Accepted Manuscript Title: Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer Author: Yi-Chao Hsu Yu-Ting Wu Ting-Hsien Yu Yau-Huei Wei PII: DOI: Reference:
S1084-9521(16)30053-2 http://dx.doi.org/doi:10.1016/j.semcdb.2016.02.011 YSCDB 1967
To appear in:
Seminars in Cell & Developmental Biology
Received date: Revised date: Accepted date:
31-12-2015 3-2-2016 5-2-2016
Please cite this article as: Hsu Yi-Chao, Wu Yu-Ting, Yu Ting-Hsien, Wei YauHuei.Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.02.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondria in mesenchymal stem cell biology and cell therapy: from cellular differentiation to mitochondrial transfer
Yi-Chao Hsu1#, Yu-Ting Wu1,2#, Ting-Hsien Yu1,2, and Yau-Huei Wei1,2
1
Institute of Biomedical Sciences, Mackay Medical College, New Taipei City 252
2
Institute of Biochemistry and Molecular Biology, National Yang-Ming University,
Taipei, Taiwan 112
*Corresponding Author: Dr. Yau-Huei Wei, Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan 252 Tel: 886-2-26360303 ext. 1100, Fax: 886-2-26361134 E-mail: [email protected]
#
These two authors made equal contributions to this work
1
Highlights 1. An integrated view on different protein kinases and sirtuins in MSCs is provided. 2. The regulation of mitochondrial biogenesis and metabolism in MSCs are summarized. 3. Recent advances in mitochondrial dynamics and transfer are discussed.
2
Abstract Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying 3
mechanisms in the regulation of mitochondrial metabolism of MSCs may ultimately improve therapeutic outcomes of stem cell therapy in the future.
Abbreviations: AMPK, AMP-activated protein kinase; BM, bone marrow; CAT, catalase;
Complex
I,
NADH-coenzyme
Q
oxidoreductase;
Complex
II,
succinate-coenzyme Q oxidoreductase; Complex III, ubiquinol-cytochrome c oxidoreductase; COX, cytochrome c oxidase; Complex V, FoF1ATPase; DAPT, N-[N-(3,5-difluor-ophenacetyl-L-alanyl)]-S-phenylglycine t-butylester; ETC, electron transport chain; ESCs, embryonic stem cells; MSCs, mesenchymal stem cells; FOXO3a, forkhead box O 3a; GPx, glutathione peroxidase; HIF-1α, hypoxia inducible factor-1α; HLA-DR, human leukocyte antigen - antigen D Related; H2O2, hydrogen peroxide; LC3, type II light chain 3; mTOR, rapamycin serine/threonine kinase; Miro1, mitochondrial Rho-GTPase 1; Mfn, mitofusin; mtTFA, mitochondrial transcription factor A; NRFs, nuclear respiratory factors; OPA1, optic atrophy protein 1; OXPHOS, oxidative phosphorylation; O2-., superoxide anion; p70S6K, p70 S6 kinase; PGC-1α, proliferator activated receptor gamma coactivator-1α; PSCs, pluripotent stem cells; PKC, protein kinase C; PDH, pyruvate dehydrogenase; PDHC, pyruvate dehydrogenase complex; PDK, PDH kinase; PSCs, pluripotent stem cells; PTM, post-translational modification; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen
species;
SOD,
superoxide
dismutase;
SREBP-1,
sterol
regulatory
element-binding protein 1; Sirt, sirtuins; TCA cycle, tricarboxylic acid cycle; TSC1/2, tuberous sclerosis complex1/2; TNTs, tunneling nanotubes.
