Regulation of the mitochondrial reactive oxygen species: Strategies to control mesenchymal stem cell fates ex vivo and in vivo

Abstract Mesenchymal stem cells (MSCs) are broadly used in cell‐based regenerative medicine because of their self‐renewal and multilineage potencies in vitro and in vivo. To ensure sufficient amounts of MSCs for therapeutic purposes, cells are generally cultured in vitro for long‐term expansion or specific terminal differentiation until cell transplantation. Although physiologically up‐regulated reactive oxygen species (ROS) production is essential for maintenance of stem cell activities, abnormally high levels of ROS can harm MSCs both in vitro and in vivo. Overall, additional elucidation of the mechanisms by which physiological and pathological ROS are generated is necessary to better direct MSC fates and improve their therapeutic effects by controlling external ROS levels. In this review, we focus on the currently revealed ROS generation mechanisms and the regulatory routes for controlling their rates of proliferation, survival, senescence, apoptosis, and differentiation. A promising strategy in future regenerative medicine involves regulating ROS generation via various means to augment the therapeutic efficacy of MSCs, thus improving the prognosis of patients with terminal diseases.

phosphorylation (OXPHOS), while mitochondrial respiration generate a certain amount of reactive oxygen species (ROS) as byproducts.
NADH-coenzyme Q oxidoreductase (Complex I) and ubiquinol-cytochrome c oxidoreductase (Complex III) release electrons for generation of superoxide anion (O 2 -.). 2 Superoxide dismutase (SOD) will be activated for generation of hydrogen peroxide (H 2 O 2 ) and then H 2 O 2 will be decomposed to O 2 and H 2 O after activation of catalase (CAT) or glutathione peroxidase (GPx). 2 In addition, other metabolic intermediates including 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase, glycerol 3-phosphate dehydrogenase also contribute to the upregulated generation of ROS in MSCs. 3  Commonly, ROS can be generated by mitochondria, 10 endoplasmic reticulum (ER), 11 cytosol, 12 peroxisomes, 13 plasma membrane, 14 and extracellular space. 15 Aerobic microenvironment brings out reduction of molecular oxygen, and then Ero1p will consequently yield stoichiometric H 2 O 2 . 11 All-trans arachidonic acid is able to improve the generation of ROS via xanthine dehydrogenase/xanthine oxidase interconversion in the in vitro liver cytosol. 12 After peroxisomes are supplemented with uric acid, H 2 O 2 will be generated at a rate which corresponds to 42%-61% of the rate of uric acid oxidation. 13 Fibroblast contains an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate a major source of ROS after tissue damage. 14 NADPH oxidase maintains endothelial cell xanthine oxidase activity for upregulating ROS generation in response to oscillatory shear stress. 15 Although ROS are generated by various organelles, the mitochondrion is the main organelle of ATP and ROS generation. 16 Respiratory chain electrons escape the mitochondrion, leading to superoxide ion and H 2 O 2 generation due to superoxide dismutation via SOD catalysis in MSCs. [17][18][19][20] Overproduction of ROS enhances autophagy and apoptosis in MSCs through activation of c-Jun NH(2)-terminal kinase (JNK) signalling. 21 In this process, colocalization of ataxia telangiectasia mutated (ATM), histone H2A.X, and p53-binding protein 1 (53BP1) results in Bcl-2-associated X protein (BAX)-Bcl-2 homologous antagonist/killer (BAK) dimerization; subsequent release of cytochrome c to the cytoplasm and caspase release triggers apoptosis. 22,23 For cellular homeostasis, endogenous scavengers help to remove excessive ROS, including the following: enzymatic proteins such as SOD, peroxiredoxins, glutathione peroxidase and lysosomal catalases; and non-enzymatic antioxidants such as vitamins, carotenoids and flavonoids. [24][25][26][27] Under physiological conditions, mitochondrially derived ROS include superoxide anion, hydroxyl radical, singlet oxygen, hydrogen peroxide, nitric oxide, and peroxynitrite. 28 However, the incomplete oxidation of oxygen to water by mitochondrial complexes leads to excessive ROS production. Various conditions including ageing, long-term culture and H 2 O 2 , adverse oxygen content, high glucose, proinflammatory cytokines and other toxic factors, abnormally up-regulated ROS production can harm MSC activities. 22,[29][30][31][32][33] On the other hand, although MSCs play critical roles on immunomodulatory properties for therapy in various diseases, the constant isolation from donors also bring out unrecoverable injury. 34 F I G U R E 1 Physiologically up-regulated ROS production is essential for MSC proliferation and differentiation In the current review, we mainly discuss the known ROS generation mechanisms and the regulatory routes for controlling MSC fates.
Some key signalling pathways for maintaining energy metabolism and ROS homeostasis play particularly important roles in regulating MSC fates ex vivo and in vivo (Table 1). Based on these regulatory pathways, it may be possible to control MSC fates in vitro and in vivo by regulating ROS levels surrounding MSCs for future regenerative medicine.

MICROE NVIRON MENTS FOR REGULATING ROS PRODUCTION IN MSCS
Stem cells have been assumed to rely on anaerobic energy metabolism, as they are always in a hypoxic microenvironment before they are isolated from the source tissues. Because MSCs cultured in vitro are sensitive to the surrounding microenvironment, optimization of culture conditions is important for long-term culture without loss of stem cell properties.
Contact culture at 100% confluence significantly increases ROS levels and promotes MSC senescence without influencing expression levels of telomerase reverse transcriptase (TERT) and p53. 35 Moreover, when added to the culture medium, multiple factors have a negative impact on MSC properties. For example, particulate matter or old rat serum inhibits MSC proliferation and increases ROS formation by attenuating signalling via AKT phosphorylation and activating Wnt/β-catenin signalling, respectively. 36,37 On the other hand, fibroblast growth factor 23 (FGF-23) promotes MSC senescence by up-regulating p53 and p21 expression levels, 33 38 In contrast, preconditioning MSCs with tumor necrosis factor alpha (TNF-α) strongly enhances their proliferative and migratory capacities; indeed, this cytokine effectively increases the survival rate and migratory capacity of MSCs even under oxidative stress in vivo. 39

DIFFE RENTIATION FATE OF MSCS
In general, undifferentiated MSCs have fewer mitochondria than differentiated MSCs, though the mitochondrial copy number, OXPHOS supercomplex, SOD expression, mitochondrial biogenesis and ROS levels are all significantly increased after specific differentiation. 51,52 ROS imbalance will lead to a significantly reduced MSC differentiation capacity, 53

| Growth factors or extracellular matrix components for ROS regulation in MSCs
To

| Extracts from natural products for ROS regulation in MSCs
In addition to drugs used in the clinic, a large number of extracts from natural products, including herbs, nutritional factors, and hor- Rb expression, consequently accelerating ageing and osteogenic differentiation defects. 124 Similarly, MSCs isolated from Tks4 knock-out mouse have reduced osteogenic and adipogenic differentiation capacities compared with those isolated from wild-type mice. 125 Although knockdown of NOX4 suppressed ROS production and adipogenic differentiation in MSCs, 126 Kim et al 43

CONF LICT OF I NTEREST
The authors declare no competing financial interests.