Stem cell‐derived mitochondria transplantation: A promising therapy for mitochondrial encephalomyopathy

Abstract Mitochondrial encephalomyopathies are disorders caused by mitochondrial and nuclear DNA mutations which affect the nervous and muscular systems. Current therapies for mitochondrial encephalomyopathies are inadequate and mostly palliative. However, stem cell‐derived mitochondria transplantation has been demonstrated to play an key part in metabolic rescue, which offers great promise for mitochondrial encephalomyopathies. Here, we summarize the present status of stem cell therapy for mitochondrial encephalomyopathy and discuss mitochondrial transfer routes and the protection mechanisms of stem cells. We also identify and summarize future perspectives and challenges for the treatment of these intractable disorders based on the concept of mitochondrial transfer from stem cells.


| INTRODUC TI ON
Mitochondria are multifunctional cellular organelles that have a critical role in energy production via oxidative phosphorylation. 1 process, but also contribute to many other processes, such as cell survival and autophagy. 3 The OXPHOS complexes in mitochondria are dually encoded by the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA). Mutations in mtDNA or mitochondrial nDNA can cause fatal or severely debilitating disorders, 4 such as mitochondrial encephalomyopathies, which occur in the neuromuscular system. The traditional "mitochondrial cocktail" has little therapeutic effect on mitochondrial encephalomyopathies. 2 Mitochondria dysfunction have unusual characteristics that may be treated from the cell level to the molecular level. 2 Gene therapy prior to conception and reproductive technology to uncouple the inheritance of mtDNA from nDNA may offer possible solutions for mitochondrial encephalomyopathies. However, treatments for current mitochondrial patients still face numerous challenges.
By understanding the molecular pathogenesis of mitochondrial diseases, it has been possible to develop some targeting therapy approaches, such as DNA manipulation and small-molecule pharmaceuticals. 2,5,6 However, the heterogeneity of mitochondrial encephalomyopathies and double-membrane structure make these therapy approaches difficult to materialize. 2,7 The development of stem cell therapy may offer great promise for mitochondrial encephalomyopathies. 2,[8][9][10][11][12][13][14][15] The therapeutic mechanism includes paracrine cytokines, modulation of the immune system, and transdifferentiation effects. 2,16 Recently, stem cells have been found to donate healthy mitochondria to injured cells to rescue aerobic respiration and recover their metabolism capability. This is being considered as a new therapeutic strategy for tissue damage, 17,18 especially for mitochondrial diseases. 15 Here, we summarize and discuss the current research on mitochondrial transfers and their protection mechanisms to provide an update on stem cell therapy targeting mitochondrial encephalomyopathy.

| CLINIC AL AND PRECLINIC AL E VIDEN CE FOR S TEM CELL THER APIE S IN MITOCHONDRIAL EN CEPHALOMYOPATHY
More and more research evidence supports the effects of stem cell therapy in some neurological diseases with mitochondrial dysfunction. 19 One of the most representative mitochondrial diseases that benefits from stem cells therapy is mitochondrial neurogastrointestinal encephalopathy (MNGIE), which is due to thymidine phosphorylase (TP) gene mutations, leading to secondary mitochondrial DNA damage. 20 Stem cell therapies can recover TP enzyme function and improve the prognosis of MNGIE patients, which provides the initial evidence to support the effects of stem cell therapy in mitochondrial encephalomyopathy. [20][21][22][23] The relative lack of mtDNA-based animal models limits stem cell research on mtDNA-related mitochondrial encephalomyopathies. 24 Encouragingly, some in vitro studies have demonstrated stem cells donate healthy mitochondria to replace dysfunctional mitochondria and recover energy metabolism in different types of recipient cells. 18,[25][26][27][28][29][30][31][32][33][34][35][36] Mesenchymal stem cells (MSCs) are shown to transfer their own mitochondria into mtDNA-depleted cells. 37 Moreover, Wharton's jelly MSCs can also transfer mitochondria to stressed mitochondrial encephalopathy fibroblasts to eliminate mutation burden, rescue mitochondrial functions, and resist against apoptotic stress, which demonstrates the protective effects of stem cell-derived mitochondria transplantation in an in vitro model of mitochondrial encephalomyopathy. 15,38 Recently, in an in vivo study, transplanted pluripotent stem cell-derived MSCs can transfer their own mitochondria to recipient cells to protect against damaged retinal ganglion cell. 35 These findings pave the way for clinical therapy study on mtDNA-related mitochondrial encephalomyopathies through stem cell-derived mitochondrial transplantation.