Keywords: mitochondrial metabolism, mitochondrial transfer, mesenchymal stem cells, sirtuins. 4
1. Introduction Stem cells are capable of self-renewal and have the potential of differentiating into multiple cell lineages. In light of the rapid progress in stem cell research in the last decade, stem cell therapy has hold great promise in regenerative medicine. The role of mitochondria in the maintenance and differentiation of stem cells has attracted increasing attention. Mesenchymal stem cells (MSCs) are adherent fibroblast-like multipotent cells, which can differentiate into mesoderm cell lineages such as osteoblasts, chondrocytes, myoblasts and adipocytes [1]. Recently, it was demonstrated that MSCs could differentiate into cells in different germ layers, such as ectodermal neuronal-like cells as well as endodermal hepatocyte-like cells [2, 3]. MSCs were first derived from bone marrow (BM) [1], and were identified and characterized by Friedenstein [4]. MSCs have been isolated from the adipose tissue [4], umbilical cord blood [5, 6], amniotic fluid [7], placenta [8-10] and dental pulp [11]. The Mesenchymal and Tissue Stem Cell Committee of International Society for Cellular Therapy has defined human MSCs based on the following four criteria: (1) plastic-adherent and fibroblast-like cells; (2) the ability of osteogenic, chondrogenic and adipogenic differentiation; (3) simultaneous expression of CD105, CD73, CD90 and CD105; (4) lack of expression of CD11b, CD14, CD19, CD34, CD45, CD79a and human leukocyte antigen-DR [12]. MSCs are full of potential for cell therapies due to their multipotency and the therapeutic advantage of hypo-immunogenicity [13], low tumorigenicity [14] and the capacity of mitochondrial transfer [15, 16]. During tissue injury, MSCs can migrate preferentially to the injured sites in the organs such as the heart [17], brain [18, 19], skeletal muscle [20] and kidney [21]. The therapeutic potential of MSCs have been demonstrated in different diseases such as myocardial infarction [22-26], acute renal failure [27], pulmonary hypertension [28-32], sepsis [33, 34], pulmonary fibrosis [28, 35], emphysema [36, 37], diabetes 5
[38-41], asthma [42-45], bone defects [46], graft-versus-host disease [47], ischemic stroke [48] and hepatic failure [49]. In light of the potential of applications of MSCs in regenerative medicine and cell therapy, the roles of mitochondrial metabolism and dynamics in the maintenance and differentiation of MSCs have received increasing attention from clinicians and life scientists [50]. The switch of energy source from glycolysis to aerobic metabolism is a hallmark of the differentiation of MSCs, which is referred to as the metabolic shift. Accumulating evidence has revealed that the metabolic shift and upregulation of mitochondrial function is essential for successful differentiation of MSCs [51]. In this review, we synthesize and discuss recent progress in the studies of metabolism and dynamics of mitochondria in the differentiation and therapeutic application of MSCs.
2. Mitochondrial biogenesis and upregulation of antioxidant enzymes occur concurrently in the differentiation of MSCs Mitochondria produce the majority of ATP through respiration and oxidative phosphorylation (OXPHOS). The electron transport chain (ETC) is composed of NADH-coenzyme
Q
oxidoreductase
(Complex
I),
succinate-coenzyme
Q
oxidoreductase (Complex II), ubiquinol-cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (COX, Complex IV). The electron flow through the ETC establishes a proton gradient across the inner membrane, which is utilized to drive ATP synthesis by the FoF1ATPase (Complex V) [52]. Upregulation of mitochondrial biogenesis [53] and aerobic metabolism [51] are the hallmarks of the differentiation of MSCs. Mitochondrial biogenesis is regulated by extracellular stimuli such as cold exposure, fasting, exercise, oxidative stress and cell differentiation. It is established that proliferator activated receptor gamma coactivator-1α (PGC-1α) regulates 6
