is a poor prognostic factor for immune checkpoint inhibitors in patients with melanoma or non-small-cell lung cancer. These findings reveal a previously unknown mechanism of cancer immune evasion through mitochondrial transfer and can contribute to the development of future cancer immunotherapies. Mitochondria with mutations in their DNA from cancer cells can be transferred to T cells in the tumour microenvironment, which leads to T cell dysfunction and impaired antitumour immunity.

Mitochondrial loss and dysfunction drive T cell exhaustion, representing major barriers to successful T cell-based immunotherapies. Here, we describe an innovative platform to supply exogenous mitochondria to T cells, overcoming these limitations. We found that bone marrow stromal cells establish nanotubular connections with T cells and leverage these intercellular highways to transplant stromal cell mitochondria into CD8+ T cells. Optimal mitochondrial transfer required Talin 2 on both donor and recipient cells. CD8+ T cells with donated mitochondria displayed enhanced mitochondrial respiration and spare respiratory capacity. When transferred into tumor-bearing hosts, these supercharged T cells expanded more robustly, infiltrated the tumor more efficiently, and exhibited fewer signs of exhaustion compared with T cells that did not take up mitochondria. As a result, mitochondria-boosted CD8+ T cells mediated superior antitumor responses, prolonging animal survival. These findings establish intercellular mitochondrial transfer as a prototype of organelle medicine, opening avenues to next-generation cell therapies.

Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment that commonly support cancer development and progression. Here we show that different cancer cells transfer mitochondria to fibroblasts in cocultures and xenograft tumors, thereby inducing protumorigenic CAF features. Transplantation of functional mitochondria from cancer cells induces metabolic alterations in fibroblasts, expression of CAF markers and release of a protumorigenic secretome and matrisome. These features promote tumor formation in preclinical mouse models. Mechanistically, the mitochondrial transfer requires the mitochondrial trafficking protein MIRO2. Its depletion in cancer cells suppresses mitochondrial transfer and inhibits CAF differentiation and tumor growth. The clinical relevance of these findings is reflected by the overexpression of MIRO2 in tumor cells at the leading edge of epithelial skin cancers. These results identify mitochondrial transfer from cancer cells to fibroblasts as a driver of tumorigenesis and provide a rationale for targeting MIRO2 and mitochondrial transfer in different malignancies. Cangkrama et al. show that tumor cells from various cancer types use the mitochondrial trafficking protein MIRO2 and nanotubes to transfer mitochondria into surrounding fibroblasts, thereby inducing cancer-associated fibroblast differentiation and subsequent tumor growth.

No abstract available

The blood-brain barrier (BBB) acts as the crucial physical filtration structure in the central nervous system. Here, we investigate the role of a specific subset of astrocytes in the regulation of BBB integrity. We showed that Dmp1-expressing astrocytes transfer mitochondria to endothelial cells via their endfeet for maintaining BBB integrity. Deletion of the Mitofusin 2 (Mfn2) gene in Dmp1-expressing astrocytes inhibited the mitochondrial transfer and caused BBB leakage. In addition, the decrease of MFN2 in astrocytes contributes to the age-associated reduction of mitochondrial transfer efficiency and thus compromises the integrity of BBB. Together, we describe a mechanism in which astrocytes regulate BBB integrity through mitochondrial transfer. Our findings provide innnovative insights into the cellular framework that underpins the progressive breakdown of BBB associated with aging and disease.

Macrophage pyroptosis and neutrophil extracellular traps (NETs) play a critical role in sepsis pathophysiology; however, the role of macrophage pyroptosis in the regulation of NETs formation during sepsis is unknown. Here, we showed that macrophages transfer mitochondria to neutrophils through microvesicles following pyroptosis; this process induces mitochondrial dysfunction and triggers the induction of NETs formation through mitochondrial reactive oxygen species (mtROS)/Gasdermin D (GSDMD) axis. These pyroptotic macrophage-derived microvesicles can induce tissues damage, coagulation, and NETs formation in vivo. Disulfiram partly inhibits these effects in a mouse model of sepsis. Pyroptotic macrophage-derived microvesicles induce NETs formation through mitochondrial transfer, both in vitro and in vivo. Microvesicles-mediated NETs formation depends on the presence of GSDMD-N-expressing mitochondria in the microvesicles. This study elucidates a microvesicles-based pathway for NETs formation during sepsis and proposes a microvesicles-based intervention measure for sepsis management.

Breast cancer (BC) is a complex disease, showing heterogeneity in the genetic background, molecular subtype, and treatment algorithm. Historically, treatment strategies have been directed towards cancer cells, but these are not the unique components of the tumor bulk, where a key role is played by the tumor microenvironment (TME), whose better understanding could be crucial to obtain better outcomes. We evaluated mitochondrial transfer (MT) by co-culturing Adipose stem cells with different Breast cancer cells (BCCs), through MitoTracker assay, Mitoception, confocal and immunofluorescence analyses. MT inhibitors were used to confirm the MT by Tunneling Nano Tubes (TNTs). MT effect on multi-drug resistance (MDR) was assessed using Doxorubicin assay and ABC transporter evaluation. In addition, ATP production was measured by Oxygen Consumption rates (OCR) and Immunoblot analysis. We found that MT occurs via Tunneling Nano Tubes (TNTs) and can be blocked by actin polymerization inhibitors. Furthermore, in hybrid co-cultures between ASCs and patient-derived organoids we found a massive MT. Breast Cancer cells (BCCs) with ASCs derived mitochondria (ADM) showed a reduced HIF-1α expression in hypoxic conditions, with an increased ATP production driving ABC transporters-mediated multi-drug resistance (MDR), linked to oxidative phosphorylation metabolism rewiring. We provide a proof-of-concept of the occurrence of Mitochondrial Transfer (MT) from Adipose Stem Cells (ASCs) to BC models. Blocking MT from ASCs to BCCs could be a new effective therapeutic strategy for BC treatment.

SUMMARY Ovarian cancer is characterized by early metastatic spread. This study demonstrates that carcinoma-associated mesenchymal stromal cells (CA-MSCs) enhance metastasis by increasing tumor cell heterogeneity through mitochondrial donation. CA-MSC mitochondrial donation preferentially occurs in ovarian cancer cells with low levels of mitochondria (“mito poor”). CA-MSC mitochondrial donation rescues the phenotype of mito poor cells, restoring their proliferative capacity, resistance to chemotherapy, and cellular respiration. Receipt of CA-MSC-derived mitochondria induces tumor cell transcriptional changes leading to the secretion of ANGPTL3, which enhances the proliferation of tumor cells without CA-MSC mitochondria, thus amplifying the impact of mitochondrial transfer. Donated CA-MSC mitochondrial DNA persisted in recipient tumor cells for at least 14 days. CA-MSC mitochondrial donation occurs in vivo, enhancing tumor cell heterogeneity and decreasing mouse survival. Collectively, this work identifies CA-MSC mitochondrial transfer as a critical mediator of ovarian cancer cell survival, heterogeneity, and metastasis and presents a unique therapeutic target in ovarian cancer.

The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin 43 (Cx43, encoded by GJA1) and the truncated, internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1 + and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 h in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA.

Rosamine-based mitochondrial dyes, such as Mitotracker Red, have commonly been employed to visualize mitochondrial localization within cells due to their preferential accumulation in organelles with membrane potential. Consequently, Mitotracker Red has often served as a surrogate indicator for tracking mitochondrial movement between neighboring cells. However, it is important to note that the presence of membrane potential in the cell membrane and other organelles may lead to the non-specific partial enrichment of Mitotracker Red in locations other than mitochondria. This study comprehensively investigates the reliability of mitochondrial dye as a marker for studying horizontal mitochondrial transfer (HMT). By meticulous replicating of previous experiments and comparing the efficiency of mitochondrial dye transfer with that of mito-targeted GFP, our findings confirm that HMT occurs at significantly lower efficiency than previously indicated by Mitotracker dye. Subsequent experiments involving mitochondria-deficient cells robustly demonstrates the non-specificity of mitochondrial dye as indicator for mitochondria. We advocate for a thorough reevaluation of existing literature in this field and propose exploration of alternative techniques to enhance the investigation of HMT. By addressing these pivotal aspects, we can advance our understanding of cellular dynamics and pave the way for future explorations in this captivating field. Evidence underscores the non-specificity of mitochondrial dye in assessing horizontal mitochondrial transfer, suggesting that alternative approaches should be considered for gaining nuanced insights into intercellular mitochondrial dynamics.

Intercellular mitochondrial transfer (IMT) is an intriguing biological phenomenon where mitochondria are transferred between different cells and notably, cell types. IMT is physiological, occurring in normal conditions, but also is utilized to deliver healthy mitochondria to cells in distress. Transferred mitochondria can be integrated to improve cellular metabolism, and mitochondrial function. Research on the mitochondrial transfer axis between astrocytes and brain capillaries in vivo is limited by the cellular heterogeneity of the neurovascular unit. To this end, we developed an inducible mouse model that expresses mitochondrial Dendra2 only in astrocytes and then isolated brain capillaries to remove all intact astrocytes. This method allows the visualization of in vivo astrocyte- endothelial cell (EC) and astrocyte-pericyte IMT. We demonstrate evidence of astrocyte-EC and astrocyte-pericyte mitochondrial transfer within brain capillaries. We also show that healthy aging enhances mitochondrial transfer from astrocytes to brain capillaries, revealing a potential link between brain aging and cellular mitochondrial dynamics. Finally, we observe that astrocyte-derived extracellular vesicles transfer mitochondria to brain microvascular endothelial cells, showing the potential route of in vivo IMT. These results represent a breakthrough in our understanding of IMT in the brain and a new target in brain aging and neurovascular metabolism.

To assess the efficacy of a novel 3D biomimetic hydrogel scaffold with immunomodulatory properties in promoting fracture healing. Immunomodulatory scaffolds were used in cell experiments, osteotomy mice treatment, and single-cell transcriptomic sequencing. In vitro, fluorescence tracing examined macrophage mitochondrial transfer and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Scaffold efficacy was assessed through alkaline phosphatase (ALP), Alizarin Red S (ARS) staining, and in vivo experiments. The scaffold demonstrated excellent biocompatibility and antioxidant-immune regulation. Single-cell sequencing revealed a shift in macrophage distribution towards the M2 phenotype. In vitro experiments showed that macrophage mitochondria promoted BMSCs’ osteogenic differentiation. In vivo experiments confirmed accelerated fracture healing. The GAD/Ag-pIO scaffold enhances osteogenic differentiation and fracture healing through immunomodulation and promotion of macrophage mitochondrial transfer.

Mitochondria (MT) participate in most metabolic activities of mammalian cells. A near-unidirectional mitochondrial transfer from T cells to cancer cells was recently observed to "metabolically empower" cancer cells while "depleting immune cells," providing new insights into tumor-T cell interaction and immune evasion. Here, we leverage single-cell RNA-seq technology and introduce MERCI, a statistical deconvolution method for tracing and quantifying mitochondrial trafficking between cancer and T cells. Through rigorous benchmarking and validation, MERCI accurately predicts the recipient cells and their relative mitochondrial compositions. Application of MERCI to human cancer samples identifies a reproducible MT transfer phenotype, with its signature genes involved in cytoskeleton remodeling, energy production, and TNF-α signaling pathways. Moreover, MT transfer is associated with increased cell cycle activity and poor clinical outcome across different cancer types. In summary, MERCI enables systematic investigation of an understudied aspect of tumor-T cell interactions that may lead to the development of therapeutic opportunities.

The use of exogenous mitochondria to replenish damaged mitochondria has been proposed as a strategy for the treatment of pulmonary fibrosis. However, the success of this strategy is partially restricted by the difficulty of supplying sufficient mitochondria to diseased cells. Herein, we report the generation of high-powered mesenchymal stem cells with promoted mitochondrial biogenesis and facilitated mitochondrial transfer to injured lung cells by the sequential treatment of pioglitazone and iron oxide nanoparticles. This highly efficient mitochondrial transfer is shown to not only restore mitochondrial homeostasis but also reactivate inhibited mitophagy, consequently recovering impaired cellular functions. We perform studies in mouse to show that these high-powered mesenchymal stem cells successfully mitigate fibrotic progression in a progressive fibrosis model, which was further verified in a humanized multicellular lung spheroid model. The present findings provide a potential strategy to overcome the current limitations in mitochondrial replenishment therapy, thereby promoting therapeutic applications for fibrotic intervention.

One of the most critical issues to be solved in reproductive medicine is the treatment of patients with multiple failures of assisted reproductive treatment caused by low-quality embryos. This study investigated whether mitochondrial transfer to human oocytes improves embryo quality and provides subsequent acceptable clinical results and normality to children born due to the use of this technology. We transferred autologous mitochondria extracted from oogonia stem cells to mature oocytes with sperm at the time of intracytoplasmic sperm injection in 52 patients with recurrent failures (average 5.3 times). We assessed embryo quality using the following three methods: good-quality embryo rates, transferable embryo rates, and a novel embryo-scoring system (embryo quality score; EQS) in 33 patients who meet the preset inclusion criteria for analysis. We also evaluated the clinical outcomes of the in vitro fertilization and development of children born using this technology and compared the mtDNA sequences of the children and their mothers. The good-quality embryo rates, transferable embryo rates, and EQS significantly increased after mitochondrial transfer and resulted in 13 babies born in normal conditions. The mtDNA sequences were almost identical to the respective maternal sequences at the 83 major sites examined. Mitochondrial transfer into human oocytes is an effective clinical option to enhance embryo quality in recurrent in vitro fertilization-failure cases.

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 H_2O_2-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 H_2O_2-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.

Mitochondria are essential organelles for maintaining intracellular homeostasis. Their dysfunction can directly or indirectly affect cell functioning and is linked to multiple diseases. Donation of exogenous mitochondria is potentially a viable therapeutic strategy. For this, selecting appropriate donors of exogenous mitochondria is critical. We previously demonstrated that ultra-purified bone marrow-derived mesenchymal stem cells (RECs) have better stem cell properties and homogeneity than conventionally cultured bone marrow-derived mesenchymal stem cells. Here, we explored the effect of contact and noncontact systems on three possible mitochondrial transfer mechanisms involving tunneling nanotubes, connexin 43 (Cx43)-mediated gap junction channels (GJCs), and extracellular vesicles (Evs). We show that Evs and Cx43-GJCs provide the main mechanism for mitochondrial transfer from RECs. Through these two critical mitochondrial transfer pathways, RECs could transfer a greater number of mitochondria into mitochondria-deficient (ρ0) cells and could significantly restore mitochondrial functional parameters. Furthermore, we analyzed the effect of exosomes (EXO) on the rate of mitochondrial transfer from RECs and recovery of mitochondrial function. REC-derived EXO appeared to promote mitochondrial transfer and slightly improve the recovery of mtDNA content and oxidative phosphorylation in ρ0 cells. Thus, ultrapure, homogenous, and safe stem cell RECs could provide a potential therapeutic tool for diseases associated with mitochondrial dysfunction.

Mitochondrial transfer is emerging as a promising therapeutic strategy for tissue repair, but whether it protects against pulpitis remains unclear. Here, we show that hyperactivated nucleotide‐binding domain and leucine‐rich repeat protein3 (NLRP3) inflammasomes with pyroptotic cell death was present in pulpitis tissues, especially in the odontoblast layer, and mitochondrial oxidative stress (OS) was involved in driving this NLRP3 inflammasome‐induced pathology. Using bone marrow mesenchymal stem cells (BMSCs) as mitochondrial donor cells, we demonstrated that BMSCs could donate their mitochondria to odontoblasts via tunnelling nanotubes (TNTs) and, thus, reduce mitochondrial OS and the consequent NLRP3 inflammasome‐induced pyroptosis in odontoblasts. These protective effects of BMSCs were mostly blocked by inhibitors of the mitochondrial function or TNT formation. In terms of the mechanism of action, TNF‐α secreted from pyroptotic odontoblasts activates NF‐κB signalling in BMSCs via the paracrine pathway, thereby promoting the TNT formation in BMSCs and enhancing mitochondrial transfer efficiency. Inhibitions of NF‐κB signalling and TNF‐α secretion in BMSCs suppressed their mitochondrial donation capacity and TNT formation. Collectively, these findings demonstrated that TNT‐mediated mitochondrial transfer is a potential protective mechanism of BMSCs under stress conditions, suggesting a new therapeutic strategy of mitochondrial transfer for dental pulp repair.

Although mesenchymal stem cell transplantation has been successful in the treatment of ischemic cardiomyopathy, the underlying mechanisms remain unclear. Herein, we investigated whether mitochondrial transfer could explain the success of cell therapy in ischemic cardiomyopathy. Mitochondrial transfer in co-cultures of human adipose-derived mesenchymal stem cells and rat cardiomyocytes maintained under hypoxic conditions was examined. Functional recovery was monitored in a rat model of myocardial infarction following human adipose-derived mesenchymal stem cell transplantation. We observed mitochondrial transfer in vitro, which required the formation of cell-to-cell contacts and synergistically enhanced energy metabolism. Rat cardiomyocytes exhibited mitochondrial transfer 3 days following human adipose-derived mesenchymal stem cell transplantation to the ischemic heart surface post-myocardial infarction. We detected donor mitochondrial DNA in the recipient myocardium concomitant with a significant improvement in cardiac function. Mitochondrial transfer is vital for successful cell transplantation therapies and improves treatment outcomes in ischemic cardiomyopathy.

We recently reported the benefit of the IV transferring of active exogenous mitochondria in a short-term pharmacological AD (Alzheimer’s disease) model. We have now explored the efficacy of mitochondrial transfer in 5XFAD transgenic mice, aiming to explore the underlying mechanism by which the IV-injected mitochondria affect the diseased brain. Mitochondrial transfer in 5XFAD ameliorated cognitive impairment, amyloid burden, and mitochondrial dysfunction. Exogenously injected mitochondria were detected in the liver but not in the brain. We detected alterations in brain proteome, implicating synapse-related processes, ubiquitination/proteasome-related processes, phagocytosis, and mitochondria-related factors, which may lead to the amelioration of disease. These changes were accompanied by proteome/metabolome alterations in the liver, including pathways of glucose, glutathione, amino acids, biogenic amines, and sphingolipids. Altered liver metabolites were also detected in the serum of the treated mice, particularly metabolites that are known to affect neurodegenerative processes, such as carnosine, putrescine, C24:1-OH sphingomyelin, and amino acids, which serve as neurotransmitters or their precursors. Our results suggest that the beneficial effect of mitochondrial transfer in the 5XFAD mice is mediated by metabolic signaling from the liver via the serum to the brain, where it induces protective effects. The high efficacy of the mitochondrial transfer may offer a novel AD therapy.

No abstract available

Mitochondrial transfer regulates intercellular communication, and mitochondria regulate cell metabolism and cell survival. However, the role and mechanism of mitochondrial transfer in Cd-induced nonalcoholic fatty liver disease (NAFLD) are unclear. The present study shows that mitochondria can be transferred between hepatocytes via microtubule-dependent tunneling nanotubes. After Cd treatment, mitochondria exhibit perinuclear aggregation in hepatocytes and blocked intercellular mitochondrial transfer. The different movement directions of mitochondria depend on their interaction with different motor proteins. The results show that Cd destroys the mitochondria-kinesin interaction, thus inhibiting mitochondrial transfer. Moreover, Cd increases the interaction of P62 with Dynactin1, promotes negative mitochondrial transport, and increases intracellular lipid accumulation. Mitochondria and hepatocyte co-culture significantly reduced Cd damage to hepatocytes and lipid accumulation. Thus, Cd blocks intercellular mitochondrial transfer by disrupting the microtubule system, inhibiting mitochondrial positive transport, and promoting their negative transport, thereby promoting the development of NAFLD. Graphical Abstract 1. Mitochondrial transfer between hepatocytes can be achieved through microtubule-dependent tunneling nanotubes 2. Cadmium inhibits the intercellular mitochondrial transfer 3. Inhibition of intercellular mitochondrial transfer mediates cadmium-induced nonalcoholic fatty liver disease. 4. Motor protein-dependent mitochondrial mobility is a target for cadmium to inhibit intercellular mitochondrial transfer

Mitochondria are the powerhouse of eukaryotic cells, which regulate cell metabolism and differentiation. Recently, mitochondrial transfer between cells has been shown to direct recipient cell fate. However, it is unclear whether mitochondria can translocate to stem cells and whether this transfer alters stem cell fate. Here, mesenchymal stem cell (MSC) regulation is examined by macrophages in the bone marrow environment. It is found that macrophages promote osteogenic differentiation of MSCs by delivering mitochondria to MSCs. However, under osteoporotic conditions, macrophages with altered phenotypes, and metabolic statuses release oxidatively damaged mitochondria. Increased mitochondrial transfer of M1‐like macrophages to MSCs triggers a reactive oxygen species burst, which leads to metabolic remodeling. It is showed that abnormal metabolism in MSCs is caused by the abnormal succinate accumulation, which is a key factor in abnormal osteogenic differentiation. These results reveal that mitochondrial transfer from macrophages to MSCs allows metabolic crosstalk to regulate bone homeostasis. This mechanism identifies a potential target for the treatment of osteoporosis.