| TRIGG ERING MECHANIS MS FOR MITOCHONDRIAL RELE A S E
The transfer of stem cell mitochondria is a complex and intriguing phenomenon. The intercellular communication between recipient cells and stem cells may set up a specific "find-me" and "rescue me" signal connection in the local injured regions. 18 Mitochondrial damage appears to be the main trigger for release of the mitochondria. 39 For the mitochondrial encephalomyopathies, injured mitochondrial components and other molecules are secreted to the periplasmic space as triggering signals by stressed cells. 40 42 The loss of cytochrome c can trigger mitochondrial transport from stem cells to the injured cells. 43 In addition, mitochondrial components and mtDNA also play a role in damage-associated molecular patterns. [44][45][46][47][48] The cytokines interleukin-1 (IL-1), IL-4, IL-10, and tumor necrosis factor alpha (TNFα) can be perceived by stem cells and can also act as triggering signals. 49 Jiang et al. found that the high production of TNFα from the retinal ganglion cells results in mitochondrial release from stem cells. 35 The translocation of p53 in neurotoxic recipient cells sends  50 When stem cells receive these triggering signals from cells with mitochondrial dysfunction, the intrinsic mechanisms in stem cell begin to regulate the mitochondria transfer. [49][50][51] The CD38/CADPR/Ca 2+ pathway is also shown to mediate astrocytes to provide their mitochondria to the damaged neurons. 52 It is necessary to explore whether the CD38/CADPR/Ca 2+ pathway also works in stem cells. These findings suggest that stem cells might perceive some degree of metabolic dysfunction in adjacent cells with mitochondrial disorders and prepare to initiate mitochondrial transfer.

| PATHWAYS OF MITOCHONDRIAL DELIVERY
Several different routes have been found to participate in mitochondrial transmission from stem cells to recipient cells, which include tunneling nanotube (TNT) formation, release of extracellular microvesicles, cellular fusion, and mitochondrial extrusion ( Figure 1A).
The molecular mechanisms mediating different intercellular transmission routes are complex. A clear understanding of these routes and mechanisms will be a benefit to stem cell therapy in mitochondrial diseases.

| Tunneling nanotubes
Tunneling nanotubes are actin-based cytoplasmic extensions connecting cells as intercellular channels 50-1000 nm in diameter in a wide variety of cell types. Ramírez-Weber initially described a kind of membrane nanotube when studying drosophila wing imaginal disks. 53 The tunneling nanotubes were then defined by Rustom et al. in a rat PC12 cell-human 293 cell co-culture. 54  Connexin 43 (Cx43) has also been demonstrated to mediate intercellular communication through TNTs. 67 Overexpression of Cx43 facilitated mitochondrial transmission from stem cells to epithelial cells through the upregulation of tunnel tube formation. 68,69 The stress caused by rotenone or TNFα has also been shown to induce nanotube formation. 57 The TNFα/NF-κB/TNFαip2 pathway is upregulated in response to TNFα 70 ; then, stem cells further promote the formation of TNT. 71 Inflammation by interferonγ or lipopolysaccharide has also been shown to promote the expression of M-Sec proteins associated with TNT formation. 72 [76][77][78][79][80] Due to the small size, exosomes are unlikely to carry intact mitochondria. 56 Instead, they are able to transfer organelle fragments and genetic components. 56 As larger EVs (50-1000 nm), microvesicles can contain both intact mitochondria and mtDNA. 52,81 Microvesicles are more heterogeneous structures independent of cell origin. 56 The mechanisms of microvesicle biogenesis are associated with TSG101 protein recruitment to the cell surface. 82  shown that astrocytes can produce extracellular mitochondria that enter neurons to improve neuronal activity after ischemic stroke. 52 These studies suggest that intercellular mitochondria transmission through microvesicles is an important route to rescue mitochondrial function in the damaged cells. 3 Recently, apoptotic bodies generated from cells undergoing apoptosis have been demonstrated to be rich in mitochondria and mitochondrial components. 83

| PROTEC TION MECHANIS MS ON RECIPIENT CELL S
After the process of intercellular delivery, stem cell-derived healthy mitochondria able to enter the recipient cells with defective mitochondria and corporate with the endogenous energy metabolism network. 39 Existent data suggest that stem cell-derived mitochondria might improve survival of recipient cells through rescuing aerobic respiration and energy metabolism, regulating mitophagy and mitochondrial biogenesis, optimizing mitochondrial dynamics, and decreasing the mtDNA mutation load ( Figure 1B).

| Rescuing aerobic respiration and energy metabolism directly
Spees et al. firstly show that the mitochondria transfer from adult stem cells can directly rescue aerobic respiration in recipient cells. 25 It has also been demonstrated that the stem cells able to rescue cybrid cells of myoclonic epilepsy with ragged red fibers (MERRF) through providing intact mitochondria and improving mitochondrial bioenergetics. 15 Likewise, bone marrow-derived MSCs able to rescue energy metabolism of the cells under oxidative stress through transporting healthy mitochondria in vitro. 93 MSCs can also transfer intact mitochondria to protect against acute central nervous system 17 or lung injury in vivo. 30 Therefore, it is a quick and direct way that stem cell-derived mitochondria incorporate into the endogenous mitochondrial network to repair metabolic machinery.