mitochondrial biogenesis by increasing the expression of mitochondrial biogenesisassociated genes, such as nuclear respiratory factors (NRFs) [54]. NRF-1 and NRF-2 are transcription factors, which regulate nuclear DNA-encoded proteins involved in mitochondrial respiration and OXPHOS. In addition, NRF-1 can activate the expression of mitochondrial transcription factor A (mtTFA), which binds to the D-loop of mtDNA and coordinates with DNA polymerase γ to replicate mtDNA [55]. Many studies have revealed that defects in mitochondrial biogenesis or OXPHOS are involved in the pathophysiology of mitochondrial disease, metabolic syndrome, heart disease, and type 2 diabetes [56]. We have demonstrated that upon osteogenic induction of MSCs, proteins involved in mitochondrial biogenesis are all increased, which include mtTFA, DNA polymerase γ and PGC-1α, and some of the enzymes of TCA cycle and protein subunits of respiratory enzymes [53]. Furthermore, it was reported that osteogenic differentiation of MSCs is accompanied by cristae development and increase in the expression levels of the constituent subunits and activity of respiratory enzyme complexes [57, 58]. Moreover, it was found that the respiration rate, mitochondrial membrane potential and intracellular ATP content were also increased, indicating an upregulation of oxidative metabolism in mitochondria during the differentiation of MSCs [53]. ATP synthesis is coupled with respiration in normal mitochondria, which also produce a certain amount of reactive oxygen species (ROS) as byproducts. During respiration of mitochondria, the electrons may leak out from Complex I and Complex III to react with O2 to generate the superoxide anion (O2-.) [59], which can be converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) and further decomposed to O2 and H2O by catalase (CAT) or glutathione peroxidase (GPx). Excess ROS can cause DNA damage, lipid peroxidation and oxidative modification of proteins, which are detrimental to cell function [60]. Accumulated evidence has 7
substantiated the notion that oxidative stress can lead to a wide spectrum of human diseases such as cardiovascular disease, neurological disorders, diabetes and cancers [61]. Therefore, the intracellular levels of ROS should be effectively controlled by the antioxidant defense system, including SOD, CAT and GPx. MSCs express significant levels of active forms of CAT, GPx and SOD, which confer the differentiating cells with the ability to cope with ROS-mediated oxidative stress and avoid damage [62]. It was reported that the mitochondrial ROS generated from Complex III during respiration is also involved in the regulation of adipogenic differentiation [63]. We found that MnSOD, CAT, GPx and glutathione reductase are up-regulated during adipogenic differentiation of human mesenchymal stem cells [64]. Furthermore, we found that forkhead box O 3a (FOXO3a), an upstream factor that regulates MnSOD transcription, is involved in the upregulation of antioxidant enzymes at the early stage of adipogenic differentiation of MSCs [64]. It has been shown that adipogenic differentiation is associated with the up-regulation of FOXO1 to counteract the increase in the ROS production, which has a pivotal role in maintaining cellular redox homeostasis and regulate the expression of antioxidant enzymes (e.g., MnSOD and CAT) [65]. Moreover, it was reported that activation of FOXO1 is involved in osteogenic differentiation [66].
3. Metabolic switch during the differentiation of MSCs In a previous study, we demonstrated that oxidative metabolism is activated by induction of the expression of respiratory enzymes and increase of the OXPHOS of mitochondria during osteogenic differentiation of MSCs [53]. In addition, glycolysis is reduced during osteogenic differentiation of MSCs as revealed by a dramatic decrease in the production of lactate [67]. These findings suggest a metabolic switch upon the osteogenic differentiation of MSCs. A similar change in metabolic profile 8
was also observed during adipogenic differentiation. A remarkable increase in mitochondrial respiration and decrease in glycolytic flux were observed in adipocytes differentiated form MSCs (Fig. 1). In particular, the metabolic state of stem cells may depend on their niche. Most differentiated cells are located around the blood vessels for ample supply of oxygen to support oxidative metabolism. By contrast, the stem cells or transplanted MSCs in the injured tissues are usually exposed to low oxygen tension or hypoxic stress that disturbs aerobic metabolism. Under such conditions, cells utilize anaerobic glycolysis to supply the majority of energy needed for the execution of cellular functions. It was demonstrated that hypoxia inducible factor-1α (HIF-1α) can suppress oxidative metabolism through inhibition of pyruvate dehydrogenase (PDH) by PDH kinase (PDK) and activate the expression of glycolytic enzymes [68]. We further showed that hypoxia could increase HIF-1α expression and inhibit osteogenic differentiation by blocking the metabolic switch of MSCs [69, 70]. In addition, it was observed that adipogenic differentiation of MSCs was inhibited by HIF-1α and enhanced by knockdown of HIF-1α under both normoxia and hypoxia [71].