No abstract available

Simple Summary Conventional natural killer (NK)-based anticancer immunotherapy has a limitation: a culture period of approximately 2 weeks is required to increase the number and activity of NK cells. By transferring functional allogeneic mitochondria into NK cells, we demonstrated that the activity of NK cells and their cytotoxicity were significantly enhanced. This approach could potentially offer a timely therapeutic strategy for cancer treatment without the need for in vitro culture, which can be time-consuming and costly. Abstract An in vitro culture period of at least 2 weeks is required to produce sufficient natural killer (NK) cells for immunotherapy, which are the key effectors in hematological malignancy treatment. Mitochondrial damage and fragmentation reduce the NK cell immune surveillance capacity. Thus, we hypothesized that the transfer of healthy mitochondria to NK cells could enhance their anticancer effects. Allogeneic healthy mitochondria isolated from WRL-68 cells were transferred to NK cells. We evaluated NK cells’ proliferative capacity, cell cycle, and cytotoxic capacity against various cancer cell types by analyzing specific lysis and the cytotoxic granules released. The relationship between the transferred allogenic mitochondrial residues and NK cell function was determined. After mitochondrial transfer, the NK cell proliferation rate was 1.2-fold higher than that of control cells. The mitochondria-treated NK cells secreted a 2.7-, 4.1-, and 5-fold higher amount of granzyme B, perforin, and IFN-γ, respectively, when co-cultured with K562 cells. The specific lysis of various solid cancer cells increased 1.3–1.6-fold. However, once allogeneic mitochondria were eliminated, the NK cell activity returned to the pre-mitochondrial transfer level. Mitochondria-enriched NK cells have the potential to be used as a novel solid cancer treatment agent, without the need for in vitro cytokine-induced culture.

OBJECTIVES This study aimed to investigate the efficacy of mitochondrial transplantation as a therapeutic intervention for idiopathic inflammatory myopathy (IIM). This study used a comprehensive approach, incorporating both in vitro and in vivo IIM models, and conducted a first-in-human clinical trial to assess the effectiveness and safety of mitochondria isolated from human umbilical cord mesenchymal stem cells (PN-101). METHODS Mitochondria isolated from umbilical cord mesenchymal stem cells were designated as PN-101. The efficacy of PN-101 was assessed using myoblasts derived from patients with IIM and C2C12 mouse perforin/granzyme B-treated myoblasts as an in vitro IIM model. PN-101's effect on IIM was examined using C protein-induced myositis (CIM) mice as an in vivo model. The efficacy and safety of PN-101 were evaluated in a phase 1/2a clinical trial involving 9 adult patients with refractory polymyositis or dermatomyositis. RESULTS The myoblasts derived from patients with IIM exhibited defects in mitochondrial function and myogenesis. PN-101 transplantation enhances muscle differentiation and mitochondrial function in IIM myoblasts. PN-101 also enhanced intracellular adenosine triphosphate content, cell viability, and myogenesis in C2C12 perforin/granzyme B-treated myoblasts. In an in vivo model, PN-101 reduced myositis severity by exhibiting anti-inflammatory effects and restoring the CIM-induced metabolic shift. In a phase 1/2a prospective clinical trial involving adult patients with refractory IIM, PN-101 demonstrated no severe adverse drug reactions and showed at least minimal improvement in the International Myositis Assessment and Clinical Studies Group (IMACS)-Total Improvement Scores (TISs) compared with baseline. CONCLUSIONS PN-101 transplantation could serve as a novel treatment for IIM by enhancing mitochondrial repair and reducing inflammation in muscle tissues.

The immune‐inflammatory responses in the brain represent a key therapeutic target to ameliorate brain injury following intracerebral hemorrhage (ICH), where pro‐inflammatory microglia and its mitochondrial dysfunction plays a pivotal role. Mitochondrial transplantation is a promising strategy to improve the cellular mitochondrial function and thus modulate their immune properties. However, the transplantation of naked mitochondria into the brain has been constrained by the peripheral clearance and the difficulty in achieving selective access to the brain. Here, a novel strategy for mitochondrial transplantation via intravenous injection of magnetically responsive artificial cells (ACs) are proposed. ACs can protect the loaded mitochondria and selectively accumulate around the lesion under an external magnetic field (EMF). In this study, mitochondria released from ACs can effectively improve microglial mitochondrial function, attenuate their pro‐inflammatory attributes, and elevate the proportion of immunosuppressive microglia. In this way, microglia immune homeostasis in the brain is reestablished, and inflammation is attenuated, ultimately promoting functional recovery. This study presents an effective approach to transplant mitochondria into the brain, offering a promising alternative to modulate the immune‐inflammatory cascade in the brain following ICH.

Mitochondrial transplantation is a significant therapeutic approach for addressing mitochondrial dysfunction in patients with spinal cord injury (SCI), yet it is limited by rapid mitochondrial deactivation and low transfer efficiency. Here, high-quality mitochondria microfactories (HQ-Mitofactories) were constructed by anchoring Prussian blue nanoenzymes onto mesenchymal stem cells for effective mitochondrial transplantation to treat paralysis from SCI. Notably, the results demonstrated that HQ-Mitofactories could continuously produce vitality-boosting mitochondria with highly interconnected and elongated network structures under oxidative stress by scavenging excessive ROS. Furthermore, HQ-Mitofactories enabled efficient transfer of therapeutic mitochondria to injured neurons primarily via gap junctions, resulting in the restoration of mitochondrial homeostasis and thereby suppressing intracellular ROS burst and facilitating neuronal repair. After i.v. administration, HQ-Mitofactories migrated to the injured spinal cords of SCI mice and subsequently promoted neuronal regeneration and remyelination. Consequently, HQ-Mitofactory-treated mice successfully recovered locomotor function within 4 weeks, with 40% of the mice fully restoring walking after hindlimb paralysis. Conversely, untreated SCI exhibited completely abolished hindlimb movements. In light of real-time generation of vitality-boosting mitochondria even under oxidative stress and enabling targeted mitochondrial transfer, HQ-Mitofactories have promising therapeutic potential in the context of mitochondrial transplantation to reduce SCI-related paralysis, and more broadly impact the field of neuroregenerative medicine.

No abstract available

The pathologically remodeled myocardial ischemic microenvironment, characterized by sustained hypoxia, metabolic insufficiency, and accumulation of inflammatory mediators, severely disrupts mitochondrial homeostasis. This dysfunction establishes a self‐perpetuating cycle that impairs the coordinated healing cascade and compromises cardiac tissue repair following myocardial infarction (MI). To counteract these effects, a novel strategy of mitochondrial augmentation is proposed, whereby healthy exogenous mitochondria are introduced into macrophages to generate mitochondria‐transplanted macrophages (Mito‐T‐Macros or MTMs), which can resist post‐MI stress. Mitochondrial transplantation (MT) effectively induces macrophage polarization toward a reparative M2‐like phenotype, thereby enhancing pro‐healing functions, including migration, invasion, and phagocytosis. In vivo, MTM therapy enhances cardiac function after MI and attenuates left ventricular remodeling by reducing fibrosis, limiting apoptosis, and promoting angiogenesis. Mechanistically, MT accelerates the phenotypic transition of macrophages to a reparative state and prolongs their activity during the healing phase. Notably, a portion of the transplanted mitochondria are released from MTMs and subsequently internalized by cardiomyocytes, suggesting an additional mechanism of myocardial support. Overall, MT enhances the reparative capabilities of macrophages and contributes to the therapeutic efficacy of MTMs in ameliorating post‐MI cardiac remodeling. These findings support MTM therapy as a promising and innovative approach for repairing myocardial injury following MI.

Mitochondria are essential organelles for cell survival that manage the cellular energy supply by producing ATP. Mitochondrial dysfunction is associated with various human diseases, including metabolic syndromes, aging, and neurodegenerative diseases. Among the diseases related to mitochondrial dysfunction, Parkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by dopaminergic neuronal loss and neuroinflammation. Recently, it was reported that mitochondrial transfer between cells occurred naturally and that exogenous mitochondrial transplantation was beneficial for treating mitochondrial dysfunction. The current study aimed to investigate the therapeutic effect of mitochondrial transfer on PD in vitro and in vivo. The results showed that PN-101 mitochondria isolated from human mesenchymal stem cells exhibited a neuroprotective effect against 1-methyl-4-phenylpyridinium, 6-hydroxydopamine and rotenone in dopaminergic cells and ameliorated dopaminergic neuronal loss in the brains of C57BL/6J mice injected 30 mg/kg of methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intraperitoneally. In addition, PN-101 exhibited anti-inflammatory effects by reducing the expression of pro-inflammatory cytokines in microglial cells and suppressing microglial activation in the striatum. Furthermore, intravenous mitochondrial treatment was associated with behavioral improvements during the pole test and rotarod test in the MPTP-induced PD mice. These dual effects of neuroprotection and anti-neuroinflammation support the potential for mitochondrial transplantation as a novel therapeutic strategy for PD.

Mitochondria, often referred to the powerhouse of the cell, are essential for cellular energy production, and their dysfunction can profoundly affect various organs. Transplantation of healthy mitochondria can restore the bioenergetics in diseased cells and address multiple conditions, but more potentials of this approach remain unclear. In this study, I demonstrated that the source of transplanted mitochondria is not limited by species, as exhibit no significant responses to mitochondria derived from different germlines. Moreover, I identified that metabolic compatibility between the recipient and exogenous mitochondria as a crucial factor in mitochondrial transplantation, which confers unique metabolic properties to recipient cells, enabling them to combat diseases. Additionally, my findings indicated competitive interactions among mitochondria with varying functions, with more bioenergetic-active mitochondria yielded superior therapeutic benefits. Notably, no upper limit for the bio-enhancement provided by exogenous mitochondria has been identified. Based on these insights, I proposes a novel therapeutic approach—adaptive bio-enhancement through mitochondrial transplantation.

BACKGROUND Acute ST-elevation myocardial infarction (STEMI) remains a globally significant health challenge in spite of improvement in management strategy. Being aware that mitochondrial dysfunction plays a crucial role in ischaemia-reperfusion injury (IRI) modulation, empirical evidence suggests functional mitochondrial transplantation strikes as a reliable therapeutic approach for patients with acute myocardial infarction. METHODS AND RESULTS We conducted a prospective, triple-blinded, parallel-group, blocked randomised clinical trial to investigate the therapeutic effects and clinical outcomes of platelet-derived mitochondrial transplantation in 30 patients with acute STEMI, such that the 15 subjects in the control group were given standard of care treatment, whereas the subjects in the intervention group received autologous platelet-derived mitochondria through the intracoronary injection. We observed that within 40 days, the intervention group had a slightly greater improvement in the left ventricular ejection fraction (LVEF) compared to the control group and experienced a significant enhancement in the exercise capacity (p < 0.001). Moreover, major adverse cardiac events (MACE), arrhythmia, fever, and tachycardia were compared between the groups and lack of significant difference marks the safety of mitochondrial transplantation (p > 0.05). Furthermore, the two groups were not significantly distinct as regards the average length of stay for a hospitalisation (p > 0.05). CONCLUSION We suggest platelet-derived mitochondrial transplantation appears as a beneficial and highly promising therapeutic option for patients of ischaemic heart disease (IHD); however, we are aware that further in-depth studies with larger sample sizes along with longer follow-up periods are necessary for validating the clinical implications of our findings.

Duchenne muscular dystrophy is caused by loss of the dystrophin protein. This pathology is accompanied by mitochondrial dysfunction contributing to muscle fiber instability. It is known that mitochondria-targeted in vivo therapy mitigates pathology and improves the quality of life of model animals. In the present work, we applied mitochondrial transplantation therapy (MTT) to correct the pathology in dystrophin-deficient mdx mice. Intramuscular injections of allogeneic mitochondria obtained from healthy animals into the hind limbs of mdx mice alleviated skeletal muscle injury, reduced calcium deposits in muscles and serum creatine kinase levels, and improved the grip strength of the hind limbs and motor activity of recipient mdx mice. We noted normalization of the mitochondrial ultrastructure and sarcoplasmic reticulum/mitochondria interactions in mdx muscles. At the same time, we revealed a decrease in the efficiency of oxidative phosphorylation in the skeletal muscle mitochondria of recipient mdx mice accompanied by a reduction in lipid peroxidation products (MDA products) and reduced calcium overloading. We found no effect of MTT on the expression of mitochondrial signature genes (Drp1, Mfn2, Ppargc1a, Pink1, Parkin) and on the level of mtDNA. Our results show that systemic MTT mitigates the development of destructive processes in the quadriceps muscle of mdx mice.

BACKGROUND AIMS Wound healing is a multistage process that requires a concerted effort of various cell types. The intricate processes involved in the healing of wounds result in high energy requirements. Furthermore, mitochondria play a crucial role in the healing process because of their involvement in neo angiogenesis, growth factor synthesis, and cell differentiation. It is unclear how mitochondria transplantation, a promising new approach, influences wound healing. METHODS In this study, healthy autologous mitochondria obtained from skeletal muscle were injected into chronic pressure wounds as an intervention to promote wound healing. RESULTS Mitochondrial transplantation accelerated wound healing by reducing wound size, increasing granulation tissue, and hastening epithelialization. CONCLUSIONS This study is the first to demonstrate the therapeutic efficacy of mitochondrial transplantation in wound healing.

The mitochondria are essential organelles not only providing cellular energy in the form of ATP, but also regulating the inflammatory response and the cell death program. Mitochondrial dysfunction has been associated with various human diseases, including metabolic syndromes as well as inflammatory and neurodegenerative diseases. Acute respiratory distress syndrome (ARDS) is an acute pulmonary disorder characterized by uncontrolled alveolar inflammation, apoptotic lung epithelial/endothelial cells, and pulmonary edema. Despite the high mortality of ARDS, an effective pharmacotherapy to treat this disease has not been established yet. Therefore, identifying a novel targeted therapy for ARDS is important. Recently, exogenous mitochondrial transplantation was reported to be beneficial for treating mitochondrial dysfunction. The current study aimed to investigate the therapeutic effect of mitochondrial transplantation on ARDS in vitro and in vivo. Mitochondria were isolated from human stem cells. For in vitro efficacy of mitochondrial transplantation on the inflammation and cell death, murine alveolar macrophages MH-S and human pulmonary microvascular endothelial cells HPMECs were exposed to LPS, respectively. The ARDS mice model established by a single intratracheal instillation of LPS was used for in vivo efficacy of intravenously treated mitochondria. Our results showed that the mitochondria isolated from human stem cells exhibited an anti-inflammatory effect against alveolar macrophages and an anti-apoptotic effect against the alveolar epithelial cells. Furthermore, intravenous mitochondrial treatment was associated with the attenuation of lung injury in the LPS-induced ARDS mice. Dual effects of mitochondria on anti-inflammation and anti-apoptosis support the potential of mitochondrial transplantation as a novel therapeutic strategy for ARDS.

Heart transplantation remains the ultimate treatment strategy for neonates and children with medically refractory end-stage heart failure and utilization of donors after circulatory death (DCD) can expand th donor pool. We have previously shown that mitochondrial transplantation preserves myocardial function and viability in neonatal swine DCD hearts to levels similar to that observed in donation after brain death (DBD). Herein, we sought to investigate the transcriptomic and proteomic pathways implicated in these phenotypic changes using ex situ perfused swine hearts. Pathway analysis showed that ATP binding, voltage-gated K channel activity involved in cardiac cell muscle contraction and ribosomal RNA biogenesis were upregulated in the mitochondrial transplantation group, while mitochondria were the predicted source. Promotion of ribosome biogenesis and downregulation of apoptosis were the overlapping mechanisms between transcriptomic and proteomic alterations. Moreover, we showed that mitochondrial transplantation modulates ischemic transcriptomic and proteomic profiles to that of non-ischemia through the mitochondria. Replication of these findings in human in vivo experiments is warranted.

Ischaemic heart diseases (IHD) are among the major causes of mortality in the elderly population. Although timely reperfusion is a common treatment for IHD, it causes additional damage to the ischaemic myocardium known as ischaemia–reperfusion (IR) injury. Considering the importance of preventing reperfusion injuries, we aimed to examine the combination effect of mitochondrial transplantation (MT) and coenzyme Q10 (CoQ10) in myocardial IR injury of aged male rats. Seventy‐two aged male Wistar rats were randomly divided into six groups: Sham, IR, CoQ10, MT, combination therapy (MT + CoQ10) and vehicle. Myocardial IR injury was established by occlusion of the left anterior descending coronary artery followed by reopening. Young male Wistar rats were used as mitochondria donors. Isolated mitochondria were injected intraventricularly (500 µL of a respiration buffer containing 6 × 106 ± 5 × 105 mitochondria/mL) in MT‐receiving groups at the onset of reperfusion. CoQ10 (10 mg/kg/day) was injected intraperitoneally for 2 weeks before IR induction. Twenty‐four hours after reperfusion, haemodynamic parameters, myocardial infarct size (IS), lactate dehydrogenase (LDH) release and cardiac mitochondrial function (mitochondrial reactive oxygen species (ROS) generation and membrane potential) were measured. The combination of MT and CoQ10 improved haemodynamic index changes and reduced IS and LDH release (P < 0.05). It also decreased mitochondrial ROS generation and increased membrane potential (P < 0.05). CoQ10 also showed a significant cardioprotective effect. Combination therapy displayed greater cardioprotective effects than single treatments. This study revealed that MT and CoQ10 combination treatment can be considered as a promising cardioprotective strategy to reduce myocardial IR injury in ageing, in part by restoring mitochondrial function.

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Idiopathic pulmonary fibrosis (IPF) is a pulmonary disease characterized by excessive extracellular matrix protein deposition in the lung interstitium, subsequently causing respiratory failure. IPF still has a high medical unmet requirement due to the lack of effective treatments to inhibit disease progression. The etiology of IPF remains unclear, but mitochondrial dysfunction is considered to be associated with IPF development. Therefore, targeting mitochondrial abnormalities would be a promising strategy for treating IPF. Recently, exogenous mitochondrial transplantation has been beneficial for treating mitochondrial dysfunction. The current study aimed to examine the therapeutic effect of mitochondrial transplantation on IPF in vitro and in vivo. Mitochondria were isolated from human umbilical cord mesenchymal stem cells, referred to as PN-101. Human lung fibroblasts and human bronchial epithelial cells were exposed to transforming growth factor-β, followed by PN-101 treatment to determine the in vitro efficacy of mitochondrial transplantation. An IPF mouse model established by a single intratracheal instillation of bleomycin was utilized to determine the in vivo efficacy of the intravenously treated mitochondria. PN-101 attenuated mitochondrial damage, inhibited EMC production, and suppressed epithelial-to-mesenchymal transition in vitro. Additionally, intravenous PN-101 administration alleviated bleomycin-induced fibrotic processes in the IPF mouse model with a therapeutic context. Our data indicate that PN-101 is a novel and potential therapeutic agent for IPF.

BACKGROUND Mitochondria have emerged as a promising target for ischemic disease. A previous study reported the application of mitochondrial transplantation in focal cerebral ischemia/reperfusion injury, but it is unclear whether exogenous mitochondrial transplantation could be a therapeutic strategy for global ischemia/reperfusion injury induced by cardiac arrest. METHODS We hypothesized that transplantation of autologous mitochondria would rescue hippocampal cells and alleviate neurological impairment after cardiac arrest. In this study, we employed a rat cardiac arrest-global cerebral ischemia injury model (CA-GCII) and transplanted isolated mitochondria intravenously. Behavior test was applied to assess neurological deficit. Apoptosis and mitochondria permeability transition pore opening in hippocampus was determined using immunoblotting and swelling assay, respectively. RESULTS Transplanted mitochondria distributed throughout hippocampal cells and reduced oxidative stress. An improved neurological outcome was observed in rats receiving autologous mitochondria. In the hippocampus, mitophagy was enhanced while cell apoptosis was induced by ischemia/reperfusion insult was downregulated by mitochondrial transplantation. Mitochondrial permeability transition pore (MPTP) opening in surviving hippocampal cells was also suppressed. CONCLUSIONS These results indicated that transplantation of autologous mitochondria rescued hippocampal cells from ischemia/reperfusion injury and ameliorated neurological impairment caused by cardiac arrest.

Background: Mitochondria are the ‘powerhouses of cells’ and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level of mobility. Moreover, non-exercise alternatives (i.e., nutraceuticals or pharmacological agents) to improve skeletal muscle bioenergetics require time to be effective in the target tissue and have another limitation in that they act systemically and not locally where needed. Mitochondrial transplantation represents a novel directed therapy designed to enhance energy production of tissues impacted by defective mitochondria. To date, no studies have used mitochondrial transplantation as an intervention to attenuate aging-induced skeletal muscle mitochondrial dysfunction. The purpose of this investigation, therefore, was to determine whether mitochondrial transplantation can enhance skeletal muscle bioenergetics in an aging rodent model. We hypothesized that mitochondrial transplantation would result in sustained skeletal muscle bioenergetics leading to improved functional capacity. Methods: Fifteen female mice (24 months old) were randomized into two groups (placebo or mitochondrial transplantation). Isolated mitochondria from a donor mouse of the same sex and age were transplanted into the hindlimb muscles of recipient mice (quadriceps femoris, tibialis anterior, and gastrocnemius complex). Results: The results indicated significant increases (ranging between ~36% and ~65%) in basal cytochrome c oxidase and citrate synthase activity as well as ATP levels in mice receiving mitochondrial transplantation relative to the placebo. Moreover, there were significant increases (approx. two-fold) in protein expression of mitochondrial markers in both glycolytic and oxidative muscles. These enhancements in the muscle translated to significant improvements in exercise tolerance. Conclusions: This study provides initial evidence showing how mitochondrial transplantation can promote skeletal muscle bioenergetics in an aging rodent model.

