A promising strategy for repairing tissue damage: mitochondria transfer from mesenchymal stem cells

Mesenchymal stem cells (MSCs) are gaining the spotlight in research due to their abundant sources, immune privileges


Background
The mitochondria is the main site for intracellular oxidative phosphorylation and the production of adenosine triphosphate (ATP) which is very important in energy provision, signal transmission and cell survival [1][2][3].Previous studies have reported that, the pathological results of most diseases are related to impaired mitochondrial function [4][5].Hence, restoring mitochondrial function is a necessity for cell survival.The mitochondria are highly dynamic organelles that can fuse, divide, and move along the cytoskeleton [6].Their ability to translocate from healthy stem cells into impaired cells is chiefly aided by mitochondria transfer mechanisms.
The MSCs contain more mitochondria than any other differentiated cells.Hence, making MSCs the preferred choice by researchers for multipotent related studies due to their low immunogenicity, rapid proliferation and differentiation capabilities [7][8].Recent studies have shown that mitochondria derived from MSCs can be transferred to damaged cells, consequently promoting functional recovery of damaged cells.This mitochondrial transfer based on MSCs is considered to be an emerging treatment for cell/tissue damage.However, the conditions for mitochondrial transfer of MSCs and the underlying mechanism of transfer remain to be clarified.Therefore, based on the summary of MSCs mitochondrial metastasis, this review discusses the conditions and the underlying mechanism of MSCs' mitochondrial transfer, aiming to have a deeper understanding of the emerging cell/tissue therapy of mitochondrial transfer derived from MSCs while providing reference basis for future clinical application of this novel therapy.Spees et al. demonstrated for the first time in 2006 that, mitochondria derived from MSCs can be transferred to lung adenocarcinoma A549 cells with mitochondrial DNA (mitochondria DNA, mtDNA) defects, consequently promoting the recovery of aerobic respiration [9].This exciting discovery provided a new direction for the treatment of mitochondrial-related diseases.As an emerging cell therapy method, mitochondrial transfer between cells has been deeply studied in different cell models.Chuang et al. demonstrated that when Wharton's Jelly Mesenchymal Stem Cell (WJMSC) and Myoclonus epilepsy associated with ragged-red fibers (MERRF) (MERRF is a maternal inherited mitochondrial disease that affects neuromuscular function) were co-cultured, mitochondrial transfer derived from WJMSC partially reduced the mtDNA mutation and oxidative stress level of MERRF, thereby improving the mitochondrial biological function of MERRF [10].The findings of the result were translated successfully in-vivo and patients suffering from mitochondrial diseases such as mitochondrial myopathy and stroke-like episodes were rescued coupled with intriguing therapeutic benefits such as improved bioenergetics and morphology of the mitochondria with increased apoptotic resistance following the tunnel nanotube (TNT) transfer of WJMSC to their rotenone-stressed fibroblast.This indicates that WJMSC transfer could be therapeutically used in our clinical settings to eliminate the potential mutation burden associated with mt.3243A>G point mutation in tRNALeu (UUR) gene of the mitochondria.Liu et al. showed that, when damaged umbilical vein received mitochondria transferred from MSCs using TNT, the mitochondrial activity of the damaged vein improved with a significant protective effect on injured endothelial cells, thus enhancing the formation of blood vessels [11].They showed that by co-culturing damaged human umbilical vein endothelial cell with MSCs in vitro.Their findings somehow provided an alternative treatment to mitochondrial dysfunction related diseases as well as challenging the classical view of transplantation therapy involving stem cells.Furthermore, Feng et al. showed that mitochondria can be transferred from human bone marrow mesenchymal stem cells (hBMMSCs) to human umbilical vein endothelial cells (HUVECs), to restore the capillary formation and migration ability of the damaged HUVECs [12].Their study reinforced the crosstalk of MSC and endothelial cells in promoting cell proliferation, reducing apoptosis and supporting the capillary angiogenic capacity.Furthermore, in adversities such as myeloablative and myelosuppresive injuries, MSCs could be used to direct strategies for improving hematopoietic system and endothelial function.