4. Post-translational protein modifications in the regulation of mitochondrial function and metabolic shift during MSCs differentiation Although human tissue-derived MSCs hold great promise for cell-based regenerative therapy of various diseases, it remains an open question as to how the differentiation capacity of MSCs can be maintained in vivo. Epigenetic mechanisms regulating gene expressions are important elements that affect differentiation and function of stem cells. In addition to the study on epigenetic and transcriptomic characteristics, systemic proteomic approaches have been applied to identify and quantify functional alterations and post-translational modifications (PTMs) of proteins in the maintenance 9
of stemness of MSCs [72-74]. In recent years, PTMs of certain target proteins have been demonstrated to play important roles in the regulation of multipotency and differentiation of MSCs. Emerging evidence shows that PTMs such as phosphorylation, glycosylation and acetylation play critical roles in the regulation of protein and enzyme functions, signaling networks and cell fates in human pluripotent stem cells (PSCs) [74-76].
4.1 Role of protein kinase in MSCs differentiation A recent study on phospho-proteome of human embryonic stem cells (ESCs) revealed that dynamic change in protein phosphorylation events occurs in the early stage of differentiation [77]. It has been shown that activation of multiple kinases is associated with different signaling pathways during stem cell differentiation. Accumulating evidence suggests that the evolutionarily conserved protein kinases act as major effectors of stem cell homeostasis and lineage differentiation via regulation of metabolism. The mammalian target of rapamycin serine/threonine kinase (mTOR) has emerged as a cellular sensor of energy status that can regulate metabolic processes, protein synthesis and cell proliferation [78]. It contains two distinct multi-protein complexes, mTORC1 (rapamycin-sensitive complex) and mTORC2. mTORC1 has a significant impact on the maintenance of metabolic homeostasis via the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway [79]. Activation of mTORC1 phosphorylates its downstream targets to up-regulate the expression levels of genes involved in glycolysis and lipid synthesis. It has been demonstrated that overexpression of mTORC1 can induce the differentiation of MSCs into adipocytes, osteoblasts and neuronal-like cells [80-82]. In adipocytes, mTOR is critical for lipid metabolism and serves as a sensor of nutrient availability to regulate the expression of PPAR. mTORC1 signaling promotes fatty acids and triacylglycerol synthesis via the 10
activation of the transcriptional factor SREBP-1 and induction of the expression of lipogenic genes [83]. It has been reported that mTORC1 signaling promotes adipogenic differentiation by activation of PPAR [84]. It has been demonstrated that activation of mTORC1 is involved in insulin-stimulated adipogenesis through PI3K/AKT-mediated inhibition of the mTOR inhibitory proteins and tuberous sclerosis complex1/2 (TSC1/2) [80, 84]. Moreover, it has been reported that transcriptional activation of CREB by p70 S6 kinase (p70S6K), the downstream target of mTOR, is involved in adipogenic differentiation of BM-derived MSCs (BM-MSCs) [80]. Inhibition of mTORC with rapamycin or shRNA silencing has been shown to reduce the expression levels of adipogenic markers and lipid accumulation in 3T3-L1 preadipocytes [85]. Negative effect of rapamycin on adipogenesis was also observed in human pre-adipocytes, human MSCs [86] and murine MSCs [78]. mTOR-mediated signaling pathways are also involved in the proliferation and differentiation of osteoblasts. Unlike the effect on adipogenesis, the effect of mTOR on osteogenic differentiation is controversial. Emerging data indicate that PI3K/mTOR signaling is critical for the maintenance of ESCs pluripotency via suppressing the induction of differentiation-related transcription factors [87, 88]. Although inhibitory effects of rapamycin on osteogenic gene expression and ALP activity were observed in a murine model, most studies