Mitochondrial transplantation prevented liver ischemia/reperfusion-induced hepatocellular injury and inflammation. In vivo intravital microscopy demonstrated that liver resident macrophages, namely Kupffer cells, rapidly sequestered, internalized and acidified transplanted mitochondria through the CRIg immunoreceptor. Mechanistically, both Kupffer cells and CRIg were necessary for the hepatoprotective and anti-inflammatory effects of mitochondrial transplantation. STRUCTURED ABSTRACT Objective To investigate the hepatoprotective effects of mitochondrial transplantation in a murine liver ischemia/reperfusion (I/R) model. Summary background data Sequential liver ischemia followed by reperfusion (I/R) is a pathophysiological process underlying hepatocellular injury in a number of clinical contexts, such as hemorrhagic shock/resuscitation, major elective liver surgery and organ transplantation. A unifying pathogenic consequence of I/R is mitochondrial dysfunction. Restoration of mitochondria via transplantation (MTx) has emerged as potential therapeutic in I/R. However, its role in liver I/R and its mechanisms of action remain poorly defined. Methods We investigated the hepatoprotective effects of MTx in an in vivo mouse model of liver I/R and used in vivo imaging and various knockout and transgenic mouse models to determine the mechanism of protection. Results We found that I/R-induced hepatocellular injury was prevented by MTx, as measured by plasma ALT, AST and liver histology. Additionally, I/R-induced pro-inflammatory cytokine release (IL-6, TNFα) was dampened by MTx, and anti-inflammatory IL-10 was enhanced. Moreover, MTx lowered neutrophil infiltration into both the liver sinusoids and lung BALF, suggesting a local and distant reduction in inflammation. Using in vivo intravital imaging, we found that I/R-subjected Kupffer cells (KCs), rapidly sequestered transplanted mitochondria, and acidified mitochondria within lysosomal compartments. To specifically interrogate the role of KCs, we depleted KCs using the diphtheria toxin-inducible Clec4f/iDTR transgenic mouse, then induced I/R, and discovered that KCs are necessary for the beneficial effects of MTx. Finally, we induced I/R in complement receptor of the immunoglobulin superfamily (CRIg) knockout mice and found that CRIg was required for mitochondria capture by KCs and mitochondrial-mediated hepatoprotection. Conclusions In this study, we demonstrated that CRIg-dependent capture of mitochondria by I/R-subjected Kupffer cells is a hepatoprotective mechanism in vivo. These data progress knowledge on the mechanisms of MTx and opens new avenues for clinical translation.

Background Mitochondrial transplantation (MTx) is an emerging but poorly understood technology with the potential to mitigate severe ischemia–reperfusion injuries after cardiac arrest (CA). To address critical gaps in the current knowledge, we test the hypothesis that MTx can improve outcomes after CA resuscitation. Methods This study consists of both in vitro and in vivo studies. We initially examined the migration of exogenous mitochondria into primary neural cell culture in vitro. Exogenous mitochondria extracted from the brain and muscle tissues of donor rats and endogenous mitochondria in the neural cells were separately labeled before co-culture. After a period of 24 h following co-culture, mitochondrial transfer was observed using microscopy. In vitro adenosine triphosphate (ATP) contents were assessed between freshly isolated and frozen-thawed mitochondria to compare their effects on survival. Our main study was an in vivo rat model of CA in which rats were subjected to 10 min of asphyxial CA followed by resuscitation. At the time of achieving successful resuscitation, rats were randomly assigned into one of three groups of intravenous injections: vehicle, frozen-thawed, or fresh viable mitochondria. During 72 h post-CA, the therapeutic efficacy of MTx was assessed by comparison of survival rates. The persistence of labeled donor mitochondria within critical organs of recipient animals 24 h post-CA was visualized via microscopy. Results The donated mitochondria were successfully taken up into cultured neural cells. Transferred exogenous mitochondria co-localized with endogenous mitochondria inside neural cells. ATP content in fresh mitochondria was approximately four times higher than in frozen-thawed mitochondria. In the in vivo survival study, freshly isolated functional mitochondria, but not frozen-thawed mitochondria, significantly increased 72-h survival from 55 to 91% ( P  = 0.048 vs. vehicle). The beneficial effects on survival were associated with improvements in rapid recovery of arterial lactate and glucose levels, cerebral microcirculation, lung edema, and neurological function. Labeled mitochondria were observed inside the vital organs of the surviving rats 24 h post-CA. Conclusions MTx performed immediately after resuscitation improved survival and neurological recovery in post-CA rats. These results provide a foundation for future studies to promote the development of MTx as a novel therapeutic strategy to save lives currently lost after CA.

The mammary microenvironment has been shown to suppress tumor progression by redirecting cancer cells to adopt a normal mammary epithelial progenitor fate in vivo. However, the mechanism(s) by which this alteration occurs has yet to be defined. Here, we test the hypothesis that mitochondrial transfer from normal mammary epithelial cells to breast cancer cells plays a role in this redirection process. We evaluate mitochondrial transfer in 2D and 3D organoids using our unique 3D bioprinting system to produce chimeric organoids containing normal and cancer cells. We demonstrate that breast cancer tumoroid growth is hindered following interaction with mammary epithelial cells in both 2D and 3D environments. Furthermore, we show mitochondrial transfer occurs between donor mammary epithelial cells and recipient cancer cells primarily through tunneling nanotubes (TNTs) with minimal amounts seen from extracellular transfer of mitochondria, likely via extracellular vesicles (EVs). This organelle exchange results in various cellular and metabolic alterations within cancer cells, reducing their proliferative potential, and making them susceptible to microenvironmental control. Our results demonstrate that mitochondrial transfer contributes to microenvironmental redirection of cancer cells through alteration of metabolic and molecular functions of the recipient cancer cells. To the best of our knowledge, this is the first description of a 3D bioprinter‐assisted organoid system for studying mitochondrial transfer. These studies are also the first mechanistic insights into the process of mammary microenvironmental redirection of cancer and provide a framework for new therapeutic strategies to control cancer.

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Significance Mitochondrial dysfunction plays a key role in many diseases, yet treatments to restore function remain limited. Cells naturally transfer mitochondria to help repair damage, but this process is inefficient. Here, we use molybdenum disulfide (MoS2) nanoflowers to boost mitochondrial production, turning donor cells into mitochondrial biofactories. These cells transfer more mitochondria to damaged cells, significantly improving energy production and function. In disease models, this approach restores cell health, offering a strategy for treating mitochondrial-related disorders. By enhancing the body’s own repair mechanisms, this nanomaterial-based method could pave the way for innovative therapies in regenerative medicine.

Colonic smooth muscle cell (CSMC) hypertrophy and hyperplasia have been described in both human ulcerative colitis (UC) and animal models. The deletion of smooth muscle (SM) 22α induces the phenotypic switching of SMCs. Here, we report that Sm22α-deficient mice develop spontaneous colitis, which is characterized by radical S-adenosyl-methionine domain-containing 2 (RSAD2)-driven mitochondrial dysfunction and inflammation in CSMCs and ferroptosis in the colonic mucosa. Mechanistically, RSAD2 mediates YTH m6A RNA-binding protein 1 (YTHDF1) methylation and activation, thereby increasing the mRNA N6-methyladenosine (m6A) modification and translation of dynamin-related protein 1 (DRP1), resulting in mitochondrial fragmentation in CSMCs. Inflammatory CSMC-derived mitochondrial extracellular vesicles trigger intestinal epithelial ferroptosis by inducing ROS production. The ablation of RSAD2 in mice with SMC-specific Sm22α knockout alleviates colitis severity in this experimental model. Importantly, increases in both RSAD2 expression and the ferroptotic signature are observed in serum and/or colonic samples from UC patients. Overall, this study shows a mitochondrial mechanism underlying the ability of dysfunctional smooth muscle to drive colitis and highlights the potential of targeting the RSAD2-YTHDF1 axis as an innovative therapeutic strategy for colitis. Colonic smooth muscle cell (CSMC) hypertrophy and hyperplasia have been described in human ulcerative colitis (UC) and animal models. Here the authors show that mice lacking smooth muscle (SM) 22a develop spontaneous colitis which has RSAD2-driven mitochondrial dysfunction and inflammation in CSMC and show dependence on RSAD2 for colitis alleviation.

The communication between neural stem cells (NSCs) and surrounding astrocytes is essential for the homeostasis of the NSC niche. Intercellular mitochondrial transfer, a unique communication system that utilizes the formation of tunneling nanotubes for targeted mitochondrial transfer between donor and recipient cells, has recently been identified in a wide range of cell types. Intercellular mitochondrial transfer has also been observed between different types of cancer stem cells (CSCs) and their neighboring cells, including brain CSCs and astrocytes. CSC mitochondrial transfer significantly enhances overall tumor progression by reprogramming neighboring cells. Despite the urgent need to investigate this newly identified phenomenon, mitochondrial transfer in the central nervous system remains largely uncharacterized. In this study, we found evidence of intercellular mitochondrial transfer from human NSCs and from brain CSCs, also known as brain tumor-initiating cells (BTICs), to astrocytes in co-culture experiments. Both NSC and BTIC mitochondria triggered similar transcriptome changes upon transplantation into the recipient astrocytes. In contrast to NSCs, the transplanted mitochondria from BTICs had a significant proliferative effect on the recipient astrocytes. This study forms the basis for mechanistically deciphering the impact of intercellular mitochondrial transfer on recipient astrocytes, which will potentially provide us with new insights into the mechanisms of mitochondrial retrograde signaling.

Mesenchymal stem cell (MSC) transplantation alleviates metabolic defects in diseased recipient cells by intercellular mitochondrial transport (IMT). However, the effect of host metabolic conditions on IMT and thereby on the therapeutic efficacy of MSCs has largely remained unexplored. Here we found impaired mitophagy, and reduced IMT in MSCs derived from high-fat diet (HFD)-induced obese mouse (MSC-Ob). MSC-Ob failed to sequester their damaged mitochondria into LC3-dependent autophagosomes due to decrease in mitochondrial cardiolipin content, which we propose as a putative mitophagy receptor for LC3 in MSCs. Functionally, MSC-Ob exhibited diminished potential to rescue mitochondrial dysfunction and cell death in stress-induced airway epithelial cells. Pharmacological modulation of MSCs enhanced cardiolipin-dependent mitophagy and restored their IMT ability to airway epithelial cells. Therapeutically, these modulated MSCs attenuated features of allergic airway inflammation (AAI) in two independent mouse models by restoring healthy IMT. However, unmodulated MSC-Ob failed to do so. Notably, in human (h)MSCs, induced metabolic stress associated impaired cardiolipin-dependent mitophagy was restored upon pharmacological modulation. In summary, we have provided the first comprehensive molecular understanding of impaired mitophagy in obese-derived MSCs and highlight the importance of pharmacological modulation of these cells for therapeutic intervention. A MSCs obtained from (HFD)-induced obese mice (MSC-Ob) show underlying mitochondrial dysfunction with a concomitant decrease in cardiolipin content. These changes prevent LC3-cardiolipin interaction, thereby reducing dysfunctional mitochondria sequestration into LC3-autophagosomes and thus impaired mitophagy. The impaired mitophagy is associated with reduced intercellular mitochondrial transport (IMT) via tunneling nanotubes (TNTs) between MSC-Ob and epithelial cells in co-culture or in vivo. B Pyrroloquinoline quinone (PQQ) modulation in MSC-Ob restores mitochondrial health, cardiolipin content, and thereby sequestration of depolarized mitochondria into the autophagosomes to alleviate impaired mitophagy. Concomitantly, MSC-Ob shows restoration of mitochondrial health upon PQQ treatment (MSC-ObPQQ). During co-culture with epithelial cells or transplantation in vivo into the mice lungs, MSC-ObPQQ restores IMT and prevents epithelial cell death. C Upon transplantation in two independent allergic airway inflammatory mouse models, MSC-Ob failed to rescue the airway inflammation, hyperactivity, metabolic changes in epithelial cells. D PQQ modulated MSCs restored these metabolic defects and restored lung physiology and airway remodeling parameters.

Iron oxide nanoparticles could significantly promote the mitochondrial transfer from mesenchymal stem cells to the injured cells.

COPYRIGHT © 2023 Chen and Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Barriers and opportunities: Intercellular mitochondrial transfer for cardiac protection—Delivery by extracellular vesicles

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Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury.

BACKGROUND Recent studies have demonstrated that mesenchymal stem cells (MSCs) can rescue injured target cells via mitochondrial transfer. However, it has not been fully understood how bone marrow-derived MSCs repair glomeruli in diabetic kidney disease (DKD). AIM To explore the mitochondrial transfer involved in the rescue of injured glomerular endothelial cells (GECs) by MSCs, both in vitro and in vivo. METHODS In vitro experiments were performed to investigate the effect of co-culture with MSCs on high glucose-induced GECs. The transfer of mitochondria was visua lized using fluorescent microscopy. GECs were freshly sorted and ultimately tested for apoptosis, viability, mRNA expression by real-time reverse transcri ptase-polymerase chain reaction, protein expression by western blot, and mitochondrial function. Moreover, streptozotocin-induced DKD rats were infused with MSCs, and renal function and oxidative stress were detected with an automatic biochemical analyzer and related-detection kits after 2 wk. Kidney histology was analyzed by hematoxylin and eosin, periodic acid-Schiff, and immunohistochemical staining. RESULTS Fluorescence imaging confirmed that MSCs transferred mitochondria to injured GECs when co-cultured in vitro. We found that the apoptosis, proliferation, and mitochondrial function of injured GECs were improved following co-culture. Additionally, MSCs decreased pro-inflammatory cytokines [interleukin (IL)-6, IL-1β, and tumor necrosis factor-α] and pro-apoptotic factors (caspase 3 and Bax). Mitochondrial transfer also enhanced the expression of superoxide dismutase 2, B cell lymphoma-2, glutathione peroxidase (GPx) 3, and mitofusin 2 and inhibited reactive oxygen species (ROS) and dynamin-related protein 1 expression. Furthermore, MSCs significantly ameliorated functional parameters (blood urea nitrogen and serum creatinine) and decreased the production of malondialdehyde, advanced glycation end products, and ROS, whereas they increased the levels of GPx and superoxide dismutase in vivo. In addition, significant reductions in the glomerular basement membrane and renal interstitial fibrosis were observed following MSC treatment. CONCLUSION MSCs can rejuvenate damaged GECs via mitochondrial transfer. Additionally, the improvement of renal function and pathological changes in DKD by MSCs may be related to the mechanism of mitochondrial transfer.

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The efficacy of drug delivery, particularly for solid tumors, is severely hampered by a cascade of biological barriers-including dense extracellular matrix, high interstitial fluid pressure, and inefficient vascularization-that limit therapeutic penetration and distribution. Biological materials, such as cells, extracellular vesicles and organelles, serve as biocompatibility and targeted delivery vehicles, offering significant therapeutic potential. However, most current strategies emphasize multifunctional and biomimetic delivery systems designed to traverse the extracellular stroma, often overlooking intracellular transport pathways. Here, we demonstrate that mitochondria and their hitchhiked cargos are transported via tunneling nanotubes (TNTs), contiguous cytoplasmic bridges that interconnect cells. Oxidative stress plays a pivotal role in stimulating both TNTs formation and mitochondrial transfer. By leveraging TNTs as an intracellular highway, we achieved intercellular transport and deep tissue penetration of the photosensitizer IR780, which was hitchhiked onto mitochondria (designated as IR780/Mito). The intensity of near-infrared (NIR) light governs TNTs dynamics, either promoting formation or inducing cleavage, by modulating oxidative stress levels generated upon IR780 excitation. Under mild NIR irradiation, moderate oxidative stress enhances TNTs formation and facilitates IR780/Mito transfer, enabling efficient delivery directly into tumor cells. Conversely, intense NIR irradiation triggers excessive reactive oxygen species (ROS) production, leading to TNT disruption and subsequent blockade of IR780/Mito transport. These findings present emerging opportunities for exploration into the use of TNTs transshipment channel to realize controllable intracellular transport of different types of cargos, with broad and promising applications in the diagnosis and treatment of multiple diseases in the future. STATEMENT OF SIGNIFICANCE: This study introduces a bioinspired drug delivery platform that utilizes tunneling nanotubes (TNTs)-natural intercellular connections-for efficient transport of mitochondria-loaded photosensitizer IR780. By leveraging near-infrared (NIR) light, we precisely modulate oxidative stress levels to control TNT formation and integrity. Under mild irradiation, moderate ROS enhancement promotes TNT-mediated intercellular transfer, facilitating deep tumor penetration; intense irradiation generates excessive ROS, disrupting TNTs and halting drug delivery. This mitochondria-driven approach enables spatiotemporally regulated drug distribution and overcomes extracellular barriers. The strategy offers a highly biocompatible and tunable alternative to synthetic systems, with promising applications in precision cancer therapy and diseases where intercellular communication is pivotal.

In the nervous system, mitochondria can be transferred between neural cells through intercellular tunneling nanotubes (TNTs), microvesicles, or as free organelles. This transfer not only alters the mitochondrial content and respiration of recipient neural cells but also triggers a profound rewiring of their physiology, with glial cells and immune responses playing key roles in this reconfiguration.

Natural intercellular mitochondrial transfer has been recognized as a pivotal mechanism in the treatment of various diseases. Bone marrow mesenchymal stem cells (BMSCs), owing to their low bioenergetic demands and inherent homing capacity, are considered highly promising mitochondrial donor cells. However, this strategy is limited in senile osteoporosis (SOP) because large amounts of ROS produced by mitochondrial oxidative stress in senescent BMSCs (S-BMSCs) impairs their viability and function. Here, we report that in-situ treatment of senescent bone marrow-derived macrophages (S-BMDMs) with a cerium-based nanosystem (CNS) composed of antioxidant and energy-active units, which exhibits superior autophagy-activating capability, effectively restores the viability and osteogenic function of S-BMSCs by promoting mitochondrial biogenesis and transfer. Transcriptomic profiling revealed that the SIRT1-PGC-1α axis, significantly associated with autophagy activation, drives mitochondrial biogenesis in S-BMDMs. The efficient intercellular mitochondrial transfer ameliorates the senescent bone microenvironment, rescues S-BMSCs functionality, and enhances bone formation. In conclusion, the autophagy-activating CNS, by effectively rejuvenating S-BMDMs and promoting mitochondrial biogenesis and transfer, provides an innovative therapeutic strategy for SOP-associated bone regeneration.

Dental pulp stem cells (DPSCs) have demonstrated remarkable potential in enhancing peripheral nerve regeneration, though the precise mechanisms remain largely unknown. This study investigates how DPSCs alleviate Schwann cell pyroptosis and restore mitochondrial homeostasis through intercellular mitochondrial transfer. In a crab-eating macaque model, we first observed that DPSC-loaded nerve conduits significantly promoted long-term nerve regeneration, facilitating tissue proliferation and myelin recovery. We further established a rat facial nerve injury (FNI) model and found that DPSC treatment reduced pyroptosis and mitochondrial ROS production in Schwann cells. A pivotal mitochondrial protective mechanism, resembling the effects of a ROS-targeted inhibitor, involved the transfer of mitochondria from DPSCs to pyroptosis-induced Schwann cells via tunneling nanotubes, while blocking intercellular junctions or mitochondrial function diminished the therapeutic effects. TNFα secreted by pyroptosis-induced Schwann cells activated the NF-κB pathway in DPSCs, enhancing mitochondrial transfer and adaptive stress responses, thereby promoting mitochondrial protection against pyroptosis in Schwann cells, as reflected in the improved therapeutic efficacy of TNFα-preconditioned DPSCs in the FNI model. These findings unveil a mechanism through which DPSCs foster nerve regeneration via mitochondrial transfer, presenting a promising strategy for enhancing stem cell-based therapies for nerve injuries.

Myocardial fibrosis is the villain of sudden cardiac death. Myocardial ischemia/reperfusion (MI/R) injury induces cardiomyocyte damage or even death, which in turn stimulates fibroblast activation and fibrosis, but the intercellular communication mechanism remains unknown. Recent studies have shown that small extracellular vesicles (sEVs) significantly contribute to intercellular communication. Whether and how sEV might mediate post-MI/R cardiomyocyte/fibroblasts communication remain unknown. Here, in vivo and in vitro MI/R models were established. We demonstrate that sEVs derived from cardiomyocyte (Myo-sEVs) carry mitochondrial components, which enter fibroblasts to initiate myocardial fibrosis. Based on bioinformatics screening and experimental verification, the activating molecule in Beclin1-regulated autophagy protein 1 (autophagy/beclin-1 regulator 1, Ambra1) was found to be a critical component of these sEV and might be a new marker for Myo-sEVs. Interestingly, release of Ambra1+-Myo-sEVs was caused by secretory rather than canonical autophagy after MI/R injury and thereby escaped degradation. In ischemic and peripheral areas, Ambra1+-Myo-sEVs were internalized by fibroblasts, and the delivered mtDNA components to activate the fibroblast cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to promote fibroblast activation and proliferation. In addition, our data show that Ambra1 is expressed on the EV surface and cardiac-specific Ambra1 down regulation inhibits the Ambra1+-Myo-sEVs release and fibroblast uptake, effectively inhibiting ischemic myocardial fibrosis. This finding newly provides the evidence that myocardial secretory autophagy plays a role in intercellular communication during cardiac fibrosis. Ambra-1 is a newly characterized molecule with bioactivity and might be a marker for Myo-sEVs, providing new therapeutic targets for cardiac remodeling.

Cancer cells with severe defects in mitochondrial DNA (mtDNA) can import mitochondria via horizontal mitochondrial transfer (HMT) to restore respiration. Mitochondrial respiration is necessary for the activity of dihydroorotate dehydrogenase (DHODH), an enzyme of the inner mitochondrial membrane that catalyzes the fourth step of de novo pyrimidine synthesis. Here, we investigated the role of de novo synthesis of pyrimidines in driving tumor growth in mtDNA-deficient (ρ0) cells. While ρ0 cells grafted in mice readily acquired mtDNA, this process was delayed in cells transfected with alternative oxidase (AOX), which combines the functions of mitochondrial respiratory complexes III and IV. The ρ0 AOX cells were glycolytic but maintained normal DHODH activity and pyrimidine production. Deletion of DHODH in a panel of tumor cells completely blocked or delayed tumor growth. The grafted ρ0 cells rapidly recruited tumor-promoting/stabilizing cells of the innate immune system, including pro-tumor M2 macrophages, neutrophils, eosinophils, and mesenchymal stromal cells (MSCs). The ρ0 cells recruited MSCs early after grafting, which were potential mitochondrial donors. Grafting MSCs together with ρ0 cancer cells into mice resulted in mitochondrial transfer from MSCs to cancer cells. Overall, these findings indicate that cancer cells with compromised mitochondrial function readily acquire mtDNA from other cells in the tumor microenvironment to restore DHODH-dependent respiration and de novo pyrimidine synthesis. The inhibition of tumor growth induced by blocking DHODH supports targeting pyrimidine synthesis as a potential widely applicable therapeutic approach.