Mitochondrial transfer of MSCs mediates cells/tissues repair
The "rescue" effect of mitochondria derived from MSCs on damaged cells, do not only occur in in vitro co-culture environment, but also in complex internal environment.In lung diseases related studies, the bone marrow-derived stromal cells (BM-MSCs) were transferred to the alveolar epithelium of mice with lipopolysaccharide induced acute lung injury (ALI), thereby increasing the level of ATP in the alveolar epithelium and the survival rate of ALI mice [13].They observed the formation of connexin 43 (Cdx) gap junction channel by wild-type BM-MSCs and with that the mediated transfer of the mitochondria to improve alveolar bioenergetics was made possible in the lung.This indicates that not all BM-MSCs could be used in the treatment of alveolar leukocytosis and often the mutated or incompetent BM-MSCs with mitochondria dysfuntion fail to rescue.In neurological diseases, Li et al. found that mitochondria transferred from BM-MSCs to neurons under oxygen-glucose deprivation (OGD) conditions improved the biological functions of motor neurons and promoted the survival of neuronal cells [14].The BM-MSCs were able to transfer mitochondria to the injured neurons of the spinal cord via the gap junction intercellular communication (GJIC) and promoted cell survival coupled with refined bioenergetics states.Conversely, blocking the GJIC with 18β glycyrrhetinic acid decreased mitochondria transfer.This evidence suggests the importance of the role of the transfer phenomenon and suggests a novel therapeutic strategy of employing mitochondria transfer for the treatment of patients with spinal cord injury.The repair effect of mitochondrial metastasis derived from MSCs was also reflected in the reduced number of early apoptotic cells in spinal cord injury (SCI) rats coupled with the reduction in the space of late SCI lesions and the area of glial scars [15].
In an ocular disease model, the results of Jiang et al. showed that, induced pluripotent stem cell-derived MSC (iPSC-MSCs) transplanted into the vitreous of mice effectively donated functional mitochondria to retinal ganglion cells (RGCs), downregulated proinflammatory cytokines and prevented the loss of RGCs caused by mitochondrial damage [16].This indicated that MSCs could rescue most diseased cell condition including retinal ganglion cell degeneration which is often difficult to treat.In the treatment of rat streptozotocin-induced diabetic nephropathy with BM-MSCs transplantation, the transfer of mitochondria from BM-MSCs to damaged proximal tubular epithelial cells (PETCs) was observed to significantly inhibit the growth of PETCs, apoptosis and the production of reactive oxygen species (ROS) [17].Furthermore, cancer (lung cancer, ovarian cancer and human mesothelioma) continues to be a real world challenge as their resistance against chemotherapy has been reported to be bolstered by Tunnel nanotubes and Gap junctions which mediate mitochondria transfer for their survival [18][19][20].Hence, therapeutically blocking the formation of tunnel nanotube with metformin and Everolimus (mTor inhibitor) and gap junctions with 18β glycyrrhetinic acid have been reported to be an effective therapeutic strategy against cancer [14,18].
We have summarized the mitochondrial transfer phenomenon between MSCs and the recipient cells and the corresponding transfer effects in Table 1 below.

Mitochondrial transfer conditions of MSCs
As mentioned above, a large number of researchers have observed and proved that mitochondria derived from MSCs can translocate to different types of damaged cells/tissues and promote the functional recovery of damaged cells/tissues.Many studies have shown that, when target cells or tissues are injured, the phenomenon of mitochondrial transfer is more obvious.The unresolved question is, Submit a manuscript: https://www.tmrjournals.com/bmec[30][31][32][33].These damaged molecules trigger the anti-inflammatory response and sensitivity to oxidative stress in MSCs [34][35].Some released or exposed mitochondrial DAMPs could enter MSCs through internalization pathways to activate MSCs cell surface receptors (such as P2X7R or FPR) or intracellular receptors (such as TLR9 or NLRP3) to initiate innate or adaptive immune responses [31].
The interaction between stem cells and damaged cells activates the protective function of stem cells against damaged cells.When potential target cells are damaged or under stress, the mitochondrial transfer of MSCs becomes more obvious.Mahrouf-Yorgov et al. reported that, when MSCs were co-cultured with damaged cardiomyocytes/endothelial cells and other somatic cells, mitochondria derived from damaged somatic cells passed through the cytoprotective heme oxygenase-1 (HO-1), and stimulated mitochondrial biogenesis of MSCs to exert the anti-apoptotic effect of MSCs [36].They also demonstrated that ROS, produced by injured somatic cells, stimulated their own mitochondria to transmit to MSCs.This "help" signal initiated the adaptive cytoprotective response of MSCs.This finding is also supported by other studies [37][38].In addition, studies have shown that co-cultivation of epithelial cells treated with MSCs and rotenone (a respiratory chain inhibitor) could reverse the ATP levels of damaged cells and activate the activity of mitochondrial complex I and IV of damaged cells, consequently reducing mitochondrial ROS production [23].The impaired respiratory function of the damaged cells possibly served a retrograde signal that triggered changes in the ratio of ROS, Ca 2+ , AMP/ATP and NAD + /NADH, which in turn allowed the recipient cells to receive mitochondria from the donor cells [39].MSCs mitochondrial transfer conditions are shown in Figure 1.