support that the disruption of mTOR contributes to osteogenesis of ESCs and MSCs [82, 89]. The above-mentioned observations strongly suggest that mTOR plays a critical role in the regulation of stem cell differentiation. In recent years, numerous studies suggest that AMPK-mediated signaling pathways favor osteogenic differentiation and play a role in the lineage commitment of MSCs. AMPK is a master regulator of cellular metabolism and energy homeostasis. It can sense cellular AMP/ATP ratio and regulate metabolic activity and maintain cell survival under insufficient oxygen supply. Moreover, AMPK regulates mitochondrial 11
biogenesis through increasing the expression level or phosphorylation of PGC-1 [90]. Indeed, AMPK activation was shown to increase the expression of PGC-1α and mtTFA in MSCs [91]. One of our previous studies also showed that PGC-1 and mitochondrial biogenesis are up-regulated during osteogenic differentiation of human MSCs [53]. Interestingly, crosstalk of AMPK with Wnt-β-catenin signaling, a master pathway that determines the differentiation of BM-MSCs into osteoblasts and adipocytes, has also been demonstrated to contribute to osetogenesis of MSCs [92, 93]. It was found that the expression and phosphorylation levels of AMPK were increased during osteogenic differentiation of MSCs [94]. It has been shown that inhibition of AMPK signaling by its inhibitor, compound C, or knockdown by shRNA suppress osteogenesis of MSCs and increase the lipid droplet formation upon osteogenic induction. Conversely [95], activation of AMPK with metformin promotes osteogenesis and inhibits adipogenesis of MSCs [95]. It has been established that AMPK activation inhibits mTORC1 by phosphorylation of TSC1/2 [96]. Coordination of AMPK and AKT/mTOR signaling modulate osteogenic differentiation of MSCs. AMPK has been shown to induce autophagy through inhibition of mTOR at the early stage of osteogenesis in MSCs. Protein kinase C (PKC) is a family of serine/threonine protein kinases that is known to be involved in the regulation of glucose metabolism and mitochondrial biogenesis. Several studies have indicated that specific PKC isoforms are associated with embryonic bone formation. PKCλ/ι was reported to be involved in the upregulation of the biogenesis and function of mitochondria in stem cell differentiation [97]. Under differentiation condition, loss of PKCλ/ι impairs mitochondrial function and induces a metabolic shift to glycolysis through the HIF-1α-mediated inhibition of PDH by PDK, which increases the pluripotency of mouse ESCs [97]. In this process, stabilization of HIF-1α increases the expression of pluripotency genes while repressing the expression of PGC-1α. Interestingly, similar to the role of AMPK in MSCs 12
differentiation, PKC has a positive effect on osteogenesis. The expression and phosphorylation levels of PKC were found to increase in osteogenic differentiation of BM-MSCs [98].
4.2 Inhibitory regulation of Sirt1 and Sirt2 on adipogenic differentiation of MSCs Numerous studies have substantiated the importance of protein deacetylation catalyzed by sirtuins in the control of the function and differentiation capacity of stem cells [75, 76, 99]. Mammalian sirtuins belong to a conserved family of proteins with NAD+-dependent deacylase activities, which catalyze lysine deacylation by coupling with NAD+ hydrolysis and ADP-ribosylation [100, 101]. Seven sirtuins (Sirt1-Sirt7) are involved in the regulation of longevity, energy homeostasis, stress response and cell development in mammals. It has been thought that these enzymes affect stem cell differentiation through deacetylation of histone and non-histone proteins associated with the regulations of gene expression, cytoskeleton remodeling and metabolic reprogramming during the differentiation process [72-75]. Here, we summarize the roles of different sirtuins in the regulation of MSCs differentiation (Table 1). Among all the sirtuins, Sirt1 is the most widely studied sirtuin that is involved in epigenetic regulation of the expression of tissue-specific genes through histones deacetylation during differentiation of MSCs [102-104]. Up-regulation of Sirt1 was found to promote osteogenic differentiation of MSCs by silencing the inhibitory gene of bone formation [102, 105, 106]. In addition to acting on histones, deacetylation of transcription factors, PPAR-γ, p53, NFκB and FOXO1 has also been suggested to be involved in Sirt1-mediated regulation of lineage determination of MSCs [103]. Simic et al. [104] showed that the MSCs obtained from MSCs-specific Sirt1 knockout mice exhibited a reduction in the efficiency of osteogenic and chondrogenic differentiation, but no such difference was found in adipogenesis. Accumulated evidence suggests a 13