Background: Rotator cuff muscle degeneration leads to poor clinical outcomes for patients with rotator cuff tears. Fibroadipogenic progenitors (FAPs) are resident muscle stem cells with the ability to differentiate into fibroblasts as well as white and beige adipose tissue. Induction of the beige adipose phenotype in FAPs has been shown to improve muscle quality after rotator cuff tears, but the mechanisms of how FAPs exert their beneficial effects have not been fully elucidated. Purpose: To study the horizontal transfer of mitochondria from FAPs to myogenic cells and examine the effects of β-agonism on this novel process. Study Design: Controlled laboratory study. Methods: In mice that had undergone a massive rotator cuff tear, single-cell RNA sequencing was performed on isolated FAPs for genes associated with mitochondrial biogenesis and transfer. Murine FAPs were isolated by fluorescence-activated cell sorting and treated with a β-agonist versus control. FAPs were stained with mitochondrial dyes and cocultured with recipient C2C12 myoblasts, and the rate of transfer was measured after 24 hours by flow cytometry. PdgfraCreERT/MitoTag mice were generated to study the effects of a rotator cuff injury on mitochondrial transfer. PdgfraCreERT/tdTomato mice were likewise generated to perform lineage tracing of PDGFRA+ cells in this injury model. Both populations of transgenic mice underwent tendon transection and denervation surgery, and MitoTag-labeled mitochondria from Pdgfra+ FAPs were visualized by fluorescent microscopy, spinning disk confocal microscopy, and 2-photon microscopy; overall mitochondrial quantity was compared between mice treated with β-agonists and dimethyl sulfoxide. Results: Single-cell RNA sequencing in mice that underwent rotator cuff tear demonstrated an association between transcriptional markers of adipogenic differentiation and genes associated with mitochondrial biogenesis. In vitro cocultures of murine FAPs with C2C12 cells revealed that treatment of cells with a β-agonist increased mitochondrial transfer compared to control conditions (17.8% ± 9.9% to 99.6% ± 0.13% P < .0001). Rotator cuff injury in PdgfraCreERT/MitoTag mice resulted in a robust increase in MitoTag signal in adjacent myofibers compared with uninjured mice. No accumulation of tdTomato signal from PDGFRA+ cells was seen in injured fibers at 6 weeks after injury, suggesting that FAPs do not fuse with injured muscle fibers but rather contribute their mitochondria. Conclusion: The authors have described a novel process of endogenous mitochondrial transfer that can occur within the injured rotator cuff between FAPs and myogenic cells. This process may be leveraged therapeutically with β-agonist treatment and represents an exciting target for improving translational therapies available for rotator cuff muscle degeneration. Clinical Relevance: Promoting endogenous mitochondrial transfer may represent a novel translational strategy to address muscle degeneration after rotator cuff tears.

Transmissible cancers are clonal lineages of neoplastic cells able to infect multiple hosts, spreading through populations in the environment as an infectious disease. Transmissible cancers have been identified in Tasmanian devils, dogs, and bivalves. Several lineages of bivalve transmissible neoplasias (BTN) have been identified in multiple bivalve species. In 2019 in Puget Sound, Washington, USA, disseminated neoplasia was observed in basket cockles (Clinocardium nuttallii), a species that is important to the culture and diet of the Suquamish Tribe as well as other tribes with traditional access to the species. To test whether disseminated neoplasia in cockles is a previously unknown lineage of BTN, a nuclear locus was amplified from cockles from Agate Pass, Washington, and sequences revealed evidence of transmissible cancer in several individuals. We used a combination of cytology and quantitative PCR to screen collections of cockles from eleven locations in Puget Sound and along the Washington coastline to identify the extent of contagious cancer spread in this species. Two BTN lineages were identified in these cockles, with one of those lineages (CnuBTN1) being the most prevalent and geographically widespread. Within the CnuBTN1 lineage, multiple nuclear loci support the conclusion that all cancer samples form a single clonal lineage. However, the mitochondrial alleles in each cockle with CnuBTN1 are different from each other, suggesting mitochondrial genomes of this cancer have been replaced multiple times during its evolution, through horizontal transmission. The identification and analysis of these BTNs are critical for broodstock selection, management practices, and repopulation of declining cockle populations, which will enable continued cultural connection and dietary use of the cockles by Coast Salish Tribes.

Recent research has shown that mtDNA-deficient cancer cells (ρ0 cells) acquire mitochondria from tumor stromal cells to restore respiration, facilitating tumor formation. We investigated the role of Miro1, an adaptor protein involved in movement of mitochondria along microtubules, in this phenomenon. Inducible Miro1 knockout (Miro1KO) mice markedly delayed tumor formation after grafting ρ0 cancer cells. Miro1KO mice with fluorescently labeled mitochondria revealed that this delay was due to hindered mitochondrial transfer from the tumor stromal cells to grafted B16 ρ0 cells, which impeded recovery of mitochondrial respiration and tumor growth. Miro1KO led to the perinuclear accumulation of mitochondria and impaired mobility of the mitochondrial network. In vitro experiments revealed decreased association of mitochondria with microtubules, compromising mitochondrial transfer via tunneling nanotubes (TNTs) in mesenchymal stromal cells. Here we show the role of Miro1 in horizontal mitochondrial transfer in mouse melanoma models in vivo and its involvement with TNTs.

Two recently introduced fungal plant pathogens (Ceratocystis lukuohia and Ceratocystis huliohia) are responsible for Rapid ‘ōhi‘a Death (ROD) in Hawai‘i. Despite being sexually incompatible, the two pathogens often co-occur in diseased ‘ōhi‘a sapwood, where genetic interaction is possible. We sequenced and annotated 33 mitochondrial genomes of the two pathogens and related species, and investigated 35 total Ceratocystis mitogenomes. Ten mtDNA regions [one group I intron, seven group II introns, and two autonomous homing endonuclease (HE) genes] were heterogeneously present in C. lukuohia mitogenomes, which were otherwise identical. Molecular surveys with specific primers showed that the 10 regions had uneven geographic distribution amongst populations of C. lukuohia. Conversely, identical orthologs of each region were present in every studied isolate of C. huliohia regardless of geographical origin. Close relatives of C. lukuohia lacked or, rarely, had few and dissimilar orthologs of the 10 regions, whereas most relatives of C. huliohia had identical or nearly identical orthologs. Each region included or worked in tandem with HE genes or reverse transcriptase/maturases that could facilitate interspecific horizontal transfers from intron-minus to intron-plus alleles. These results suggest that the 10 regions originated in C. huliohia and are actively moving to populations of C. lukuohia, perhaps through transient cytoplasmic contact of hyphal tips (anastomosis) in the wound surface of ‘ōhi‘a trees. Such contact would allow for the transfer of mitochondria followed by mitochondrial fusion or cytoplasmic exchange of intron intermediaries, which suggests that further genomic interaction may also exist between the two pathogens.

Autonomous replication and segregation of mitochondrial DNA (mtDNA) creates the potential for evolutionary conflict driven by emergence of haplotypes under positive selection for ‘selfish’ traits, such as replicative advantage. However, few cases of this phenomenon arising within natural populations have been described. Here, we survey the frequency of mtDNA horizontal transfer within the canine transmissible venereal tumour (CTVT), a contagious cancer clone that occasionally acquires mtDNA from its hosts. Remarkably, one canine mtDNA haplotype, A1d1a, has repeatedly and recently colonised CTVT cells, recurrently replacing incumbent CTVT haplotypes. An A1d1a control region polymorphism predicted to influence transcription is fixed in the products of an A1d1a recombination event and occurs somatically on other CTVT mtDNA backgrounds. We present a model whereby ‘selfish’ positive selection acting on a regulatory variant drives repeated fixation of A1d1a within CTVT cells. The competitive dynamics of mitochondrial haplotypes juxtaposed within the same cell are poorly studied. Here the authors show, in the context of a transmissible cancer, that one haplotype has recurrently entered cancer cells by horizontal transfer and appears to have a ‘selfish’ selective advantage.

Recently, we showed that generation of tumours in syngeneic mice by cells devoid of mitochondrial (mt) DNA (ρ0 cells) is linked to the acquisition of the host mtDNA. However, the mechanism of mtDNA movement between cells remains unresolved. To determine whether the transfer of mtDNA involves whole mitochondria, we injected B16ρ0 mouse melanoma cells into syngeneic C57BL/6Nsu9-DsRed2 mice that express red fluorescent protein in their mitochondria. We document that mtDNA is acquired by transfer of whole mitochondria from the host animal, leading to normalisation of mitochondrial respiration. Additionally, knockdown of key mitochondrial complex I (NDUFV1) and complex II (SDHC) subunits by shRNA in B16ρ0 cells abolished or significantly retarded their ability to form tumours. Collectively, these results show that intact mitochondria with their mtDNA payload are transferred in the developing tumour, and provide functional evidence for an essential role of oxidative phosphorylation in cancer. DOI: http://dx.doi.org/10.7554/eLife.22187.001

Alterations in cancer cell metabolism have recently gained considerable attention as a possible cause of adaptation and resistance to therapy. However, the underlying molecular mechanisms, particularly in leukemia resistance occurring in the bone marrow microenvironment, remain unclear. Here, we explore the role of direct stroma-leukemia interactions and transfer of membrane vesicles along with proteins as a mechanism of stroma-driven protection. K562 CML leukemia cells and primary CD34 + CML blasts were cultured alone or co-cultured with HS-5 stromal cells to mimic the bone marrow microenvironment conditions. Imatinib treatment was used experimentally as it is a standard first-line treatment in CML. Assessment of vesicles transfer, metabolic parameters, mitochondrial function phenotyping, Trans-SILAC proteomics and metabolomics, together with apoptosis assessment, verified the influence of stroma on metabolic plasticity, protein transfer and adaptation to imatinib in leukemic cells. Trans-system evaluated necessity of direct cell-cell contact. Data from single-cell atlas of diagnostic CML bone marrow were used to correlate gene expression profiles with clinical outcome. Telaglenastat was used to validate the clinical potential of our findings. Stromal cells enhanced metabolic plasticity and oxidative capacity in leukemia, thereby protecting against metabolic decline and oxidative stress caused by imatinib. Direct stroma-leukemia contact was necessary for vesicles transfer, metabolic rearrangement and protection from imatinib-induced apoptosis. This was accompanied with shift towards OXPHOS activity, associated with increased utilization of non-glucose substrates. We found the presence of stromal TCA-related proteins in leukemic cells, associated with higher TCA cycle dynamics and activity, increased glutamine and reduced oxidative stress. The gene expression profiles correlated with clinical resistance to TKIs. Targeting the glutamine-TCA axis by telaglenastat in combination with imatinib reversed the stroma-driven protection, leading to increased apoptosis. This study describes a novel mechanism of direct bone marrow-mediated protection of leukemic cells from imatinib/TKI, related to transfer of metabolic proteins leading to higher activity of TCA cycle, metabolic plasticity and adaptation. Targeting the stroma-driven TCA cycle-related metabolism combined with imatinib presents a promising strategy to achieve therapeutic efficacy to overcome bone marrow microenvironment-mediated protection in CML.

Significance Increasing evidence suggests that extracellular vesicles (EVs) can transfer genetic material to recipient cells. However, the mechanism and role of this phenomenon are largely unknown. Here we have made a remarkable discovery: EVs can harbor the full mitochondrial genome. These extracellular vesicles can in turn transfer their mtDNA to cells with impaired metabolism, leading to restoration of metabolic activity. We determined that hormonal therapy induces oxidative phosphorylation-deficient breast cancer cells, which can be rescued via the transfer of mtDNA-laden extracellular vesicles. Horizontal transfer of mtDNA occurred in cancer stem-like cells and was associated with increased self-renewal potential of these cells, leading to resistance to hormonal therapy. We propose that mtDNA transfer occurs in human cancer via EVs. The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.

No abstract available

This work elucidates the mechanisms of horizontal genome transfer and uncovers a new pathway of intercellular transport. Recent work has revealed that both plants and animals transfer genomes between cells. In plants, horizontal transfer of entire plastid, mitochondrial, or nuclear genomes between species generates new combinations of nuclear and organellar genomes, or produces novel species that are allopolyploid. The mechanisms of genome transfer between cells are unknown. Here, we used grafting to identify the mechanisms involved in plastid genome transfer from plant to plant. We show that during proliferation of wound-induced callus, plastids dedifferentiate into small, highly motile, amoeboid organelles. Simultaneously, new intercellular connections emerge by localized cell wall disintegration, forming connective pores through which amoeboid plastids move into neighboring cells. Our work uncovers a pathway of organelle movement from cell to cell and provides a mechanistic framework for horizontal genome transfer.

Summary Cells transmit their genomes vertically to daughter cells during cell divisions. Here, we demonstrate the occurrence and extent of horizontal mitochondrial (mt)DNA acquisition between cells that are not in a parent-offspring relationship. Extensive single-cell sequencing from various tissues and organs of adult chimeric mice composed of cells carrying distinct mtDNA haplotypes showed that a substantial fraction of individual cardiomyocytes, neurons, glia, intestinal, and spleen cells captured donor mtDNA at high levels. In addition, chimeras composed of cells with wild-type and mutant mtDNA exhibited increased trafficking of wild-type mtDNA to mutant cells, suggesting that horizontal mtDNA transfer may be a compensatory mechanism to restore compromised mitochondrial function. These findings establish the groundwork for further investigations to identify mtDNA donor cells and mechanisms of transfer that could be critical to the development of novel gene therapies.

Autologous fat grafting is a widely used technique in plastic and reconstructive surgery, but its efficacy is often limited by the poor survival rate of transplanted adipose tissue. This study aims to enhance the survival of fat grafts by investigating the role of thymosin beta-4 (Tβ4) in facilitating mitochondrial transfer from adipose-derived stem cells (ADSCs) to adipocytes and newly formed blood vessels within the grafts via tunneling nanotubes (TNTs). We demonstrate that Tβ4 upregulates the Rac/F-actin pathway, leading to an increased formation of TNTs and subsequent transfer of mitochondria from ADSCs. This process mitigates oxidative stress, reduces apoptosis, and promotes revascularization, thereby improving the quality and volume retention of fat grafts. Our findings provide a novel mechanistic insight into the enhancement of fat graft survival and suggest Tβ4 as a potential therapeutic agent to improve clinical outcomes in autologous fat transfer procedures.

Mesenchymal stromal cells (MSCs) have shown promise in treating various diseases, and optimizing their therapeutic potential is a crucial objective in MSCs-based clinical applications. The microenvironment, particularly three-dimensional (3D) culture systems, plays a pivotal role in regulating the fate determination and enhancing the therapeutic potential of MSCs. Currently, the mechanisms governing the interactions between MSCs cultured in a dynamic 3D system and host recipient cells remain incompletely understood. MSCs transfer mitochondria to influence the fate of recipient cells, with tunneling nanotubes (TNTs) being the primary method. However, whether MSCs cultured under dynamic 3D conditions transfer mitochondria via TNTs to exert therapeutic effects remains to be elucidated. This study developed a dynamic 3D culture system for stem cells from human exfoliated deciduous teeth (SHED), a type of MSCs, utilizing gelatin microcryogel microcarriers and stirred tank bioreactor. A mouse model of full-thickness skin defects was employed to validate the enhanced therapeutic efficacy of SHED cultured under dynamic 3D conditions. Co-culture experiments with SHED and endothelial cells demonstrated that the dynamic 3D culture conditions empower the MSCs to transfer mitochondria via TNTs, thereby promoting angiogenesis. This research provides novel insights into the mechanisms underlying wound healing acceleration by SHED cultured under dynamic 3D conditions and offers a new strategy for developing MSCs transplantation applications.

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods. We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.

The repair of bone defects in the elderly individuals is significantly delayed due to cellular senescence and dysfunction, which presents a challenge in clinical settings. Furthermore, there are limited effective methods available to promote bone repair in older individuals. Herein, melatonin-loaded mesoporous bioactive glasses microspheres (MTBG) were successfully prepared based on their mesoporous properties. The repair of bone defects in aged rats was significantly accelerated by enhancing mitochondrial function through the sustained release of melatonin and bioactive ions. MTBG effectively rejuvenated senescent bone marrow mesenchymal stem cells (BMSCs) by scavenging excessive reactive oxygen species (ROS), stabilizing the mitochondrial membrane potential (ΔΨm), and increasing ATP synthesis. Analysis of the underlying mechanism revealed that the formation of tunneling nanotubes (TNTs) facilitated the intercellular transfer of mitochondria, thereby resulting in the recovery of mitochondrial function. This study provides critical insights into the design of new biomaterials for the elderly individuals and the biological mechanism involved in aged bone regeneration.

Rationale: Tunnel nanotube (TNT)-mediated mitochondrial transport is crucial for the development and maintenance of multicellular organisms. Despite numerous studies highlighting the significance of this process in both physiological and pathological contexts, knowledge of the underlying mechanisms is still limited. This research focused on the role of the ROCK inhibitor Y-27632 in modulating TNT formation and mitochondrial transport in retinal pigment epithelial (RPE) cells. Methods: Two types of ARPE19 cells (a retinal pigment epithelial cell line) with distinct mitochondrial fluorescently labeled, were co-cultured and treated with ROCK inhibitor Y-27632. The formation of nanotubes and transport of mitochondria were assessed through cytoskeletal staining and live cell imaging. Mitochondrial dysfunction was induced by light damage to establish a model, while mitochondrial function was evaluated through measurement of oxygen consumption rate. The effects of Y-27632 on cytoskeletal and mitochondrial dynamics were further elucidated through detailed analysis. Results: Y-27632 treatment led to an increase in nanotube formation and enhanced mitochondrial transfer among ARPE19 cells, even following exposure to light-induced damage. Our analysis of cytoskeletal and mitochondrial distribution changes suggests that Y-27632 promotes nanotube-mediated mitochondrial transport by influencing cytoskeletal remodeling and mitochondrial movement. Conclusions: These results suggest that Y-27632 has the ability to enhance mitochondrial transfer via tunneling nanotubes in retinal pigment epithelium, and similarly predict that ROCK inhibitor can fulfill its therapeutic potential through promoting mitochondrial transport in the retinal pigment epithelium in the future.

Recent therapeutic advances have improved survival rates for patients with lung cancer. However, drug resistance remains a significant challenge and is closely linked to mitochondrial alterations in lung cancer cells. Magnolol, a compound extracted from Magnolia officinalis, has anti-cancer properties. However, its impact on mitochondria in cancer cells remains poorly understood. This study aimed to explore the therapeutic potential of Magnolol in lung cancer and elucidate its effects on mitochondria in lung cancer cells. The effects of Magnolol on lung cancer were studied using xenograft mouse studies and in vitro analyses. Magnolol promotes the production of reactive oxygen species (ROS) and inhibits the antioxidant pathway in cancer cells. This disruption further impairs redox interactions between mitochondria and peroxisome, leading to mitochondrial dysfunction and mitocytosis. Additionally, Magnolol activates oxeiptosis, facilitating intercellular transport of damaged mitochondria and peroxisomes via tunneling nanotubes. The increased fusion of mitochondria may contribute to mitochondrial dysfunction, promote the accumulation of dysfunctional peroxisomes in recipient cells, and elevate ROS levels. In turn, this process enhances oxeiptosis, ultimately inhibiting tumour progression. These findings suggest that Magnolol may serve as a promising targeted therapeutic for disrupting mitochondrial function in lung cancer.

Background: Glioblastoma (GBM) has a median survival of <2 years and generally recurs within 6 months of treatment due to the development of chemo- and radiotherapy (RT) resistance. Tunneling nanotubes (TNTs) serve as intercellular conduits for establishing robust, tumor-promoting networks within the hypoxic tumor microenvironment. TNTs are provoked by hypoxia and can passage organelles like mitochondria, i.e., between astrocytes and stem-like brain tumor-initiating cells (BTICs). This is theorized to expand resistance properties to other cell types, as well as facilitate metabolic rescue in damaged cancer cells. We investigated the mitochondrial uptake (MU) abilities of RT-sensitive or RT-resistant BTICs from normal human astrocytes (NHAs) under hypoxic conditions. Methods: We used a Cytation5 Cell Imager to quantify MU by BTICs from NHAs in direct contact under normal (20%) or hypoxic oxygen tensions (5%). We obtained RT-sensitive (JX14P) patient-derived xenograft BTICs and generated a paired acquired-resistant (JX14P-RT) line. This was achieved by implanting primary tumors into flanks of athymic nude mice and serially treating with 6 fractions of 2Gy over 14 days for multiple passages until the median doubling time was halved. Cells were plated at a 1:1 ratio on Geltrex for 18h and exposed to 5% or 20% oxygen in serum-free media. NHAs were pre-labeled with a GFP-mitochondria tracker and BTICs were infected with a mCherry lentivirus. BTIC-MU was determined by quantifying double-positive cells in whole-well images. Viability was determined using CellTiterGlo, n = 4. Results: Time-lapse imaging revealed GFP-mitochondria transfer from NHAs to BTIC cells via TNTs stimulated by hypoxic conditions. We measured overall MU and cell viability in both BTIC lines. JX14P co-cultured with NHAs trended toward an increased MU (cell fraction) in hypoxia (Hyp) compared to Normoxia (Norm) (Norm = 32.61 ± 13, Hyp = 44.83 ± 5, P>0.167). JX14P exhibit higher cell viability (RLU) in hypoxia when mono- or co-cultured with NHAs (Mono: Norm = 10275 ± 901, Hyp = 12599 ± 579, P<0.0039, Co: Norm = 5415 ± 664, Hyp = 8341 ± 700, P<0.0001). JX14P-RT co-cultured with NHAs show a trend for more MU in hypoxia compared to Normoxia (Norm = 25.90 ± 12, Hyp = 38.12 ± 12, P>0.167). Compared to monoculture, JX14PRT exhibits higher cell viability in co-culture with NHAs under hypoxia (Mono: Norm = 9721 ± 255, Hyp = 10011 ± 1462, P>0.998, Co: Norm = 4928 ± 664, Hyp = 7805 ± 944, P<0.005). Conclusions: RT-sensitive or -resistant BTICs cocultured with NHAs exhibit increased cell viability under acute hypoxia compared to Normoxia with a trend toward increased MU. Results indicate a potential protective effect following direct interaction with NHAs under hypoxia. We are further exploring metabolic changes in each cell type following mitochondrial exchange. Citation Format: Lauren C. Nassour-Caswell, Nicholas J. Eustace, Christian T. Stackhouse, Hasan Alrefai, Patricia H. Hicks, Taylor L. Schanel, Joshua C. Anderson, Andee M. Beierle, Christopher D. Willey. Glioblastoma brain tumor-initiating cells are protected from hypoxia when co-cultured with normal human astrocytes revealing a potential role for mitochondrial transfer via tunneling nanotubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1273.