Figure 1 Mitochondrial transfer conditions of MSCs
The effect of mitochondrial transfer from MSCs for the repair of damaged cells/tissues brings new hope for stem cell-based Protected against oxidative stress-induced mitochondrial dysfunction in the cornea.
[29] Submit a manuscript: https://www.tmrjournals.com/bmecregenerative medicine treatments.Some of the notable mechanism of mitochondrial delivery include TNT, Cx3, macrophage phagocytosis and Miro1.These transfer mechanisms occur in normal physiological states such as tissue homeostasis and are the gateway of survival for most aberrant cells including cancer and stressed cells [40].For example, cancer cells share healthy mitochondria via the transfer mechanism for their survival in the presence of chemotherapy while stressed cells employ same mechanism to reduce impaired mitochondria burden [40].Although these mediated transfer mechanisms may synergistically interact to promote mitochondria transfer in some cells such as the neuronal cells, other studies suggest the dispensability of some of the mechanisms following a knockout study [20].For example, Miro 1 have been reported to facilitate mitochondria transfer by interacting with microtubules which happens to be found in thicker microtubule containing TNTs whiles other studies revealed no effect at all on mitochondrial transfer following Miro1 knockout in cancer cells (neuroblastoma and leukemia) [20].This indicates that, in a normal physiological state the absence of Miro 1 could slow down the mitochondria transfer rate than in diseased states.
Furthermore, TNT and Gap junctions are closely related as they either occur when two cells (donor and recipient) lie close together and move in opposite direction or by fusion of protruded membrane from one or both cells which lie separately and not together [20].The formation of TNT between cells comprise of actin containing membranes, takes several minutes to establish, capable of moving cargos (mitochondria, miRNA and organelles) to damaged cells and are often temporarily regulated due to TNT breach or tear [20,41].The TNT formation could establish both close-ended or open-ended (direct connection of cytoplasm of two close cells) structures with the later more associated with Gap junction.Gap junctions are intercellular channels which partake in the transfer of mitochondria as well as small molecules and ions from a healthy cell to an aberrant cell [20,41].
Among the current types of mitochondrial transfer mechanisms of MSCs, we discussed the main mechanisms mediating their transfer in details as follows:

TNTs-mediated mitochondrial transfer of MSCs
Tunnel nanotubes (TNTs) is the most reported mediator for mitochondrial transfer in so many studies.It is a highly sensitive nanotube structure with a diameter of 50-1500nm and length spanning between tens to hundreds of microns, connecting two cells together [42].This cell-to-cell communication model, interacts with neighboring cells through direct contact with cell surface receptors and intercellular bridges, thus serving as a channel to transmit MSCs mitochondria for functional recovery of damaged cells [43].
Research by Feng et al. showed that co-culturing with hBMMSCs reduced the apoptosis rate of HUVECs caused by chemotherapy, and blocking the formation of TNTs impeded mitochondrial transfer of hBMMSCs, thereby eliminating the rescue effect of hBMMSCs.The results indicated that, TNTs-mediated mitochondrial transfer of hBMMSCs is an important part of the therapeutic effect [12].In the co-culture system of MSCs and alveolar macrophages, Jackson et al. proved that, the transfer of mitochondrial derived from MSCs promoted the oxidative phosphorylation of macrophages and enhanced the phagocytic ability of macrophages.However, when TNTs were destroyed by cytochalasin B (An actin polymerization inhibitor), the effect of MSCs on the phagocytosis of macrophages was completely eliminated [21].Recent studies have shown that tumor necrosis factor alpha (TNF-a) promoted the expression of NF-κB subunits p-IκB, p-P65 and TNF-aip2, while the inhibitors of NF-κB and SC-514 significantly reduced the expression of p-P65 and TNF-aip2, thereby inhibiting the formation of TNTs.In the study of Zhang et al., it was found that iPSC-MSCs were more sensitive to TNTs-mediated transfer of mitochondria to cardiomyocytes (CMs) due to the high level of TNFaIP2 expression [44].
It was also proved that the formation of TNTs induced by TNF-a by iPSC-MSCs was regulated by the TNF-a/NF-kB/TNFaIP2 signal transduction pathway, and inhibiting the expression of TNFaIP2 in iPSC-MSCs would reduce the mitochondrial transfer of iPSC-MSCs.Inhibition of the expression of TNFaIP2 in iPSC-MSCs reduced the efficiency of iPSC-MSC mitochondrial transfer, thereby reducing the protective effect of iPSC-MSC on CMs [29,44].The expression of immune marker CD38 was reported to correlate with TNT formation and blockage of CD38 expression impeded mitochondria transfer from BM-MSC, reduced size of tumor and increased survival of myeloma mice [20].

Cx43-mediated mitochondrial transfer of MSCs
Junction proteins (also known as gap junction proteins, GJPs) are a large class of transmembrane proteins that have channel-dependent and independent functions, and play an important role in cell growth, differentiation and signal transduction [45].In previous reports, connexin 43 (Cx43) participated in the migration of many types of cells [46][47], but recent reports have found that Cx43 played an important role in the mitochondrial transfer of MSCs.Islam et al. found that BM-MSCs mitochondrial transfer increased the alveolar ATP in mice with acute lung injury induced by lipopolysaccharide in a Cx43-dependent manner, while BM-MSCs with dysfunctional Cx43 lost alveolar ATP [13].Yao et al. also proved that Cx43-mediated mitochondrial transfer of iPSC-MSCs to epithelial cells protected mice from ovalbumin-induced allergic airway inflammation [48].

Macrocytosis-mediated mitochondrial transfer of MSCs
Macropinocytosis is a non-clathrin-driven pinocytosis process driven by actin.Cells move through the plasma membrane to engulf a relatively large amount of extracellular fluid solutes, including nutrients and other substances [49][50][51].Giant pinocytosis is closely related to actin cytoskeleton dynamics.Cells can form plasma membrane protrusions through actin polymerization to complete pinocytosis [51][52].Recouvreux et al. found that, intact mitochondria isolated from human uterine endometrial gland-derived mesenchymal cells (EMCs) was phagocytosed by cultured cardiomyocytes through giant pinocytosis and significantly improved mtDNA function in mitochondria deprived cardiomyocytes [52].The addition of cytochalasin D (actin polymerization inhibitor) and nocodazole (microtubule assembly inhibitor) in the co-culture environment impeded the transfer of mitochondria.However, the addition of chlorpromazine (an inhibitor of clathrin-mediated endocytosis) did not impede the transfer of mitochondria.Furthermore, it has been established that, cardiomyocytes obtained exogenous mitochondria through pinocytosis rather than clathrin-mediated endocytosis, which ultimately promoted the functional recovery of their own mitochondria [53].