potential role of Sirt1 in fate decision of MSCs based on the findings that Sirt1 activation favors osteogenic differentiation while inhibits adipogenic differentiation [104, 107]. In contrast to its positive regulation of osteogenesis, Sirt1 negatively regulates adipogenic differentiation. Down-regulation of Sirt1 was observed during adipogenic induction of mouse MSCs cell lines [108-110]. We also found that the protein expression level of Sirt1 was decreased during adipogenic differentiation of adipose-derived human MSCs. However, the mechanism by which Sirt1 inhibits adipogenesis has remained unclear. Inhibition of Sirt1 by nicotinamide stimulates adipogenesis through increase of the number of adipocytes and expression of adipocyte markers. Sirt1 was shown to reduce fat and delay adipogenesis through inhibiting PPARγ signaling and stimulating lipolysis in 3T3-L1 preadipocytes [111]. It has been reported that Sirt1 regulates lipolysis and fat mobilization through the deacetylation and activation of FOXO1 transcription factor in adipose tissues [112]. Recent studies revealed that the deacetylation of FOXO1 at the forkhead DNA binding domain by Sirt1 could increase its transcriptional activity, and in turn inhibit PPARγ expression, which might contribute to the inhibitory effect of FOXO1 on adipogenic differentiation at the early stage [113, 114]. Therefore, inactivation of Sirt1 during adipogenic induction of MSCs could maintain the gene expression of PPARγ and induce adipogenic gene expression. This is in line with the observation that resveratrol blocked adipocyte development and increased the expression of osteogenic markers in MSCs [107]. Furthermore, miR-146b repressed the transcription of Sirt1 and increased adipogenic differentiation of 3T3-L1 preadipocytes, which was mediated by inhibition of the Sirt1-FOXO1 cascade [99]. The above-mentioned findings suggest that Sirt1 may serve as a key regulator to determine the adipogenic and osteogenic differentiation, respectively, of MSCs. Like 14
Sirt1, the Sirt2 protein level was also decreased at the early stage of adipogenic differentiation of MSCs [115]. Sirt2 localizes to cytosol and acts on the deacetyltion of -tubulin and regulates microtubule stability to control the cell morphology change during differentiation [116, 117]. Acetylated α-tubulin was demonstrated to facilitate the transport of mitochondria on the microtubules [118]. Acetylation of α-tubulin is increased during adipogenesis, and impairment in adipogenic differentiation was observed in the preadipocytes expressing an acetylation-resistant α-tubulin mutant in the mouse [116]. Moreover, it was shown that hyperacetylation of α-tubulin can initiate cytoskeleton remodeling by activating microtubule-severing protein, katanin, which in turn facilitates the expansion of lipid droplets and controls morphological transition towards mature adipocytes [117]. Decrease of Sirt2 expression promotes the differentiation of 3T3L1 preadipocytes via enhanced acetylation and phosphorylation of FOXO1, which promotes its cytoplasmic translocation and inactivation [119]. It was shown that FOXO1 deacetylation by Sirt2 suppressed adipogenic differentiation of MSCs through down-regulation of PPARγ [120, 121]. On the other hand, regulation of GSK3β by Sirt2 was reported to be required for early cell lineage commitment of mouse ESCs [121]. However, whether inactivation of Sirt2 is associated with lineage determination between adipogenesis and osteogenesis remains to be elucidated.