Tunneling nanotubes (TNTs) are newly discovered tubular structures between two distant cells that facilitate the intercellular exchange of signals and components. Recent reports show that mesenchymal stem cells (MSCs) can rescue injured target cells and promote recovery from a variety of stresses via TNT-mediated mitochondrial transfer. In this study, we explored how TNTs form between bone marrow MSCs and endothelial cells (ECs) by using a human umbilical cord vein endothelial cell (HUVEC) model. TNT formation between MSCs and HUVECs could be induced by treating HUVECs with cytarabine (Ara-C), and human bone marrow mesenchymal stem cells (hBMMSCs) could transfer mitochondria to injured HUVECs through TNTs. Mitochondrial transfer from hBMMSCs to HUVECs via TNTs rescued the injured HUVECs by reducing apoptosis, promoting proliferation and restoring the transmembrane migration ability as well as the capillary angiogenic capacity of HUVECs. This study provides novel insights into the cell–cell communication between MSCs and ECs and supports the results of prior studies indicating that ECs promote hematopoietic regeneration. An improved understanding of MSC-EC cross-talk will promote the development of MSC-directed strategies for improving EC function and hematopoietic system regeneration following myelosuppressive and myeloablative injuries.

Mesenchymal stromal cells (MSC) have been reported to improve bacterial clearance in preclinical models of Acute Respiratory Distress Syndrome (ARDS) and sepsis. The mechanism of this effect is not fully elucidated yet. The primary objective of this study was to investigate the hypothesis that the antimicrobial effect of MSC in vivo depends on their modulation of macrophage phagocytic activity which occurs through mitochondrial transfer. We established that selective depletion of alveolar macrophages (AM) with intranasal (IN) administration of liposomal clodronate resulted in complete abrogation of MSC antimicrobial effect in the in vivo model of Escherichia coli pneumonia. Furthermore, we showed that MSC administration was associated with enhanced AM phagocytosis in vivo. We showed that direct coculture of MSC with monocyte‐derived macrophages enhanced their phagocytic capacity. By fluorescent imaging and flow cytometry we demonstrated extensive mitochondrial transfer from MSC to macrophages which occurred at least partially through tunneling nanotubes (TNT)‐like structures. We also detected that lung macrophages readily acquire MSC mitochondria in vivo, and macrophages which are positive for MSC mitochondria display more pronounced phagocytic activity. Finally, partial inhibition of mitochondrial transfer through blockage of TNT formation by MSC resulted in failure to improve macrophage bioenergetics and complete abrogation of the MSC effect on macrophage phagocytosis in vitro and the antimicrobial effect of MSC in vivo. Collectively, this work for the first time demonstrates that mitochondrial transfer from MSC to innate immune cells leads to enhancement in phagocytic activity and reveals an important novel mechanism for the antimicrobial effect of MSC in ARDS. Stem Cells 2016;34:2210–2223

Abstract Broad applications of cobalt nanoparticles (CoNPs) have raised increased concerns regarding their potential toxicity. However, the underlining mechanisms of their toxicity have yet to be characterized. Here, we demonstrated that CoNPs reduced cell viability and induced membrane leakage. CoNPs induced oxidative stress, as indicated by the generation of reactive oxygen species (ROS) secondary to the increased expression of hypoxia-induced factor 1 alpha. Moreover, CoNPs led to mitochondrial damage, including generation of mitochondrial ROS, reduction in ATP content, morphological damage and autophagy. Interestingly, exogenous mitochondria were observed between neurons and astrocytes upon CoNPs exposure. Concomitantly, tunneling nanotubes (TNTs)-like structures were observed between neurons and astrocytes upon CoNPs exposure. These structures were further verified to be TNTs as they were found to be F-actin rich and lacking tubulin. We then demonstrated that TNTs were utilized for mitochondrial transfer between neurons and astrocytes, suggesting a novel crosstalk phenomenon between these cells. Moreover, we found that the inhibition of TNTs (using actin-depolymerizing drug latrunculin B) intensified apoptosis triggered by CoNPs. Therefore, we demonstrate, for the first time, that the inhibition of intercellular mitochondrial transfer via TNTs aggravates CoNPs-induced cellular and mitochondrial toxicity in neuronal cells, implying a novel intercellular protection mechanism in response to nanoparticle exposure.

Tunneling nanotubes (TNTs) are small membranous tubes of 50–1000 nm diameter observed to connect cells in culture. Transfer of subcellular organelles through TNTs was observed in vitro and in vivo, but the formation and significance of these structures is not well understood. A polydimethylsiloxane biochip-based coculture model was devised to constrain TNT orientation and explore both TNT-formation and TNT-mediated mitochondrial transfer. Two parallel microfluidic channels connected by an array of smaller microchannels enabled localization of stem cell and cardiomyocyte populations while allowing connections to form between them. Stem cells and cardiomyocytes were deposited in their respective microfluidic channels, and stem cell-cardiomyocyte pairs were formed via the microchannels. Formation of TNTs and transfer of stained mitochondria through TNTs was observed by 24 h real-time video recording. The data show that stem cells are 7.7 times more likely to initiate contact by initial extension of filopodia. By 24 h, 67% of nanotube connections through the microchannels are composed of cardiomyocyte membrane. Filopodial extension and retraction by stem cells draws an extension of TNTs from cardiomyocytes. MitoTracker staining shows that unidirectional transfer of mitochondria between stem cell-cardiomyocyte pairs invariably originates from stem cells. Control experiments with cardiac fibroblasts and cardiomyocytes show little nanotube formation between homotypic or mixed cell pairs and no mitochondrial transfer. These data identify a novel biological process, unidirectional mitochondrial transfer, mediated by heterotypic TNT connections. This suggests that the enhancement of cardiomyocyte function seen after stem-cell injection may be due to a bioenergetic stimulus provided by mitochondrial transfer.

Zika virus (ZIKV) is unique among orthoflaviviruses in its vertical transmission capacity in humans, yet the underlying mechanisms remain incompletely understood. Here, we show that ZIKV induces tunneling nanotubes (TNTs) in placental trophoblasts which facilitate transfer of viral particles, proteins, mitochondria, and RNA to neighboring uninfected cells. TNT formation is driven exclusively via ZIKV non-structural protein 1 (NS1). Specifically, the N-terminal 1-50 amino acids of membrane-bound ZIKV NS1 are necessary for triggering TNT formation in host cells. Trophoblasts infected with TNT-deficient ZIKVΔTNT mutant virus elicited a robust antiviral IFN-λ 1/2/3 response relative to WT ZIKV, suggesting TNT-mediated trafficking allows ZIKV cell-to-cell transmission camouflaged from host defenses. Using affinity purification-mass spectrometry of cells expressing wild-type NS1 or non-TNT forming NS1, we found mitochondrial proteins are dominant NS1-interacting partners. We demonstrate that ZIKV infection or NS1 expression induces elevated mitochondria levels in trophoblasts and that mitochondria are siphoned via TNTs from healthy to ZIKV-infected cells. Together our findings identify a stealth mechanism that ZIKV employs for intercellular spread among placental trophoblasts, evasion of antiviral interferon response, and the hijacking of mitochondria to augment its propagation and survival and offers a basis for novel therapeutic developments targeting these interactions to limit ZIKV dissemination. Michita et al. show that Zika virus (ZIKV) NS1 induces tunneling nanotubes (TNTs) in placental cells, which facilitate viral spread and transport of mitochondria in placental cells. Infection with a NS1 mutant ZIKV not inducing TNTs results in a higher interferon response than wild-type ZIKV infection.

Tyrosine kinase inhibitors (TKIs) are highly effective in treating chronic myeloid leukemia (CML), but drug resistance remains a significant challenge. Our study aims to explore the impact of bone marrow microenvironment (BMM) mesenchymal stem cells (MSCs) on TKI therapy. Using a CML mouse model and cell lines, we found that CML cells extensively form tunneling nanotubes (TNTs) with MSCs, with unidirectional mitochondrial transfer from CML cells to MSCs via TNTs. Additionally, CML cells co-cultured with MSCs exhibited increased drug resistance. Following TKI treatment, CML cell metabolism was suppressed, and reactive oxygen species (ROS) levels were significantly reduced; however, with continued treatment, metabolism and ROS levels gradually recovered. In co-culture experiments with MSCs and CML cells, we similarly observed a significant reduction in CML cell ROS levels, accompanied by increased TKI resistance, regardless of prior TKI exposure, indicating that MSCs support CML cells by influencing their metabolism. Compared to normal cells, CML cells have higher ROS levels, and treatment with the ROS inhibitor N-acetylcysteine (NAC) increased CML cell resistance. However, in the MSC-CML co-culture system, NAC treatment significantly enhanced CML cell sensitivity to TKI drugs. We also observed the gradual formation of TNTs between CML cells and MSCs, facilitating mutual mitochondrial transfer. Therefore, we propose that during CML treatment, MSCs modulate CML cell metabolism and restore TKI sensitivity by transferring mitochondria via TNTs. Our study reveals the critical role of BMM cells in supporting CML cells through TNTs, suggesting that targeting this interaction could improve therapeutic outcomes for CML.

Recent studies have proved the role of autophagy in mesenchymal stem cell (MSCs) function and regenerative properties. How and by which mechanism autophagy modulation can affect the juxtacrine interaction of MSCs should be addressed. Here, the role of autophagy was investigated in the formation of tunneling nanotubes (TNTs) and homotypic mitochondrial donation. MSCs were incubated with 15 µM Metformin (Met) and/or 3 µM 3-methyladenine (3-MA) for 48 h. The formation of TNTs was assessed using bright-field and SEM images. The mitochondria density and ΔΨ values were monitored using flow cytometry analysis. Using RT-PCR and protein array, the close interaction and shared mediators between autophagy, apoptosis, and Wnt signaling pathways were also monitored. The total fatty acid profile was assessed using gas chromatography. Data indicated the increase of TNT length and number, along with other cell projections after the induction of autophagy while these features were blunted in 3-MA-treated MSCs (p < 0.05). Western blotting revealed the significant reduction of Rab8 and p-FAK in 3-MA-treated MSCs (p < 0.05), indicating the inhibition of TNT assembly and vesicle transport. Likewise, the stimulation of autophagy increased autophagic flux and mitochondrial membrane integrity compared to 3-MA-treated MSCs. Despite these findings, protein levels of mitochondrial membrane Miro1 and 2 were unchanged after autophagy inhibition/stimulation (p > 0.05). We found that the inhibition/stimulation of autophagy can affect the protein, and transcription levels of several mediators related to Wnt and apoptosis signaling pathways involved in different cell bioactivities. Data confirmed the profound increase of mono and polyunsaturated/saturated fatty acid ratio in MSCs exposed to autophagy stimulator. In summary, autophagy modulation could affect TNT formation which is required for homotypic mitochondrial donation. Thus, the modulation of autophagy creates a promising perspective to increase the efficiency of cell-based therapies.

No abstract available

Cell-to-cell mitochondria transfer via tunneling nanotubes (TNTs) has recently been revealed as a spontaneous way to protect damaged cells. Previously, we have reported mesenchymal stem cells (MSCs) can rescue retinal ganglion cell and corneal epithelium through intercellular mitochondrial trafficking. Mitochondrial damage and oxidative stress in corneal epithelial cells are vital in dry eye disease (DED). However, whether intercellular mitochondrial transfer is involved in the pathological and repair process of DED is currently unknown. Therefore, in this study, we designed a coculture system to evaluate the role of intercellular mitochondrial transfer between human corneal epithelial cells (CEC) in DED. In addition, we successfully discovered the ROCK inhibitor, Y-27632 as an intensifier to improve the efficiency of intercellular mitochondrial transport. As expected, the enhanced mitochondrial transfer promotes the regeneration of CECs. Moreover, through further exploration of mechanisms, it was demonstrated that F-actin-mediated cell morphological changes and cytoskeletal remodeling may be potential mechanisms for Y-27632 to induce mitochondrial metastasis. In conclusion, we established a new method for cell repair in DED that healthy CEC offered mitochondria to damaged CEC, providing a new insight into the cellular mechanism of corneal epithelium homeostatic regenerative therapeutics in DED.

Microglia are crucial for maintaining brain health and neuron function. Here, we report that microglia establish connections with neurons using tunneling nanotubes (TNTs) in both physiological and pathological conditions. These TNTs facilitate the rapid exchange of organelles, vesicles, and proteins. In neurodegenerative diseases like Parkinson's and Alzheimer's disease, toxic aggregates of alpha-synuclein (α-syn) and tau accumulate within neurons. Our research demonstrates that microglia use TNTs to extract neurons from these aggregates, restoring neuronal health. Additionally, microglia share their healthy mitochondria with burdened neurons, reducing oxidative stress and normalizing gene expression. Disrupting mitochondrial function with antimycin A before TNT formation eliminates this neuroprotection. Moreover, co-culturing neurons with microglia and promoting TNT formation rescues suppressed neuronal activity caused by α-syn or tau aggregates. Notably, TNT-mediated aggregate transfer is compromised in microglia carrying Lrrk22(Gly2019Ser) or Trem2(T66M) and (R47H) mutations, suggesting a role in the pathology of these gene variants in neurodegenerative diseases.

BACKGROUND Prevention of thymus atrophy during menopause is of great significance for improving the immune function and overall health of menopausal women. Epimedin C is one of the major bioactive compounds in Herba Epimedii, a traditional herbal medicine for the treatment of menopausal syndrome in China, but the action and mechanism of Epimedin C in the treatment of menopausal thymus atrophy remains unclear. OBJECTIVE To study the effect of Epimedin C on thymus atrophy in 4-vinylcyclohexene diepoxide (4-VCD) induced mimetic-menopausal mice and explore its mechanism from new perspectives of tunneling nanotubes (TNTs) formation and mitochondrial transfer (MitoT). METHOD The effects of Epimedin C in the 4-VCD induced menopause-like phenotype in mice were observed, and the thymic output function was evaluated by the quantitative detection of T cell receptor excision circles (TRECs). The structure of the thymus was observed by H&E. The arrangement and quantity of different cell subpopulations of thymic epithelial cells (TECs) and thymocytes were detected by multiple fluorescent staining and flow cytometry. Mitochondrial morphology was observed with transmission electron microscopy. LC-MS/MS was used to analyze and identify the differential protein expression in thymus before and after Epimedin C treatment. Actin polymerization inhibitor was used to verify the possible mechanism of Epimedin C. The treadmilling-balance of actin, TNTs formation, and MitoT processes were observed by specific fluorescent probe labeling. The interaction between G-actin, Thymosin β4 (Tβ4), and Epimedin C were studied by protein cross-linking assay. RESULTS Epimedin C significantly increased the thymus weight and the area of the thymus medulla, increased the grip strength and bone strength in 4-VCD induced mimetic-menopausal mice, and enhanced ovarian secretion function. It could affect the thymus output, increase CK5 and CK8 expression, maintain the reticular structure of TECs, inhibit the differentiation of thymocytes into double positive cells (CD4+CD8+) and CD4SP (CD3+TCR β+CD4+CD8-) cells. Epimedin C promoted the conversion of G-actin to F-actin and accelerated MitoT via stimulating the TNTs formation, which related with the downregulation of Tβ4 and obstruction to the formation of Tβ4-G-actin complex. CONCLUSION Epimedin C can promote TEC activity in 4-VCD induced mimetic-menopausal mice by decreasing the expression of Tβ4, inhibiting the binding of Tβ4 to G-actin, promoting the F-actin polymerization and the TNTs-depended MitoT.

BACKGROUND Cerebral ischemic events, comprising of excitotoxicity, reactive oxygen production, and inflammation, adversely impact the metabolic-redox circuit in highly active neuronal metabolic profile which maintains energy-dependent brain activities. Therefore, we investigated neuro-regenerative potential of melatonin (Mel), a natural biomaterial secreted by pineal gland. METHODS We specifically determined whether Mel could influence tunneling nanotubes (TNTs)-mediated transfer of functional mitochondria (Mito) which in turn may alter membrane potential, oxidative stress and apoptotic factors. In vitro studies assessed the effects of Mito on levels of cytochrome C, mitochondrial transfer, reactive oxygen species, membrane potential and mass, which were all further enhanced by Mel pre-treatment, whereas in vivo studies examined brain infarct area (BIA), neurological function, inflammation, brain edema and integrity of neurons and myelin sheath in control, ischemia stroke (IS), IS + Mito and IS + Mel-Mito group rats. RESULTS Results showed that Mel pre-treatment significantly increased mitochondrial transfer and antioxidants, and inhibited apoptosis. Mel-pretreated Mito also significantly reduced BIA with improved neurological function. Apoptotic, oxidative-stress, autophagic, mitochondrial/DNA-damaged biomarkers indices were also improved. CONCLUSION Conclusively, Mel is a potent biomaterial which could potentially impart neurogenesis through repairing impaired metabolic-redox circuit via enhanced TNT-mediated mitochondrial transfer, anti-oxidation, and anti-apoptotic activities in ischemia.

Glioblastoma (GBM) is well known to interact with surrounding tumor and stromal cells in a variety of ways to maintain its pathogenicity. Emerging evidence has identified tunneling nanotubes (TNTs), dynamic actin-rich structures that mediate direct intercellular communication, as critical facilitators of GBM progression and therapeutic resistance, yet mediators of TNT formation remain poorly understood. Myristorylated Alanine-Rich C Kinase Substrate (MARCKS), an actin-binding protein regulated by PKC, may influence TNT dynamics. We hypothesized that MARCKS effector domain (ED) phosphorylation via PKC regulates TNT formation and mitochondrial transfer between GBM cells and astrocytes, contributing to chemoresistance. We employed serum-free TNT-promoting co-culture models of PTEN-null GBM cells (patient-derived xenograft JX14 cells or U87) and normal human astrocytes (NHAs), using immunofluorescence confocal microscopy to detect MARCKS localization, and quantitative assays to measure TNT number, length, and mitochondria transfer. Pharmacological modulation of PKC with phorbol ester agonist (PMA) or inhibitor (Enzastaurin), and treatment with a MARCKS ED peptide (MED2), assessed effects on TNTs. Doxycycline-inducible U87 MARCKS ED mutants (WT, pseudo-phosphorylated [PP], non-phosphorylatable [NP]) were used to dissect MARCKS phosphorylation roles. CGGA database analysis examined correlations between MARCKS and TNT-related genes in primary classical GBM. JX14 co-cultured with NHAs exhibited increased temozolomide resistance. MARCKS localized to heterotypic TNTs, and MED2 reduced TNT number, length, and mitochondrial transfer. PKC activation increased TNT formation and transfer, while inhibition produced opposite effects. U87-PP cells displayed abundant TNTs and transfer similar to PKC activation, whereas U87-NP cells showed reduced TNTs. MARCKS positively correlated with MyosinX, ACTB, ACTG1, and RHOT1 in GBM patient data. MARCKS ED phosphorylation via PKC promotes functional TNT formation and mitochondrial trafficking between GBM cells and astrocytes, contributing to chemoresistance. Targeting MARCKS or PKC may represent a novel strategy to disrupt TNT-mediated resistance mechanisms in GBM.

Lung injury has become a critical clinical problem that urgently requires resolution due to its high morbidity, high mortality, and the limitations of existing treatment methods. Mitochondrial dysfunction, as the core mechanism of lung injury, promotes disease progression through energy metabolism imbalances, oxidative stress, and exacerbated inflammatory responses. Recent studies have found that intercellular mitochondrial transfer, acting as a “transcellular rescue” mechanism, can deliver functional mitochondria through pathways such as tunneling nanotubes, exosome. This process provides a novel approach to replenish energy for damaged cells, regulate inflammation, and repair tissues. In various lung injury models, mitochondrial transfer/transplantation has been shown to improve alveolar-capillary barrier function, reduce collagen deposition, inhibit the release of inflammatory factors, and restore mitochondrial membrane potential. This is particularly evident in conditions such as acute lung injury, pulmonary fibrosis, acute respiratory distress syndrome, and chronic obstructive pulmonary disease, where it shows significant therapeutic potential. The combination of diverse delivery methods and multi-source mitochondria provide a flexible strategy for clinical application. In summary, mitochondrial transfer, as an emerging intercellular communication and rescue mechanism, provides a promising new direction for the precision treatment of lung injury.