| Regulating mitochondrial biogenesis and mitophagy
For mitochondrial encephalomyopathy, stem cell-derived healthy mitochondria are also important pathways for intracellular quality control of mitochondria. Mitophagy and biogenesis are coordinated and opposing pathways that regulate mitochondrial quality control and metabolism. 94 Mitochondrial biogenesis is intricate process that includes transcription and translation of nuclear and mitochondrial genomes, recruitment, and import of mitochondrial proteins and lipids. [94][95][96] Mitochondrial biogenesis is rigorously controlled by intracellular signaling pathways and the activation of nuclear transcription factors, such as peroxisome proliferator-activated receptor gamma, coactivator 1 α, nuclear respiratory factors, and transcription factor A. 96 The kinase pathways, second messenger molecules, and hormones participate in regulating the complex process. 95,96 The import of stem cell-derived mitochondria and components may provide a quick supplement for mitochondrial biogenesis, and the exogenous mitochondria also require coordination with intracellular network in recipient cells.
Parkin-dependent process, which is regulated by induced putative kinase 1 (PINK1), is a well-studied mechanism of mitophagy. [100][101][102] Besides, FUN14 domain containing 1, Bcl2 interacting protein 3 (BNIP3) or BNIP3L can directly recruit autophagosomes to injured mitochondria via interaction with LC3. 100 In addition to canonical mitochondrial degradation, mitochondrial-derived vesicle is one important kind of mitophagy pathway. 100,103 Mitochondrial spheroid also distincts from other autophagy pathways and represents the structural remodel of mitochondria in response to oxidative stresses. 100,[104][105][106][107] The transmitophagy has also been observed in stem cells that mitochondria released from damaged cells are engulfed by stem cells, 108,109 then stem cells can degrade the damaged mitochondria and produce healthy mitochondria against programmed cell death. 42 Except the mitochondrial transfer, the transport of progenitor cell-derived lysosomes via TNT is also observed and involved in the autophagy process of stressed cells. 110 Therefore, stem cells may involve in regulating both intra-and intercellular mitophagy and biogenesis process of mitochondria in host cells with mitochondrial disorders.

| Optimizing mitochondrial dynamics
As high dynamic organelles, mitochondria fission and fusion are crucial for quality control. The fission can segregate dysfunctional mitochondria, while fusion can share healthy mitochondrial components. 100,111,112 Mitochondrial encephalomyopathies always have the imbalance of mitochondrial dynamics in skeletal muscle or nervous system. 113 It has the possibility that stem cell-derived mitochondria may involve in the mitochondria dynamics in recipient cells of mitochondrial encephalomyopathies, especially the fission and fusion process. 108  acquiring healthy mtDNA from ambient cells. 39 It is also proposed that stem cells can partly reduce mtDNA mutation load via providing healthy mtDNA and mitochondria, which is sufficient to recover the mitochondrial respiration in MERRF cybrid cells long term. 15 The routes of mtDNA transfer not only restrict to TNTs, microvesicles, cellular fusion, and mitochondrial extrusion, [125][126][127] but also including more tiny structures such as exosomes and gap junction. 127,128 However, emerging data challenge the potential therapeutic use of EV-based delivery systems for mtDNA-based diseases. 129  Moreover, the mechanisms through which stem cell-derived mitochondria can be incorporated into the endogenous energy metabolism network remain to be elucidated. 39 Furthermore, mitochondrial damage and ROS are considered to be probably involved in the inflammation. 133 The mitochondrial dysfunction could also play an important role in chronic inflammation of the neurodegenerative disorders and mitochondrial diseases. 134 The transplantation of stem cell-derived mitochondria may be used as an effective treatment for these pathologies, which may attenuate production of ROS and have immunomodulatory effects. 133,134 Future research on the molecular mechanisms underlying the improvement of aerobic respiration, dynamics, and the quality control of transplanted mitochondria will accelerate the development of stem cell treatment in mitochondrial encephalomyopathies.
However, most current studies on mitochondrial disease remain in the in vitro stage due to a lack of mtDNA-based animal models of

| CON CLUS ION
Currently, the treatment of mitochondrial encephalomyopathy faces serious challenges. 39 Restoring the function of mitochondria and rescuing damaged mitochondria are crucial for treating mitochondrial disorders. Stem cell-derived mitochondria transplantation has been demonstrated to play a significant role not only in metabolic rescue but also in mitochondrial dynamics, quality control, and reduction of mutation load, which may eventually prevent cell apoptosis. Thus, the therapy offers great promise for mitochondrial encephalomyopathies. Meanwhile, mitochondrial integrity and mitochondrial dynamics also become dysfunctional during some neurodegenerative diseases. [135][136][137][138] The application of stem cell-derived mitochondria transplantation has also attracted attention for its potential to treat many diseases with the pathogenesis of mitochondrial dysfunction, such as cerebral vascular disease, 139,140 Parkinson's disease, dementia, amyotrophic lateral sclerosis, myocardial ischemia-reperfusion injury, and acute lung injury. 30,71,74,108,[141][142][143][144] However, as one of the most representative and intractable mitochondrial diseases, mitochondrial encephalomyopathies should be the first to see a breakthrough and benefit from this novel treatment strategy.

ACK N OWLED G EM ENTS
Study Funded by the Shandong Provincial Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.