4.3 Roles of Sirt3 and Sirt5 in mitochondrial biogenesis and oxidative metabolism during adipogenic differentiation of MSCs Mitochondrial dysfunction is one of the factors causing defects in adipogenesis and dysfunction of adipocytes, which culminate in insulin resistance and type 2 diabetes [122, 123]. In a previous study, we showed that activation of oxidative metabolism is required for adipogenic differentiation [124]. Increases in oxygen consumption and 15
activities of respiratory enzyme complexes were observed in the differentiation of adipocytes. Therefore, activation of mitochondrial function is crucial for the success of the differentiation of stem cells. Sirt3 is the major sirtuin in mitochondria and is preferentially expressed in the tissues of high metabolic demand [125]. It exhibits robust deacetylase activity and acts on numerous targets that are involved in oxidative metabolism. We and other investigators have shown that Sirt3-mediated protein deacetylation plays a crucial role in the regulation of energy homeostasis through the activation of mitochondrial enzymes involved in OXPHOS, TCA cycle, -oxidation of fatty acids, urea cycle and ketogenesis, respectively [126-129]. Recent studies revealed a decline of Sirt3 in diabetic rats and that the deficiency of Sirt3 may increase the risk of developing insulin resistance and obesity [130]. These findings suggest that Sirt3 is involved in the regulation of glucose metabolism of adipocytes. However, the roles of Sirt3 during adipogenesis of MSCs and in the function of mature adipocytes have remained poorly understood. In contrast to the suppressive effect of above-mentioned sirtuins on differentiation, Sirt3 activity was shown to be essential for brown adipogenic differentiation in vitro [131]. By using an adipogenic induction system of MSCs, we found a significant increase in the protein level of Sirt3 during adipogenic differentiation (Fig. 2A-2C). We demonstrated that the expression levels of differentiation markers and adiponectin secretion were decreased in adipocytes differentiated from Sirt3-knockdown MSCs, which suggest that Sirt3 is essential for adipogenesis of MSCs (Fig. 2D-2F). We also showed that adipocytes differentiated
from
Sirt3-knockdown
MSCs
displayed
impairment
in
the
mitochondrial biogenesis including the decrease of PGC-1 mtTFA and protein subunits of respiratory enzyme complexes (Fig. 2G-I) and respiratory dysfunction (Fig. 3A). It was also reported that up-regulation of Sirt3 could enhance myogenic differentiation by activation of mitochondrial function [132]. In this study, silencing 16
of Sirt3 was found to down-regulate PGC-1 and MnSOD, which led to abnormal differentiation of myoblasts. Likewise, increase in mtDNA content and expression of mitochondrial biogenesis-related transcription factors have been observed in neurons overexpressing Sirt3 [133, 134]. These findings together suggest that Sirt3 might be involved in the up-regulation of the mitochondrial biogenesis and respiratory function during adipogenic differentiation of MSCs. In addition to lysine acetylation, malonylation and succinylation catalyzed by Sirt5 have recently been suggested to regulate the activities of some mitochondrial proteins [135, 136]. These two types of acylation are achieved by using intermediary metabolites malonyl-CoA and succinyl-CoA as the substrate, which are generated by catabolism of fatty acids and amino acids. Although the deacetylase activity of Sirt5 is very low, it is the only sirtuin known to catalyze desuccinylation and demalonylation of mitochondrial proteins [135-137]. Recent studies of a mouse model revealed an association of Sirt5-catalyzed protein succinylation with energy metabolism [135, 137]. Like the role of Sirt3 in the regulation of lipid metabolism, loss of Sirt5 results in the impairment of -oxidation of fatty acids in liver and skeletal muscle of the Sirt5 knockout mice [137]. The protein expression level of Sirt5 was decreased during adipogenic differentiation and was dramatically reduced at the terminal stage of differentiation of MSCs [138] (Fig. 2C), whereas our unpublished data showed that the succinylation and malonylation levels of proteins in mitochondria were significantly increased in MSCs after adipogenic differentiation. Whether Sirt5 is involved in adipogenic differentiation of MSCs warrants further investigation. Sirt5 was recently shown to catalyze protein desuccinylation and down-regulate the activities of pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase [137], which resulted in subsequent impairment of aerobic metabolism in human embryonic kidney cells HEK293. Mitochondrial PDHC is the pivotal regulator of 17