Mitochondria are essential organelles that regulate various biological processes including metabolism. Beyond their intracellular functions, intercellular mitochondrial transfer has emerged as a novel mechanism of intercellular communication. Notably, an increasing number of studies have reported its occurrence in the tumor microenvironment (TME), where it contributes to tumor progression. While previous studies largely characterized cancer cells as recipients of mitochondria, Cangkrama et al. demonstrated that cancer cells donate their mitochondria to fibroblasts via tunneling nanotubes. The mitochondrial transfer to fibroblasts reprogrammed them into cancer‐associated fibroblasts exhibiting combined myofibroblastic and inflammatory characteristics, with enhanced oxidative metabolism and pro‐tumorigenic activity. Our group has identified mitochondrial ‘hijack’ from cancer cells to tumor‐infiltrating lymphocytes, leading to an impaired antitumor immunity. These insights underscore the need to recognize cancer cells as mitochondrial donors in the TME capable of reshaping the TME to their own advantage, resembling a dynastic expansion strategy that exerts influence by strategically placing lineages.

Background: The disruption of mitochondrial homeostasis in acute kidney injury (AKI) is an important factor that drives persistent renal dysfunction. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have shown great therapeutic potential in AKI, but insufficient specificity of targeting the impaired mitochondrial function. Herein, we developed an engineered nitric oxide (NO)-primed MSC-EVs (pEVs) to restore mitochondrial homeostasis for AKI therapy. Methods: A cisplatin-induced AKI model was established to investigate the therapeutic effects of MSC-EVs. Proteomic and Western blot analyses compared mitochondrial cargos and functional assays included mitochondrial complex I activity and Adenosine triphosphate (ATP) quantification. Mitochondrial transfer was tracked using flow cytometry and confocal imaging. Mitochondrial dynamics, oxidative stress, and apoptosis were evaluated through ATP measurement, western blotting and rotenone-mediated respiratory chain inhibition. Results: Our data indicated that pEVs outperformed cEVs in restoring renal function and histopathology. Additionally, a reduction in mitochondria-associated oxidative stress and cell death was observed. Proteomic profiling revealed that NO priming enriched pEVs with mitochondrial complex I components, which directly enhanced their bioenergetic capacity, as evidenced by higher mitochondrial complex I activity and elevated ATP production compared to cEVs. In vivo tracking confirmed targeted delivery of pEV-derived mitochondrial contents to renal tubular cells, reducing mitochondrial reactive oxygen species (ROS) and restoring mitochondrial mass. Crucially, mitochondria-depleted pEVs abolished these therapeutic effects, establishing mitochondrial cargos as the primary therapeutic driver. Furthermore, pEVs activated a pro-survival cascade in recipient cells, showing superior efficacy in promoting mitochondrial biogenesis, dynamics, and mitophagy, thereby restoring renal mitochondrial homeostasis. Conclusion: Our study elucidated a mitochondria-targeted therapeutic strategy enabled by engineered EVs that deliver functional cargo to restore mitochondrial homeostasis. These advances provide transformative potential for AKI and other mitochondrial disorders.

BACKGROUND Platelet activation causes the release of extracellular vesicles, of which a small proportion contain respiratory competent mitochondria. Mitochondria are integral for energy production and in the regulation of apoptotic pathways, however the existence of extracellular mitochondria highlights a potential new role in intercellular communication. Indeed, mitochondrial transfer has gained significant research interest in recent years, highlighting mechanisms through which cellular function and metabolism may be augmented. OBJECTIVE To characterise changes in neutrophil function and phenotype that occur because of interactions with mitochondria-positive or mitochondria-negative platelet extracellular vesicles. METHODS Platelet extracellular vesicle subpopulations were separated based on mitochondrial content using cell sorting and subsequently incubated with isolated neutrophils. Alterations in surface receptor repertoire were analysed by flow cytometry, and functional characterisation performed by immunofluorescence, flow cytometry and Seahorse XF metabolic flux assays. RESULTS In this work we demonstrate that platelet extracellular vesicles containing mitochondria interact with and are internalised by neutrophils, subsequently increasing their metabolic capacity. These interactions promote changes in neutrophil receptor repertoire, indicative of enhanced neutrophil activation, adhesion and migration pathways. The internalisation of platelet mitochondria renders neutrophils unable to subsequently engulf bacteria, demonstrating reduced phagocytic capacity, but enhances the formation of neutrophil extracellular traps, both alone and in the presence of additional stimuli. CONCLUSIONS Our findings show that platelet mitochondria released in extracellular vesicles can be transferred into neutrophils, altering their metabolic function and activity. This research highlights an important role for platelet mitochondria as intercellular communicators and modulators of inflammatory and immune responses.

Background/Objectives: This study aimed to explore the therapeutic potential of umbilical mesenchymal stem cell-derived apoptotic vesicles (UMSC-apoVs) in a 5-Fluorouracil (5-FU)-induced impairment in skin wound healing. Methods: UMSC-apoVs were isolated from UMSCs using differential centrifugation after the induction of apoptosis. A murine model was established by administering 5-FU via intravenous tail injection, followed by full-thickness skin wound creation. Mice received local injections of UMSC-apoVs at the lesion site. Wound healing was evaluated based on wound closure rates, histological analysis, and in vivo/in vitro functional assays. Rotenone (Rot)-pretreated UMSC-apoVs were used to explore the role of mitochondrial transfer between skin mesenchymal stem cells (SMSCs) and UMSC-apoVs in wound healing. Results: UMSC-apoVs significantly accelerated wound healing in 5-FU-treated mice, as demonstrated by enhanced wound closure rates and histological findings of reduced inflammatory infiltration and increased collagen deposition. UMSC-apoVs transferred mitochondria to SMSCs, enhancing viability, proliferation, and migration while reducing reactive oxygen species (ROS) production in SMSCs. Rot pretreatment inhibited the therapeutic effects of UMSC-apoVs on wound healing by inducing mitochondrial dysfunction in UMSC-apoVs. Conclusions: Our findings indicate that UMSC-apoVs improve 5-FU-induced impaired skin wound healing by facilitating mitochondrial transfer, suggesting a novel therapeutic strategy for alleviating chemotherapy-induced impairment in wound healing.

Platelet‐rich plasma (PRP) is a safe, autologous plasma component abundant in cytokines and extracellular vesicles, frequently applied to treat inflammatory disorders. Although PRP demonstrates potential for psoriasis therapy, its underlying mechanism remains insufficiently understood. In this study, various PRP constituents were evaluated in an imiquimod (IMQ)‐induced mouse model of psoriasis. PRP, platelet‐derived extracellular vesicles (PEVs), and platelet‐poor plasma (PPP) were isolated from mice and administered subcutaneously. The data showed that PEVs, rather than PPP, served as the principal anti‐psoriatic factor. Furthermore, RNA sequencing and flow cytometry revealed that PEVs markedly suppressed M1 polarisation of macrophages, thereby mitigating psoriatic‐like inflammation. In vitro, PEVs delivered encapsulated mitochondria to RAW264.7 cells in a concentration‐dependent manner. These functional organelles enhanced oxidative phosphorylation and suppressed glycolysis, driving a metabolic shift favouring an anti‐inflammatory phenotype and attenuating the inflammatory response. In conclusion, PEVs emerge as the primary PRP component responsible for inflammatory suppression in psoriasis. Notably, mitochondria transfer mediated by PEVs underscores a promising therapeutic avenue and provides novel insight into the role of platelet derivatives in inflammatory diseases.

Mitochondrial damage is a critical pathological factor in various forms of tissue injury, and specific therapies with high biosafety are desirable. Inspired by the natural role of extracellular vesicles (EVs) in regulating mitochondrial metabolism, we report that healthy tissue-derived mitochondria-rich EVs (Ti-mitoEVs) can boost mitochondrial biogenesis for regenerative medicine. Ti-mitoEVs that contain abundant functional mitochondria can be highly efficiently isolated from muscles via an optimized method. In vitro, Ti-mitoEV treatment increased mitochondrial biogenesis and reduced mitochondrial damage in recipient cells, and these effects occurred at least partly via mitochondrial genome transfer. In vivo, Ti-mitoEV treatment attenuated diverse types of tissue injury (e.g., muscle and kidney) by rescuing mitochondrial injury and its associated inflammation. As natural nanovesicles, the therapeutic potency of mitoEVs can be further improved by integrating them with other engineering methods. This study highlights the promising role of Ti-mitoEVs in boosting mitochondrial biogenesis, positioning them as potential therapies for treating various types of tissue injury characterized by mitochondrial damage.

Background Autoimmune hepatitis (AIH) is a serious liver disease characterized by immune disorders, particularly effector T-cell overactivation. This study aimed to explore the therapeutic effect and underlying mechanism of mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment on CD4+ T-cell overactivation and liver injury in AIH. Methods The metabolic changes of CD4+ T cells were assayed in human AIH and mouse hepatitis models. The liver protective effect of MSC-EVs was evaluated by transaminase levels, liver histopathology and inflammation. The effect of MSC-EVs on the metabolic state of CD4+ T cells was also explored. Results Enhanced glycolysis (eg, ~1.5-fold increase in hexokinase 2 levels) was detected in the CD4+ T cells of AIH patient samples and mouse hepatitis models, whereas the inhibition of glycolysis decreased CD4+ T-cell activation (~1.8-fold decrease in CD69 levels) and AIH liver injury (~6-fold decrease in aminotransferase levels). MSC-EV treatment reduced CD4+ T-cell activation (~1.5-fold decrease in CD69 levels) and cytokine release (~5-fold decrease in IFN-γ levels) by reducing glycolysis (~3-fold decrease) while enhancing mitochondrial oxidative phosphorylation (~2-fold increase in maximal respiration) in such cells. The degree of liver damage in AIH mice was ameliorated after MSC-EV treatment (~5-fold decrease in aminotransferase levels). MSC-EVs carried abundant mitochondrial proteins and might transfer them to metabolically reprogram CD4+ T cells, whereas disrupting mitochondrial transfer impaired the therapeutic potency of MSC-EVs in activated CD4+ T cells. Conclusion Disordered glucose metabolism promotes CD4+ T-cell activation and associated inflammatory liver injury in AIH models, which can be reversed by MSC-EV therapy, and this effect is at least partially dependent on EV-mediated mitochondrial protein transfer between cells. This study highlights that MSC-EV therapy may represent a new avenue for treating autoimmune diseases such as AIH.

Periodontitis is the leading cause of tooth loss in adults due to progressive bone destruction, which is closely related to the dysfunction of bone mesenchymal stem cells (BMSCs). Existing evidence suggests that mitochondrial disorders are associated with periodontitis. However, whether mitochondrial dysregulation contributes to the osteogenic impairment of BMSCs and the underlying mechanisms remain unclear. Macrophages have been shown to communicate extensively with BMSCs in periodontitis. Recent studies have reported a novel manner of cellular communication in which mitochondria-rich extracellular vesicles（MEVs） transfer mitochondria from parent cells to recipient cells, playing a role in both physiological and pathological conditions. Therefore, we aimed to investigate the role of MEVs in orchestrating the crosstalk between macrophages and BMSCs in periodontitis to formulate management strategies for bone loss. Our results revealed that macrophages underwent significant mitochondrial dysfunction and inflammation in periodontitis and that MEVs derived from these macrophages played a role in alveolar bone destruction. Furthermore, cell imaging showed that inflammatory macrophages packaged numerous damaged mitochondria into MEVs, and the entry of these impaired mitochondria into BMSCs disrupted mitochondrial dynamics and hindered donut-shaped mitochondria formation, leading to osteogenic dysfunction. Proteomic analysis revealed that the proteins enriched in macrophage-derived MEVs were largely related to mitochondria and the formation and transport of vesicles. Additionally, we found that MEVs from macrophages significantly increased lipocalin 2 (LCN2) in BMSCs in periodontitis and that LCN2 perturbed mitochondrial morphological changes in BMSCs by inducing the degradation of OMA1 and accumulation of OPA1, resulting in osteogenesis impairment in BMSCs. Inhibition of LCN2 rescued the osteogenic dysfunction of BMSCs and alveolar bone loss in periodontitis. The transfer of mitochondria to BMSCs via MEVs exacerbates alveolar bone resorption through LCN2/OMA1/OPA1 signaling in periodontitis. Inhibition of LCN2 alleviates inflammatory bone loss, suggesting a promising therapeutic strategy for periodontitis.

Abstract Extracellular vesicles (EVs) exert a significant influence not only on the pathogenesis of diseases but also on their therapeutic interventions, contingent upon the variances observed in their originating cells. Mitochondria can be transported between cells via EVs to promote pathological changes. In this study, we found that EVs derived from M1 macrophages (M1‐EVs), which encapsulate inflammatory mitochondria, can penetrate pancreatic beta cells. Inflammatory mitochondria fuse with the mitochondria of pancreatic beta cells, resulting in lipid peroxidation and mitochondrial disruption. Furthermore, fragments of mitochondrial DNA (mtDNA) are released into the cytosol, activating the STING pathway and ultimately inducing apoptosis. The potential of adipose‐derived stem cell (ADSC)‐released EVs in suppressing M1 macrophage reactions shows promise. Subsequently, ADSC‐EVs were utilized and modified with an F4/80 antibody to specifically target macrophages, aiming to treat ferroptosis of pancreatic beta cells in vivo. In summary, our data further demonstrate that EVs secreted from M1 phenotype macrophages play major roles in beta cell ferroptosis, and the modified ADSC‐EVs exhibit considerable potential for development as a vehicle for targeted delivery to macrophages.

Studies have shown that oxidative stress and its resistance plays important roles in the process of tumor metastasis, and mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage is an important molecular event in oxidative stress. In lung cancer, the normal fibroblasts (NFs) are activated as cancer-associated fibroblasts (CAFs), and act in the realms of the tumor microenvironment (TME) with consequences for tumor growth and metastasis. However, its activation mechanism and whether it participates in tumor metastasis through antioxidative stress remain unclear. The role and signaling pathways of tumor cell derived extracellular vesicles (EVs) activating NFs and the characteristic of induced CAFs (iCAFs) were measured by the transmission electron microscopy, nanoparticle tracking analysis, immunofluorescence, collagen contraction assay, quantitative PCR, immunoblotting, luciferase reporter assay and mitochondrial membrane potential detection. Mitochondrial genome and single nucleotide polymorphism sequencing were used to investigate the transport of mtDNA from iCAFs to ρ0 cells, which were tumor cells with mitochondrial dysfunction caused by depletion of mtDNA. Further, the effects of iCAFs on mitochondrial function, growth and metastasis of tumor cells were analysed in co-culture models both in vitro and in vivo, using succinate dehydrogenase, glutathione and oxygen consumption rate measurements, CCK-8 assay, transwell assay, xenotransplantation and metastasis experiments as well as in situ hybridization and immunohistochemistry. Our findings revealed that EVs derived from high-metastatic lung cancer cells packaged miR-1290 that directly targets MT1G, leading to activation of AKT signaling in NFs and inducing NFs conversion to CAFs. The iCAFs exhibit higher levels of autophagy and mitophagy and more mtDNA release, and reactive oxygen species (ROS) could further promote this process. After cocultured with the conditioned medium (CM) of iCAFs, the ρ0 cells may restore its mitochondrial function by acquisition of mtDNA from CAFs, and further promotes tumor metastasis. These results elucidate a novel mechanism that CAFs activated by tumor-derived EVs can promote metastasis by transferring mtDNA and restoring mitochondrial function of tumor cells which result in resistance of oxidative stress, and provide a new therapeutic target for lung cancer metastasis.

Mitochondria transfer is a spontaneous process that releases functional mitochondria to damaged cells via different mechanisms including extracellular vesicle containing mitochondria (EV-Mito) to restore mitochondrial functions. However, the limited EV-Mito yield makes it challenging to supply a sufficient quantity of functional mitochondria to damaged cells, hindering their application in mitochondrial diseases. Here, we show that the release of EV-Mito from mesenchymal stem cells (MSCs) is regulated by a calcium-dependent mechanism involving CD38 and IP3R signaling (CD38/IP3R/Ca2+ pathway). Activating this pathway through our non-viral gene engineering approach generates super donor MSCs which produce Super-EV-Mito with a threefold increase in yield compared to Ctrl-EV-Mito from normal MSCs. Leber’s hereditary optic neuropathy (LHON), a classic mitochondrial disease caused by mtDNA mutations, is used as a proof-of-concept model. Super-EV-Mito rescues mtDNA defects and alleviates LHON-associated symptoms in LHON male mice. This strategy offers a promising avenue for enhancing mitochondria transfer efficiency and advancing its clinical application in mitochondrial disorders. In mitochondria transplantation, the limited activity and yield of mitochondria constrain their clinical application for mitochondrial diseases. Here, authors develop a method for producing mitochondria-enriched extracellular vesicles, which offer high-quality, abundant mitochondrial material for transplantation.

The quality control network in type 2 alveolar epithelial cells (AEC2s) is essential to respond to intrinsic and extrinsic challenges. However, the mechanisms that regulate AEC2 mitochondrial homeostasis remain unclear understood. Here, we report a role of G protein-coupled receptor class C group 5 member A (GPRC5A) in mitochondrial quality control in AEC2s through promoting mitochondrial secretion in extracellular vesicles (EVs). Utilizing mice models, we demonstrate that the disruption of GPRC5A specifically in AEC2s aggravates lung injuries. We further observe that GPRC5A deficiency in AEC2s reduces secretion of mitochondrial components in small-EVs and disrupts mitochondrial functions both in vitro and in vivo. Mechanistically, we determine that the GPRC5A-MIRO2 pathway facilitates the transfer of mitochondrial fragments into late endosomes. Collectively, our findings provide evidence of the shedding of mitochondrial components dependent on GPRC5A as a pathway of mitochondrial quality control in AEC2s, which is crucial in the maintenance of epithelial physiological activities and lung tissue homeostasis. The authors provide evidence of the secretion of mitochondrial components in extracellular vesicles dependent on GPRC5A as a pathway of mitochondrial quality control in AEC2s, which is essential for lung homeostasis maintenance.

Cell-derived extracellular vesicles (EV) are mediators of intercellular communication with increased circulating levels of endothelial cell-derived EV (EC-EV) reported in cardiovascular diseases (CVD). The EC-EV ability to elicit either detrimental or restorative effects on target EC is thought to be, in part, due to horizontal transfer of their mitochondrial cargo. To understand the role of mitochondrial cargo in EC-EV paracrine effects, large EV were collected from media of cultured human EC, and the number of mitochondria-carrying EV (mitoEV), EV mitochondrial cargo mass, and mitoEV quality/polarization were quantified. EC activation with tumor necrosis factor (TNF)-α caused an increased release rate of EV (TNF-EV), including mitoEV that carried a larger and more depolarized mitochondrial cargo, compared to EV released from control EC (C-EV). EC co-treatment with TNF-α and the mitochondria-targeted antioxidant MitoTEMPO restored both the mitochondrial cargo quality and the number of mitoEV carrying polarized mitochondria to levels similar to C-EV. TNF-EV, but not C-EV, dose-dependently upregulated inflammatory gene expression in target naïve EC. Fluorescence microscopy showed the EV mitochondrial cargo to transfer and colocalize with the target EC mitochondrial network. Mitochondrial cargo depolarization of C-EV using carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone was sufficient for those EV to trigger inflammation in target naïve EC. In conclusion, the mitochondrial redox state of donor EC regulates mitoEV mitochondrial cargo quality that, at least in part, determines their capacity to cause target EC dysfunction and promote CVD. The mitochondrial membrane potential (ΔΨm) in EC-mitoEV may be a new biomarker and therapeutic target in vascular biology and medicine.