Miro1 protein-mediated mitochondrial transfer of MSCs
In the current mitochondrial transport model, a calcium-sensitive adaptor protein Miro1 was discovered to connect mitochondria and KIF5 motor protein through a set of accessory proteins such as Miro2, TRAK1, TRAK2 and Myo19 [54][55][56].Another study has reported that, kinesin and dynein motors regulated mitochondrial transfer along microtransport [57].
Recently, studies have shown that, high expression of Miro1 improved the efficiency of MSCs mitochondrial transfer and enhanced the therapeutic effect of MSCs [27,58].In recent epigenetic studies, Kalinski and colleagues revealed that histone deacetylase 6 (HDAC6) enzyme deacetylate Miro 1 in MSC and consequently led to axon growth inhibition due to impeded mitochondria transport until reversed when the enzyme was inhibited [59].Although there is not much literature information on the role of epigenetics, this evidence provides avenue for in depth investigation of epigenetic methods to achieve beneficial therapeutic effects.Ahmad et al. proved that in a mouse model of allergic airway inflammation, compared with the control group, MSCs with high expression of Miro1 had a better therapeutic effect [23].The results of Zhang et al. suggested that, the mitochondrial transfer efficiency of iPSC-MSCs is better than BM-MSCs, due to the high expression of the inherent Miro1 of Submit a manuscript: https://www.tmrjournals.com/bmeciPSC-MSCs.This was supported by another study where the inhibition of the expression of Miro1 in iPSC-MSCs, decreased the efficiency of mitochondrial transfer of iPSC-MSCs, and the protective effect on cardiomyocytes [45].In addition, Rodriguez et al. also reached the same conclusions when they compared their model with the wild-type MSCs.In their study, MSCs with high expression of Miro-1 promoted more mitochondria transfer to damaged cells and efficiently repaired damaged cells.On the contrary, inhibition of Miro-1 in MSCs, negatively affected the ability of MSCs to transfer mitochondria, hence, rescue effect was lost [60].Therefore, targeted modification of the Miro1 levels of MSCs to enhance their donation of mitochondria to damaged cells may be a new way to improve the mitochondrial transfer of MSCs to achieve better therapeutic effects.The effectiveness of Miro1 in transferring mitochondria, made Guy Las and Orian S Shirihai named it as the "wheel" of MSCs mitochondrial transfer [61].The mitochondrial transfer mechanisms of MSC are shown in Figure 2.

Conclusions and perspectives
Many evidences show that mitochondrial transfer from MSCs has a non-negligible effect on the repair of damaged cells/tissues, which brings bright prospects for early intervention and treatment of mitochondrial dysfunction diseases [62].Although the transfer of mitochondria from MSCs to damaged cells has achieved gratifying results in tissue repair, with further research, it has been found that MSCs mitochondria can be transferred to tumor cells through TNTs, and can enhance the chemoresistance of tumor cells, hence, promoting cancer cells proliferation and finally aggravating the development of diseases [24,[63][64][65][66][67].Therefore, inhibiting the transfer of mitochondria to tumor cells could be a potential therapeutic target for the treatment of cancer.The use of drugs such as vinca alkaloids or Taxanes, TNF-α inhibitors, and proposed kinases/phosphatases are some of the selective restrictors on the delivery of mitochondria with their effects targeting microtubule polymerization, TNT and GJ protein (Cx43), respectively so as to avoid unwanted metastasis [20].
A better understanding and application of MSCs mitochondrial transfer, could provide a broad prospect for regenerative medicine to develop powerful cell-derived therapies.Future research work could consider increasing the level of Miro1 of MSCs and selectively promote/inhibit the targeted mitochondrial transfer of MSCs, which will be the key considerations for the transformation of this emerging treatment method into clinical application.

Figure 2 Mechanisms of mitochondrial transfer MSCs
HUVEC, human umbilical cord vein endothelial cell; MELAS, Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes; TNT, tunneling nanotubes; CS, cigarette smoke; mBMSCs, mouse Bone marrow-derived stromal cells; ALI, acute lung injury; hBMSCs, human Bone marrow-derived stromal cells; AM, alveolar macrophages; ARDS, Acute Respiratory Distress Syndrome, OGD, oxygen-glucose deprivation; GJIC, gap junction intercellular communication; EC, epithelial cell; ATP: adenosine triphosphate; AML, acute myeloid leukemia; MMSC, multipotent mesenchymal stem cells; NSCs, neural stem cells CECs, corneal epithelial cell.what molecules in the damaged cell/tissue environment serve as signals to trigger mitochondrial transfer of MSCs?Studies have shown that, the mtDNA released by damaged cells is phagocytosed by MSCs and replaced with MSCs' mitochondria.This cytoprotective function of MSCs are triggered by retrograde signals (stress signals) to stimulate mitochondrial biogenesis of MSCs for the donation.The stress signals released by recipient cells include mtDNA and mitochondrial products characterized by damage associated molecular patterns (DAMPs)