Chronic exercise leads to systemic health benefits across tissues, including an increase in mitochondrial function in skeletal muscle. One potential mechanism is via the release of extracellular vesicles (EVs), which can transfer functional cargo to recipient cells. Previously, we have shown that using an in vitro model of exercise, chronic contractile activity (CCA)-derived EVs (CCA-EV) increased mitochondrial biogenesis in healthy myoblasts. Here, we hypothesize that CCA-EVs will have a concentration-dependent effect on mitochondrial respiration, and also regulate mitochondrial dynamics in myoblasts. C2C12 myoblasts were differentiated into myotubes, and electrically paced (3hrs/day, 4 days, 14V, C-PACE EM, IonOptix). EVs from control and CCA-stimulated myotubes were isolated from conditioned media using differential ultracentrifugation and biophysically characterized by size and concentration using TRPS (Izon). C2C12 myoblasts were treated daily for 4 days using three different concentrations: 1.0E4; 2.0E4 and 4.0E4 EVs/cell (N=4-5). Basal and maximal oxygen consumption rates (OCR) were measured using the Seahorse XFe24 (Agilent). We used live-cell fluorescence microscopy and Mitometer software to access mitochondrial movement: area/volume, displacement, distance, length, perimeter, speed and velocity. Average size was unchanged between control-EVs (123±10.8 nm) and CCA-EVs (123±11.9 nm) (p=0.9137, N=3), and 2.3-fold more CCA-EVs were released vs. control-EVs (p=0.2072, N=3). Lowest EV concentration (1.0E+4 EVs/cell, N=5) had no effect on basal (p=0.4226) and maximal (p=0.1835) OCR. Intermediate dosage of CCA-EVs (2.0E+4 EVs/cell, N=5) increased basal OCR by 20% (p=0.0037) and maximal OCR by 18% (p=0.0095), while the highest concentration (4.0E+4 EVs/cell, N=4) decreased basal OCR by 9% (p=0.0001) and maximal OCR by 29% (p=0.0009), compared to control-EVs. Mitochondrial motility (N=484-976) in myoblasts treated daily after each day of CCA showed enhanced mitochondrial dynamics with CCA-EVs: increased area/volume (p<0.0001), displacement (p<0.0001), distance (p<0.0001), mean intensity (p<0.0001), perimeter/surface area (p=0.0137) and speed (p<0.0001), compared to control-EVs. In summary, CCA-EVs induced concentration-dependent improvements in mitochondrial respiration in healthy myoblasts, concomitant with enhanced mitochondrial movement. These results open exciting avenues for the therapeutic potential of CCA-EVs in rescuing mitochondrial dysfunction. TFGS is funded by a Research Manitoba Postdoctoral Fellowship. Grants from CHRIM, CHF, DREAM and NSERC to AS funded the research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mitochondrial DNA (mtDNA) carrying certain pathogenic mutations or single nucleotide variants (SNVs) enhances the invasion and metastasis of tumor cells, and some of these mutations are homoplasmic in tumor cells and even in tumor tissues. On the other hand, intercellular transfer of mitochondria and cellular components via extracellular vesicles (EVs) and tunneling nanotubes (TNTs) has recently attracted intense attention in terms of cell-to-cell communication in the tumor microenvironment. It remains unclear whether metastasis-enhancing pathogenic mutant mtDNA in tumor cells is intercellularly transferred between tumor cells and stromal cells. In this study, we investigated whether mtDNA with the NADH dehydrogenase subunit 6 (ND6) G13997A pathogenic mutation in highly metastatic cells can be horizontally transferred to low-metastatic cells and stromal cells in the tumor microenvironment. When MitoTracker Deep Red-labeled high-metastatic Lewis lung carcinoma A11 cells carrying the ND6 G13997A mtDNA mutation were cocultured with CellLight mitochondria-GFP-labeled low-metastatic P29 cells harboring wild-type mtDNA, bidirectional transfer of red- and green-colored vesicles, probably mitochondria-related EVs, was observed in a time-dependent manner. Similarly, intercellular transfer of mitochondria-related EVs occurred between A11 cells and α-smooth muscle actin (α-SMA)-positive cancer-associated fibroblasts (CAFs, WA-mFib), macrophages (RAW264.7) and cytotoxic T cells (CTLL-2). Intercellular transfer was suppressed by inhibitors of EV release. The large and small EV fractions (L-EV and S-EV, respectively) prepared from the conditioned medium by differential ultracentrifugation both were found to contain mtDNA, although only S-EVs were efficiently incorporated into the cells. Several subpopulations had evidence of LC3-II and contained degenerated mitochondrial components in the S-EV fraction, signaling to the existence of autophagy-related S-EVs. Interestingly, the S-EV fraction contained a MitoTracker-positive subpopulation, which was inhibited by the respiration inhibitor antimycin A, indicating the presence of mitochondria with membrane potential. It was also demonstrated that mtDNA was transferred into mtDNA-less ρ0 cells after coculture with the S-EV fraction. In syngeneic mouse subcutaneous tumors formed by a mixture of A11 and P29 cells, the mitochondria-related EVs released from A11 cells reached distantly positioned P29 cells and CAFs. These results suggest that metastasis-enhancing pathogenic mtDNA derived from metastatic tumor cells is transferred to low-metastatic tumor cells and stromal cells via S-EVs in vitro and in the tumor microenvironment, inferring a novel mechanism of enhancement of metastatic potential during tumor progression.

Hepatitis B virus (HBV) is a human hepatotropic pathogen causing hepatocellular carcinoma. We recently obtained HBV‐susceptible immortalized human hepatocyte NKNT‐3 by exogenously expressing NTCP and its derived cell clones, #28.3.8 and #28.3.25.13 exhibiting different levels of HBV susceptibility. In the present study, we showed that HBV infection activated the ATM‐Chk2 signaling pathway in #28.3.25.13 cells but not in #28.3.8 cells. Both the cell culture supernatant and extracellular vesicles (EVs) derived from HBV‐infected #28.3.25.13 cells also activated the ATM‐Chk2 signaling pathway in naïve #28.3.25.13 cells. Interestingly, EVs derived from HBV‐infected #28.3.25.13 cells included higher level of mitochondrial DNA (mtDNA) than those from HBV‐infected #28.3.8 cells. Based on our results, we propose the novel model that EVs mediate the activation of ATM‐Chk2 signaling pathway by the intercellular transfer of mtDNA in HBV‐infected human hepatocyte.

BACKGROUND Autoimmune hepatitis (AIH) is an inflammatory liver disease that is associated with impaired self-tolerance. Myeloid-derived supprfessor cells (MDSCs) have been considered to exert counterregulatory effects on AIH. However, the specific mechanism underlying these effects is unclear. Herein, we investigated the efficacy and safety of MDSCs in protecting against AIH and explored the underlying mechanism. METHODS Circulating and liver MDSC expression levels in 71 AIH patients and 47 healthy control (HC) individuals were detected by flow cytometry and immunohistochemistry. The adoptive transfer of induced bone marrow-derived MDSCs (BM MDSCs) to AIH mice was used to explore the function of MDSCs. Hepatic injury and mitochondrial damage were evaluated by transaminase levels, histopathology, immunohistochemistry, transmission electron microscopy and western blotting. MDSCs were pretreated with the small extracellular vesicle (sEV) generation inhibitor GW4869 to explore the mechanism. Importantly, sEVs derived from MDSCs and MDSCs-GW4869 were injected into model mice to monitor mitochondrial function and biogenesis. RESULTS Circulating and liver MDSCs were expanded in AIH patients and mouse model. Furthermore, the follow-up data of AIH patients showed that immunosuppressive therapy further promoted the expansion of MDSCs. More importantly, the adoptive transfer of BM MDSCs to AIH mice effectively ameliorated liver injury and regulated the imbalance of the immune microenvironment. Additionally, BM MDSCs reduced liver mitochondrial damage and improved mitochondrial biogenesis. Mechanistically, sEVs derived from BM MDSCs showed the same biological effects as cells, and blocking sEV production weakened the function of BM MDSCs. Finally, multiple long-term administrations of BM MDSCs were proven to be safe in general. CONCLUSION In conclusion, MDSCs ameliorate liver mitochondrial damage to protect against autoimmune hepatitis by releasing small extracellular vesicles.

Platelets, traditionally known for their role in hemostasis, also contribute to inflammation, cancer, and intercellular communication through the release of platelet-derived extracellular vesicles (or platelet-derived microparticles; PMPs). Among these vesicles, a subpopulation containing functional mitochondria, known as mitoMPs, can be transferred to recipient cells, thereby modulating their metabolism and biological responses. This mitochondrial transfer plays a key role in various pathological processes, where it may either restore metabolic functions or enhance cancer cell proliferation, survival, and metabolic plasticity. In this study, we developed a permeabilization protocol combined with high-resolution respirometry to assess mitochondrial respiration in both platelets and PMPs. First, we found that saponin was a more effective permeabilizing agent than digitonin to measure mitochondrial respiration in these models. Moreover, our analysis revealed distinct respiratory profiles between platelets and PMPs and demonstrated that freeze-thaw cycles severely compromise mitochondrial functions in PMPs. Additionally, we performed proteomic profiling of PMPs to characterize their protein cargo, which associate with specific molecular pathways, particularly those associated with mitochondrial metabolism. These results provide novel insights into the biological functions of PMPs and their potential involvement in disease processes. Together, these findings advance the understanding of PMP-mediated mitochondrial transfer and intercellular communication and establish a foundation for future biomedical and therapeutic investigations.

No abstract available

Does maternal endometrial mitochondrial (mt) DNA cargo of EVs modulate embryo bioenergetics during embryo implantation? We demonstrate the vertical transmission of maternal mtDNA within endometrial-derived EVs and their uptake by the trophoblast which reduces mitochondrial respiration and ATP production. The release and uptake of membrane-enclosed compartments with specific cargos, commonly known as EVs, represents a novel cell-to-cell communication mechanism in physiological and pathogenic conditions. EVs are generally classified into three populations based on their biogenetic pathways, composition, and physical characteristics: apoptotic bodies (ABs), microvesicles (MVs), and exosomes (EXOs). Among other contents, EVs contain single- and double-stranded DNA, with their relative abundance varying depending on the cell and vesicle type. The vertical transmission of EV-associated DNA has been proposed as a novel genetic material transfer mechanism that may impact genome evolution and tumorigenesis. Prospective observational multicenter analysis in which EVs were obtained from endometrial fluid from healthy donors aged 18–35 years (n = 10) during the receptive phase of their natural cycle and under hormonal replacement therapy in pre-receptive (P + 2), receptive (P + 5), post-receptive (P + 8) stages (n = 13). Endometrial EVs isolated using ultracentrifugation and classified according to `parameters obtained from electron microscopy, Western blotting, and size distribution analysis. DNA copy number identified using high throughput sequencing. EV-associated DNA tagged with 5-ethynyl-2’-deoxyuridine was followed by confocal imaging after co-incubation of EVs with murine embryos (n = 200). ATP levels assessed using the FLASC luciferase reporter system, and the Seahorse XFE96 extracellular flux analyzer used to measure embryo oxygen consumption rate (OCR) (n = 400). The human endometrium secretes all three EV types - ABs, MVs, and EXOs - into the human endometrial fluid. Deep sequencing revealed that EVs encapsulated nuclear and mtDNA. When analyzing endometrial biopsies, we observed the reduced mtDNA content of endometrial cells and the activation of mitochondrial clearance mechanisms, which coincided with the time of embryo implantation together with specific enrichment in endometrial MVs secreted during the periconceptional period. EVs were internalized and DNA was transferred to the cytoplasm and nuclei of trophectoderm of murine embryos. We analyzed ATP concentrations in murine embryos and found a significant reduction in ATP levels following the coculture of embryos with a combination of all EVs types compared to control embryos cultured without endometrial EVs (p < 0.001). Finally, we demonstrated a reduction in the OCR in embryos treated with endometrial EVs obtained during the receptive phase compared with the pre-receptive phase. In conclusion, maternal EVs modulate the bioenergetics of the preimplantation embryo by increasing the embryo's metabolic rate and oxygen consumption during the periconceptional period. These results were obtained using a combination of a human endometrial model and a murine embryo model. Our results suggest that the vertical transmission of maternal mtDNA encapsulated within EVs to the trophectoderm might energetically assist the preimplantation embryo through the implantation process. N/A

Recent studies have demonstrated that synovial mesenchymal stem cell-derived small extracellular vesicles (EVs) engineered to deliver GrpE-like 1 activated PTEN-induced kinase 1-dependent mitophagy and restored chondrocyte homeostasis. This study revealed that interleukin-1β-challenged chondrocytes exhibited efficient cargo transfer, increased mitophagy signaling with reduced p62 levels, lower oxidative stress, and a shift toward matrix preservation, characterized by higher collagen II and aggrecan levels, and lower matrix metallopeptidase 13 and ADAM metallopeptidase with thrombospondin type 1 motif 5 levels. In a rat knee osteoarthritis model, intra-articular dosing preserved cartilage architecture and improved histological scores. Collectively, these findings suggest that EV-based delivery of mitochondrial regulators is a plausible disease-modifying strategy, rather than purely symptomatic care. Building on this evidence, this editorial distills key advances and outlines near-term research and translational priorities, including standardized EV characterization, pharmacokinetics, dosing, safety, and manufacturability. The suitability of GrpE-like 1-loaded small EVs for early-stage osteoarthritis was also evaluated.

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1’s EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394 to 431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1’s EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

Relocalizing the mitochondrial motor–adaptor protein Miro to peroxisomes and systematically manipulating each GTPase domain reveal the importance of the N-terminal GTPase domain of Miro1 for regulating mitochondrial transport. Mitochondrial transport relies on a motor–adaptor complex containing Miro1, a mitochondrial outer membrane protein with two GTPase domains, and TRAK1/2, kinesin-1, and dynein. Using a peroxisome-directed Miro1, we quantified the ability of GTPase mutations to influence the peroxisomal recruitment of complex components. Miro1 whose N-GTPase is locked in the GDP state does not recruit TRAK1/2, kinesin, or P135 to peroxisomes, whereas the GTP state does. Similarly, the expression of the MiroGAP VopE dislodges TRAK1 from mitochondria. Miro1 C-GTPase mutations have little influence on complex recruitment. Although Miro2 is thought to support mitochondrial motility, peroxisome-directed Miro2 did not recruit the other complex components regardless of the state of its GTPase domains. Neurons expressing peroxisomal Miro1 with the GTP-state form of the N-GTPase had markedly increased peroxisomal transport to growth cones, whereas the GDP-state caused their retention in the soma. Thus, the N-GTPase domain of Miro1 is critical for regulating Miro1’s interaction with the other components of the motor–adaptor complex and thereby for regulating mitochondrial motility.

Mesenchymal stem cells (MSCs) transfer healthy mitochondria to damaged acceptor cells via actin-based intercellular structures. In this study, we tested the hypothesis that MSCs transfer mitochondria to neural stem cells (NSCs) to protect NSCs against the neurotoxic effects of cisplatin treatment. Our results show that MSCs donate mitochondria to NSCs damaged in vitro by cisplatin. Transfer of healthy MSC-derived mitochondria decreases cisplatin-induced NSC death. Moreover, mitochondrial transfer from MSCs to NSCs reverses the cisplatin-induced decrease in mitochondrial membrane potential. Blocking the formation of actin-based intercellular structures inhibited the transfer of mitochondria to NSCs and abrogated the positive effects of MSCs on NSC survival. Conversely, overexpression of the mitochondrial motor protein Rho-GTPase 1 (Miro1) in MSCs increased mitochondrial transfer and further improved survival of cisplatin-treated NSCs. In vivo, MSC administration prevented the loss of DCX+ neural progenitor cells in the subventricular zone and hippocampal dentate gyrus which occurs as a result of cisplatin treatment. We propose mitochondrial transfer as one of the mechanisms via which MSCs exert their therapeutic regenerative effects after cisplatin treatment.

Miro proteins are universally conserved mitochondrial calcium-binding GTPases that regulate a multitude of mitochondrial processes, including transport, clearance and lipid trafficking. Miro binds a variety of client proteins involved in these functions. How this binding is operated at the molecular level and whether and how it is important for mitochondrial health, however, remains unknown. Here, we show that known Miro clients all use a similar short motif to bind the same structural element: a highly conserved hydrophobic pocket in the calcium-binding domain of Miro. Using these Miro-binding motifs, we identified direct interactors de novo, including yeast Mdm34, and mammalian MTFR1/2/1L, VPS13D and Parkin. Given the shared binding mechanism and conservation across eukaryotes, we propose that Miro is a universal mitochondrial adaptor coordinating mitochondrial health. One-Sentence Summary Functionally diverse mitochondrial proteins interact with a conserved hydrophobic pocket on the calcium-binding Miro-GTPases.

No abstract available

In the current model of mitochondrial trafficking, Miro1 and Miro2 Rho‐GTPases regulate mitochondrial transport along microtubules by linking mitochondria to kinesin and dynein motors. By generating Miro1/2 double‐knockout mouse embryos and single‐ and double‐knockout embryonic fibroblasts, we demonstrate the essential and non‐redundant roles of Miro proteins for embryonic development and subcellular mitochondrial distribution. Unexpectedly, the TRAK1 and TRAK2 motor protein adaptors can still localise to the outer mitochondrial membrane to drive anterograde mitochondrial motility in Miro1/2 double‐knockout cells. In contrast, we show that TRAK2‐mediated retrograde mitochondrial transport is Miro1‐dependent. Interestingly, we find that Miro is critical for recruiting and stabilising the mitochondrial myosin Myo19 on the mitochondria for coupling mitochondria to the actin cytoskeleton. Moreover, Miro depletion during PINK1/Parkin‐dependent mitophagy can also drive a loss of mitochondrial Myo19 upon mitochondrial damage. Finally, aberrant positioning of mitochondria in Miro1/2 double‐knockout cells leads to disruption of correct mitochondrial segregation during mitosis. Thus, Miro proteins can fine‐tune actin‐ and tubulin‐dependent mitochondrial motility and positioning, to regulate key cellular functions such as cell proliferation.

Neurodegenerative disorders, including chemotherapy-induced cognitive impairment, are associated with neuronal mitochondrial dysfunction. Cisplatin, a commonly used chemotherapeutic, induces neuronal mitochondrial dysfunction in vivo and in vitro. Astrocytes are key players in supporting neuronal development, synaptogenesis, axonal growth, metabolism and, potentially mitochondrial health. We tested the hypothesis that astrocytes transfer healthy mitochondria to neurons after cisplatin treatment to restore neuronal health. We used an in vitro system in which astrocytes containing mito-mCherry-labeled mitochondria were co-cultured with primary cortical neurons damaged by cisplatin. Culture of primary cortical neurons with cisplatin reduced neuronal survival and depolarized neuronal mitochondrial membrane potential. Cisplatin induced abnormalities in neuronal calcium dynamics that were characterized by increased resting calcium levels, reduced calcium responses to stimulation with KCl, and slower calcium clearance. The same dose of cisplatin that caused neuronal damage did not affect astrocyte survival or astrocytic mitochondrial respiration. Co-culture of cisplatin-treated neurons with astrocytes increased neuronal survival, restored neuronal mitochondrial membrane potential, and normalized neuronal calcium dynamics especially in neurons that had received mitochondria from astrocytes which underlines the importance of mitochondrial transfer. These beneficial effects of astrocytes were associated with transfer of mitochondria from astrocytes to cisplatin-treated neurons. We show that siRNA-mediated knockdown of the Rho-GTPase Miro-1 in astrocytes reduced mitochondrial transfer from astrocytes to neurons and prevented the normalization of neuronal calcium dynamics. In conclusion, we showed that transfer of mitochondria from astrocytes to neurons rescues neurons from the damage induced by cisplatin treatment. Astrocytes are far more resistant to cisplatin than cortical neurons. We propose that transfer of functional mitochondria from astrocytes to neurons is an important repair mechanism to protect the vulnerable cortical neurons against the toxic effects of cisplatin.

Abstract Mitochondria are organelles present in most eukaryotic cells, where they play major and multifaceted roles. The classical notion of the main mitochondrial function as the powerhouse of the cell per se has been complemented by recent discoveries pointing to mitochondria as organelles affecting a number of other auxiliary processes. They go beyond the classical energy provision via acting as a relay point of many catabolic and anabolic processes, to signaling pathways critically affecting cell growth by their implication in de novo pyrimidine synthesis. These additional roles further underscore the importance of mitochondrial homeostasis in various tissues, where its deregulation promotes a number of pathologies. While it has long been known that mitochondria can move within a cell to sites where they are needed, recent research has uncovered that mitochondria can also move between cells. While this intriguing field of research is only emerging, it is clear that mobilization of mitochondria requires a complex apparatus that critically involves mitochondrial proteins of the Miro family, whose role goes beyond the mitochondrial transfer, as will be covered in this review.

There has been an upsurge of interest in the bone marrow mesenchymal stem cell (BMSC) mitochondrial transfer as a potential therapeutic innovation in organ injury repair. Previous research mainly focused on its transfer routes and therapeutic effects. However, its intrinsic mechanism has not been well deciphered. The current research status needs to be summarized for the clarification of future research direction. Therefore, we review the recent significant progress in the application of BMSC mitochondrial transfer in organ injury repair. The transfer routes and effects are summarized, and some suggestions on the future research direction are provided.

Leber's Hereditary Optic Neuropathy (LHON) is the most prevalent mitochondrial inherited disorder, primarily caused by primary mitochondrial mutations. Clinically, LHON is characterized by degeneration of optic nerves that leads to acute or subacute sudden or painless central vision loss. Currently no effective treatment has been established for LHON. Recent studies have highlighted the significance of intercellular mitochondrial transfer, which facilitates communication between cells and presents a novel therapeutic avenue. In this study, we investigated the formation of tunnelling nanotubes (TNTs) and the subsequent mitochondrial transfer between Bone Marrow Mesenchymal Stem Cells (BM-MSCs) and LHON ND4 mutant cells within the coculture system. Our findings demonstrated that mitochondrial transfer from BM-MSCs to LHON mutant cells via TNTs effectively rescued the mutant LHON cells by reducing apoptosis, restoring mitochondrial membrane potential and reducing reactive oxygen species (ROS) generation. These results provide compelling evidence of cell-cell communication between mesenchymal stem cells and LHON mutant cells, indicating a potential regenerative capacity through the reduction in mitochondrial mutation load. This study would help to implement further research in this area for the protective effect of mitochondria transfer and future cell-based treatment approaches for LHON.

Angiogenesis is crucial to improving neurovascular remodeling poststroke. Therein, the transformation of endothelial cells (ECs) to tip cells is essential in initiating angiogenesis. Mitochondrial damage in ECs poststroke and associated metabolic disorder are key factors repressing angiogenesis, but the mechanisms are unknown. Here, we designed an Arg-Gly-Asp peptide (RGD)-modified, mitochondria-enriched, and extracellular vesicle mimetics (mitoEVMs) platform for mitochondrial transfer. RGD mediated the mesenchymal stem cell-derived mitochondria transfer to ECs around the lesion targetedly. We found MSC-derived mitochondria promoted tip cell transition and further stimulated angiogenesis after stroke, alleviated brain atrophy, and improved functional rehabilitation. We noticed mitochondrial transfer rescued mitochondrial function in ECs and reprogrammed glutathione metabolism to activate the mTORC1 pathway, upregulated the expression of p4E-BP1 and VEGFR2, and ultimately facilitated tip cell transition. Our work elucidates the mechanism of MSC-derived mitochondrial transfer in poststroke treatment and proposes a potential approach for rehabilitation after stroke.

Mitochondrial transfer is becoming recognized as an important immunomodulatory mechanism used by mesenchymal stem cells (MSCs) to influence immune cells. While effects on T cells and macrophages have been documented, the influence on B cells remains unexplored. This study investigates the modulation of B lymphocyte fate by MSC‐mediated mitochondrial transfer.

The aberrant cellular senescence in chronic wounds presents a significant barrier to healing. Mitochondrial dysfunction is critical in initiating and maintaining cellular senescence, underscoring therapeutic potential in restoring mitochondrial function by delivering healthy mitochondria to wound cells. However, approaches for delivering mitochondria to achieve optimized wound repair remain lacking. Herein, enucleated MSCs‐derived microvesicles containing functional mitochondria (Mito@euMVs) via simple extrusion are developed. By controlling the size of microvesicles within a small micron‐scale range, the mitochondrial encapsulation efficiency is optimized. Mito@euMVs effectively delivered mitochondria into fibroblasts and HUVECs, inhibiting and rejuvenating hyperglycemia‐induced cellular senescence. To enhance the clinical applicability, soluble PVA microneedle patches for the transdermal Mito@euMVs delivery are utilized. In diabetic rats with pressure sores, the senescence‐inhibiting and ‐rescuing properties of Mito@euMVs are further validated, along with their therapeutic efficacy, demonstrating their potential for chronic wound repair. Moreover, as a versatile delivery vehicle for mitochondria, Mito@euMVs hold promising for treating mitochondrial dysfunction and aging‐related conditions.

No abstract available

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment for acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). However post-transplant disease recurrence and alloreactive complications (chiefly graft versus host diseases - GvHD) are still associated with severe morbidity and mortality. Proliferative advantage of transformed cells as well as immune and microenvironmental dysregulations contribute both to promoting relapse and escape from graft versus leukemia (GvL) effect. Bone marrow (BM) mesenchymal stromal cells (MSC) are well known for their role in hematopoiesis and immune modulation. MSCs have been shown able to transfer mitochondria (MT) to different cell populations, including immune effectors, in order to (re)establish a healthy oxidative metabolism within the recipient cell. Here we hypothesize that these mechanisms are highly active in BM microenvironment after allo-HSCT and may contribute to alloreactivity, modifying the immune environment. On this basis we stipulate that those intercellular exchanges are deeply impacted by MSCs' senescence, that could be deployed as a useful parameter to predict post-transplant outcomes. To that end, we conducted a translational research, based on BM specimens prospectively collected at our Institution (NCT03357172, NCT03964922), involving patients undergoing allo-HSCT for high-risk MDS and AML and receiving fludarabine/busulfan-based conditioning regimens, anti-lymphoglobuline and cyclosporin/mycophenolate mofetil. MSC phenotype and functionality at the pre-transplant stage, MT capacity, immunomodulation towards T cells and phenotype were studied by multiparametric flow cytometry. Cell metabolism, in terms of oxygen consumption and extracellular acidification rate, was characterized with Seahorse XF technology. Percentages of senescent MSCs were determined with SA β-galactosidase test. First, we compared phenotypic and metabolic characteristics of MSC from MDS/AML (N=29) patients with those retrieved from a cohort of healthy controls (N=8). We observed that patient MSCs were characterized by higher expression of CD157 and CD146 and lower expression of CD200 as compared to healthy MSCs. Pre-transplant senescence quantification highlighted a certain degree of heterogeneity within the MSC compartment in leukemic patients (median = 37.42% sd=16.30 for AML patient and m=11.74% sd=6.4 for HC). In an attempt to link clinical phenotypes to this heterogeneity, we observed that patients with high degree of MSC senescence in pretransplant samples (mean= 51.6% sd=16.6 cells/sample), developed more frequently acute GvHD, as compared to those with low degree of pre-transplant MSC senescence (mean= 23.8% sd=14.3) who experienced instead a higher incidence of disease relapse (p=0,004). Assuming that the link between senescence and alloreactivity could rely to different metabolic states and MT capabilities in MSCs, we analyzed these biological features in our samples. Indeed, we observed that low degree of MSC senescence was associated with higher MT to T cells, in particular to CD4+ cells, shifting their metabolism and phenotype toward an immunosuppressive state The characterization of cellular metabolism showed in highly senescent MSCs the presence of markers of mitochondrial dysfunction with high oxidative power, but reduced coupling efficiency of respiratory chain. This metabolic profile involved a high mitochondrial mass characterized by a high maximal respiration capacity compared to HC MSCs. The in vitro modulation of MSC senescence with nicotinamide administration restored healthy mitochondrial metabolism and improved coupling efficacy of oxidative phosphorylation, possibly impacting on the immunomodulation capabilities of this microenvironment component. Here for the first time, we provide the evidence that MSC senescence plays an pivotal role in defining an altered immune state in transplanted AML possibly shifting the degree of alloreactivity from a high GvH/GvL response (high GvHD and low relapse) to a high immunosuppressive state (low GvHD and high relapse). These findings pave the way for implementing MSC senescence as a biomarker able to predict post-transplant outcomes and show new biological rationales to integrate donor-derived MSC products to better adjust GvH responses.

Mitochondria are important organelles for cell metabolism and tissue survival. Their cell-to-cell transfer is important for the fate of recipient cells. Recently, bone marrow mesenchymal stem cells (BM-MSCs) have been reported to provide mitochondria to cancer cells and rescue mitochondrial dysfunction in cancer cells. However, the details of the mechanism have not yet been fully elucidated. In this study, we investigated the humoral factors inducing mitochondrial transfer (MT) and the mechanisms. BM-MSCs produced MT in colorectal cancer (CRC) cells damaged by 5-fluorouracil (5-FU), but were suppressed by the anti-high mobility group box-1 (HMGB1) antibody. BM-MSCs treated with oxidized HMGB1 had increased expression of MT-associated genes, whereas reduced HMGB1 did not. Inhibition of nuclear factor–κB, a downstream factor of HMGB1 signaling, significantly decreased MT-associated gene expression. CRC cells showed increased stemness and decreased 5-FU sensitivity in correlation with MT levels. In a mouse subcutaneous tumor model of CRC, 5-FU sensitivity decreased and stemness increased by the MT from host mouse BM-MSCs. These results suggest that oxidized HMGB1 induces MTs from MSCs to CRC cells and promotes cancer cell stemness. Targeting of oxidized HMGB1 may attenuate stemness of CRCs.

Acute ischemic stroke (AIS) initiates secondary injuries that worsen neurological damage and hinder recovery. While peripheral immune responses play a key role in stroke outcomes, clinical results from immunotherapy have been suboptimal, with limited focus on T-cell dynamics. Umbilical mesenchymal stem cells (UMSCs) offer therapeutic potential due to their immunomodulatory properties. They can regulate immune responses and reduce neuroinflammation, potentially enhancing recovery by fostering a pro-regenerative peripheral immune environment. However, the effect of UMSCs on T-cell dynamics in AIS remains underexplored. This study investigates T-cell dynamics following AIS and examines how UMSCs may mitigate immune dysregulation to develop better treatment strategies. AIS patients (NIHSS scores 0–15) were recruited within 72 h of stroke onset, with peripheral blood samples collected on Day 0 (enrollment) and Day 7. T-cell compartments were identified by flow cytometry, and plasma cytokine levels were quantified using a cytometric bead array (CBA). Mitochondria in UMSCs were labeled with MitoTracker. Peripheral blood mononuclear cells from patients were isolated, treated with lipopolysaccharide (LPS), and cocultured with UMSCs in both direct contact and Transwell systems. Flow cytometry, CBA, RT-qPCR, and immunofluorescence assays were used to detect T-cell compartments, gene expression markers for helper T (Th) cell differentiation, cytokine profiles, mitochondrial transfer, reactive oxygen species (ROS) production, and mitochondrial membrane potential. Additionally, mitochondrial DNA in UMSCs was depleted. The effects of UMSCs and mitochondria-depleted UMSCs on ischemic stroke mice were compared through behavioral assessments and analysis of the peripheral immune microenvironment. In AIS, T-cell compartments underwent a phenotypic shift from naïve to effector or memory states, with a specific increase in Th17 cells and a decrease in regulatory T cells, leading to alterations in T-cell-mediated immune functions. In an ex vivo co-culture system, LPS stimulation further amplified these disparities, inducing mitochondrial dysfunction and oxidative stress in T cells. Notably, UMSCs restored mitochondrial function and reversed the shift in T-cell compartments through mitochondrial transfer. Critically, UMSC treatment significantly improved both neurological deficits and peripheral immune disorders in ischemic stroke mice, whereas mitochondria-depleted UMSCs failed to produce this effect. Our comprehensive insights into the key attributes of T-cell compartments in acute ischemic stroke and the immune regulatory mechanisms of UMSCs provide a crucial theoretical foundation for understanding peripheral immune disorders in ischemic stroke and the therapeutic potential of UMSC treatment.

No abstract available

. 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. . 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. . 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.

Mesenchymal stem cells (MSCs) are multipotent cells with broad immunosuppressive capacities. Recently, it has been reported that MSCs can transfer mitochondria to various cell types, including fibroblast, cancer, and endothelial cells. It has been suggested that mitochondrial transfer is associated with a physiological response to cues released by damaged cells to restore and regenerate damaged tissue. However, the role of mitochondrial transfer to immune competent cells has been poorly investigated. Here, we analyzed the capacity of MSCs from the bone marrow (BM) of healthy donors (BM-MSCs) to transfer mitochondria to primary CD4+CCR6+CD45RO+ T helper 17 (Th17) cells by confocal microscopy and fluorescent-activated cell sorting (FACS). We then evaluated the Th17 cell inflammatory phenotype and bioenergetics at 4 h and 24 h of co-culture with BM-MSCs. We found that Th17 cells can take up mitochondria from BM-MSCs already after 4 h of co-culture. Moreover, IL-17 production by Th17 cells co-cultured with BM-MSCs was significantly impaired in a contact-dependent manner. This inhibition was associated with oxygen consumption increase by Th17 cells and interconversion into T regulatory cells. Finally, by co-culturing human synovial MSCs (sMSCs) from patients with rheumatoid arthritis (RA) with Th17 cells, we found that compared with healthy BM-MSCs, mitochondrial transfer to Th17 cells was impaired in RA-sMSCs. Moreover, artificial mitochondrial transfer also significantly reduced IL-17 production by Th17 cells. The present study brings some insights into a novel mechanism of T cell function regulation through mitochondrial transfer from stromal stem cells. The reduced mitochondrial transfer by RA-sMSCs might contribute to the persistence of chronic inflammation in RA synovitis.

No abstract available

Background: Mesenchymal stem cells-conditioned medium (MSC-CM) provides a promising cell-free therapy for Alzheimer’s disease (AD) mainly due to the paracrine of MSCs, but the precise mechanisms remain unclear. Studies suggests that mitochondrial dysfunction precedes the accumulation of amyloid-β plaques and neurofibrillary tangles, and involves in the onset and development of AD. Objective: In the present study, we evaluated the protective effects and explored the related-mitochondrial mechanisms of human umbilical cord derived MSC-CM (hucMSC-CM) in an AD model in vitro. Methods: To this end, an AD cellular model was firstly established by okadaic acid (OA)-treated SH-SY5Y cells, and then treated by hucMSC-CM to assess the oxidative stress, mitochondrial function, apoptosis, AD-related genes, and signaling pathways. Results: hucMSC-CM significantly deceased tau phosphorylated at Thr181 (p181-tau) level, which was increased in AD. hucMSC-CM also alleviated intracellular and mitochondrial oxidative stress in OA-treated SH-SY5Y cells. In addition, hucMSC-CM suppressed apoptosis and improved mitochondrial function in OA-treated SH-SY5Y cells. Flow cytometric analysis indicated that hucMSC-CM exerted the protective effects relying on or partly extracellular vesicle (EV) mitochondrial transfer from hucMSCs to OA-treated SH-SY5Y cells. Moreover, RNA sequencing data further demonstrated that hucMSC-CM regulated many AD-related genes, signaling pathways and mitochondrial function. Conclusion: These results indicated that MSC-CM or MSC-EVs containing abundant mitochondria may provide a novel potential therapeutic approach for AD.

Current clinical interventions for stroke majorly involve thrombolysis or thrombectomy, however, cessation of the progressive deleterious cellular cascades post-stroke and long-term neuroprotection are yet to be explored. Mitochondria are highly vulnerable organelles and their dysfunction is one of the detrimental consequences following stroke. Mitochondria dysregulation activate unfavourable cellular events over a period of time that leads to the collapse of neuronal machinery in the brain. Hence, strategies to protect and replenish mitochondria in injured neurons may be useful and needs to be explored. Stem cell therapy in ischemic stroke holds a great promise. Past studies have shown beneficial outcomes of endovascularly delivered stem cells in both pre-clinical and clinical settings. Intra-arterial (IA) administration can provide more cells to the stroke foci and affected brain regions than intravenous administration. Supplying new mitochondria to the stroke-compromised neurons either in the core or penumbra by infused stem cells can help increase their survival and longevity. Previously, our lab has demonstrated that IA 1*105 mesenchymal stem cells (MSCs) in rats were safe, efficacious and rendered neuroprotection by regulating neuronal calcineurin, modulating sirtuin1(SIRT-1) mediated inflammasome signaling, ameliorating endoplasmic reticulum-stress, alleviation of post-stroke edema and reducing cellular apoptosis. To explore further, our present study aims to investigate the potential of IA MSCs in protecting and replenishing mitochondria in the injured neurons post-stroke and thereof involvement of SIRT-1/RHOT-1/PGC-1α loop towards mitochondria transfer, biogenesis, and neuroprotection. This study will open new avenues for using stem cells for ischemic stroke in clinics as one of the future adjunctive therapies.

No abstract available

Amid the widespread scarcity of donor livers, mitigating ischemia-reperfusion injury (IRI) of liver grafts is vital for ensuring early recovery of post-transplant liver function. Human bone marrow-derived mesenchymal stem cells (hBMSCs) have shown potential in alleviating IRI damage by regulating mitochondrial function. Hypoxia-preconditioning hBMSCs (hypo-hBMSCs) have shown considerable promise in enhancing therapeutic efficacy, yet the underlying mechanism remain to be elucidated. Therefore, this study aims to explore the role of hypo-hBMSCs in alleviating hepatic IRI and uncover their potential mechanisms, with the goal of offering new strategies for the application of hBMSCs in liver protection after transplantation. Initially, we investigated the impact of hypoxia preconditioning on the quality of hBMSCs mitochondria and whether hypo-hBMSCs can alleviate IRI damage in liver grafts by transferring mitochondria. Subsequently, by employing the enhancer RA and the inhibitor Gap26 to modulate the function of gap junctions (GJs) in vivo and in vitro, we confirmed their crucial role in the process of hypo-hBMSCs transferring mitochondria to hepatocytes. Ultimately, through bioinformatics analysis, Co-IP, siRNA and overexpression, we demonstrate that the up-regulated Cx43 and Cx32 in hypo-hBMSCs can form homotypic Cx43-GJs and Cx32-GJs with hepatocytes, thereby enhancing the transfer of mitochondria. The results indicate that hypoxia preconditioning diminishes superoxides accumulation and elevates the mitochondrial membrane potential by inducing mitophagy in hBMSCs, consequently improving mitochondrial quality. Upon administration via portal vein injection, hypo-hBMSCs significantly mitigate hepatic IRI. Compared with hBMSCs, hypo-hBMSCs are capable of transferring more mitochondria to hepatocytes through GJs. When the function of GJs is modulated by the enhancer RA or the inhibitor Gap26, the efficiency of mitochondrial transfer correspondingly shifts. Further investigation uncovers that hypo-hBMSCs prompts an upsurge in the expression of Cx43 and Cx32 (not Cx26). Nevertheless, these proteins are unable to form heterotypic GJs (Cx43-Cx32-GJs) with hepatocytes; instead, they form homotypic Cx43-GJs and Cx32-GJs, which facilitate the transfer of mitochondria between hypo-hBMSCs and hepatocytes. Hypo-hBMSCs can enhance mitochondrial quality by inducing mitophagy. Meanwhile, they can up-regulate Cx43 and Cx32 to form homotypic Cx43-GJs and Cx32-GJs with hepatocytes, thereby transferring more high-quality mitochondria to hepatocytes to exert a protective effect.

Osteoarthrosis (OA) is a leading cause of disability and early mortality, with no disease modifying treatment. Mitochondrial (MT) dysfunction and changes in energy metabolism, leading to oxidative stress and apoptosis, are main drivers of disease. In reaction to stress, mesenchymal stromal/stem cells (MSCs) donate their MT to damaged tissues. Methods: To evaluate the capacity of clinically validated MSCs to spontaneously transfer their MT to human OA chondrocytes (OA-Ch), primary cultured Ch isolated from the articular cartilage of OA patients were co-cultured with MT-labeled MSCs. MT transfer (MitoT) was evidenced by flow cytometry and confocal microscopy of MitoTracker-stained and YFP-tagged MT protein. MT persistence and metabolic analysis on target cells were assessed by direct transfer of MSC-derived MT to OA-Chs (Mitoception), through SNP-qPCR analysis, ATP measurements and Seahorse technology. The effects of MitoT on MT dynamics, oxidative stress and cell viability were gauged by western blot of fusion/fission proteins, confocal image analysis, ROS levels, Annexin V/7AAD and TUNEL assays. Intra-articular injection of MSC-derived MT was tested in a collagenase-induced murine model of OA. Results: Dose-dependent cell-to-cell MitoT from MSCs to cultured OA-Chs was detected starting at 4 hours of co-culture, with increasing MT-fluorescence levels at higher MSC:Ch ratios. PCR analysis confirmed the presence of exogenous MSC-MT within MitoT+ OA-Chs up to 9 days post Mitoception. MitoT from MSCs to OA-Ch restores energetic status, with a higher ATP production and metabolic OXPHOS/Glycolisis ratio. Significant changes in the expression of MT network regulators, increased MFN2 and decreased p-DRP1, reveal that MitoT promotes MT fusion restoring the MT dynamics in the OA-Ch. Additionally, MitoT increases SOD2 transcripts, protein, and activity levels, and reduces ROS levels, confering resistance to oxidative stress and enhancing resistance to apoptosis. Intra-articular injection of MSC-derived MT improves histologic scores and bone density of the affected joints in the OA mouse model, demonstrating a protective effect of MT transplantation on cartilage degradation. Conclusion: The Mitochondria transfer of MSC-derived MT induced reversal of the metabolic dysfunction by restoring the energetic status and mitochondrial dynamics in the OA chondrocyte, while conferring resistance to oxidative stress and apoptosis. Intra-articular injection of MT improved the disease in collagenase-induced OA mouse model. The restoration of the cellular homeostasis and the preclinical benefit of the intra-articular MT treatment offer a new approach for the treatment of OA.

Myoclonus epilepsy associated with ragged-red fibers (MERRF) is a maternally inherited mitochondrial disease affecting neuromuscular functions. Mt.8344A>G mutation in mitochondrial DNA (mtDNA) is the most common cause of MERRF syndrome and has been linked to an increase in reactive oxygen species (ROS) level and oxidative stress, as well as impaired mitochondrial bioenergetics. Here, we tested whether WJMSC has therapeutic potential for the treatment of MERRF syndrome through the transfer of mitochondria. The MERRF cybrid cells exhibited a high mt.8344A>G mutation ratio, enhanced ROS level and oxidative damage, impaired mitochondrial bioenergetics, defected mitochondria-dependent viability, exhibited an imbalance of mitochondrial dynamics, and are susceptible to apoptotic stress. Coculture experiments revealed that mitochondria were intercellularly conducted from the WJMSC to the MERRF cybrid. Furthermore, WJMSC transferred mitochondria exclusively to cells with defective mitochondria but not to cells with normal mitochondria. MERRF cybrid following WJMSC coculture (MF+WJ) demonstrated improvement of mt.8344A>G mutation ratio, ROS level, oxidative damage, mitochondrial bioenergetics, mitochondria-dependent viability, balance of mitochondrial dynamics, and resistance against apoptotic stress. WJMSC-derived mitochondrial transfer and its therapeutic effect were noted to be blocked by F-actin depolymerizing agent cytochalasin B. Collectively, the WJMSC ability to rescue cells with defective mitochondrial function through donating healthy mitochondria may lead to new insights into the development of more efficient strategies to treat diseases related to mitochondrial dysfunction.

No abstract available

Acute myeloid leukemia (AML) is characterized by abundant immature myeloid cells, relapse and refractory due to leukemia stem cells (LSCs). Bone marrow mesenchymal stem/ stromal cells (BMSCs) supported LSCs survival, meanwhile, chemotherapy improved connexin43 (CX43) expression. CX43, as the most intercellular gap junction, facilitated transmit mitochondria from BMSCs into AML. We hypothesized that increased mitochondria transferred from BMSCs supported metabolic remodeling in LSCs to sustain their stemness. Primary BMSCs from AML patients were isolated. CX43-BMSCs, overexpressing CX43, were cocultured with KG-1a cells. Fluorescence and confocal microscopy observed mitochondrial transfer. Flow cytometry, EdU assay, and clonogenicity evaluated cell cycle, proliferation, and clonogenic potential. Xenograft mouse models were used to evaluate the tumorigenicity of KG-1a in vivo. Seahorse, RNA-seq, and LC-MS assessed mitochondrial function, transcriptomes, and metabolites post-coculture. CX43-BMSCs promoted unidirectional mitochondrial transfer, enhancing KG-1a adhesion and proliferation to maintain LSCs stemness in vitro and vivo. RNA-seq revealed coculture with CX43-BMSCs upregulated genes related to adhesion, proliferation, and migration in KG-1a cells. Elevated CX43 expression strengthened BMSCs-KG-1a interaction, facilitating mitochondrial transfer and nucleoside metabolism, fueling KG-1a cells. This enhanced mitochondrial energy metabolism, promoting metabolic reprogramming and clonogenicity. CX43-mediated mitochondrial transfer from BMSCs to KG-1a enhances LSCs adhesion, proliferation, clonogenicity, and metabolic reprogramming. CX43 emerges as a potential therapeutic target for AML by sustaining LSCs stemness through metabolic remodeling.

No abstract available

No abstract available