oxidative metabolism in mammalian cells [139]. Its deficiency decreases the conversion of pyruvate to acetyl-CoA in mitochondria and impairs aerobic metabolism. Activation of PDHC [140] and down-regulation of glycolysis [51] are important for the maintenance of high aerobic metabolism during adipogenic differentiation. It was found that acetylation of PDH-E1 inhibited its activity by increasing its phosphorylation and thereby increasing the glycolytic flux [141]. Deacetylation of PDHC by Sirt3 could activate its enzyme activity and in turn enhance oxidative metabolism [142]. Actually, we observed that Sirt3 deficiency impaired mitochondrial respiration (Fig. 3A) and induced a metabolic shift to glycolysis (Fig. 3B) during adipogenic differentiation of MSCs. In contrast to the activation of PDHC by Sirt3, Sirt5 seems to negatively regulate the activity of this enzyme. It remains to be investigated whether a decline of Sirt5 is essential for the activation of PDHC in MSCs differentiation and how Sirt3 and Sirt5 regulate aerobic metabolism, in a reciprocal manner, during adipogenic differentiation of MSCs. In addition to Sirt1, a decline of mitochondrial Sirt3 was observed in the tissues of aged mice and in mammalian cells subject to oxidative stress [126]. Accumulated evidence has indicated that Sirt3 is likely associated with an extension of lifespan of humans by counteracting oxidative stress, though some studies are controversial. Sirt3 is considered a potential regulator of the ROS detoxification due to its up-regulation on the deacetylation and activation of MnSOD and isocitrate dehydrogenase 2 to enhance the antioxidant defense of target cells [143-145]. In myocardial tissues and cardiomyocytes, Sirt3-mediated activation of FOXO3a by deacetylation eliminates excess ROS through induction of the expression of antioxidant enzymes MnSOD and CAT [146]. Our unpublished data suggest that up-regulation of Sirt3 in MSCs contributes to the increase in the protein level of FOXO3a, and that Sirt3 deficiency caused an increase of mitochondrial ROS at the initial stages of adipogenic 18
differentiation. Sirt3 deficiency could lead to defects in mitochondrial respiration and accumulation of ROS, which in turn may affect the signaling pathways involved in adipogenic differentiation and function of maturate adipocytes. Recently, it was reported that overexpression of Sirt3 in human BM-MSCs improves cell resistance to oxidative stress-induced apoptosis through the up-regulation of MnSOD and CAT. Wang et al. [132] showed that the expression level of exogenous Sirt3 was decreased in BM-MSCs under severe oxidative stress induced by a high concentration of H2O2. They further demonstrated that the reduction of Sirt3 by H2O2 increased the cellular susceptibility of aged BM-MSCs to oxidative stress [132]. In consideration of the importance of Sirt3 in the mitochondrial function and antioxidant defense, we contended that oxidative stress-induced decline of Sirt3 may affect differentiation ability of aged MSCs. Brown et al. [147] showed that up-regulation of Sirt3 could improve the regenerative capacities of aged hemopoietic stem cells. Furthermore, other sirtuins, such as Sirt6 [148, 149] and Sirt7 [150, 151] have also been suggested to play important roles in the differentiation of MSCs. Thus, manipulation of sirtuins levels in MSCs by potential drugs or compounds (e.g., resveratrol) to increase mitochondrial function and antioxidant defense during adipogenic and osteogenic differentiation can be used to improve the quality of MSCs in advance for cell therapy in future clinical applications.
5. Mitochondrial dynamics Mitochondrial fusion, fission, biogenesis, degradation, movement within the cytoplasm and even intercellular transfer contribute to mitochondrial dynamics. Three GTPase proteins, including mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy protein 1 (OPA1), are involved in the regulation of mitochondrial fusion [152]. The fusion process enables the two-way exchange of mtDNA and proteins 19
between mitochondria and between mitochondria and the nucleus to maintain precise mtDNA transmission and promote overall mitochondrial functions. During mitosis, mitochondrial fission regulates the partitioning process of mitochondrial contents and further generates heterogeneity, and thereby removes damaged mitochondria [153]. Abnormal mitochondrial dynamics has been shown to cause neuromuscular disorders [154]. Furthermore, mitochondrial fission and fusion may also play important roles in the maintenance of pluripotency as inhibition of fission in mouse ESCs will result in the loss of colony morphology and pluripotency [155]. On the other hand, depletion of fusion proteins, mitofusins 1 and 2 (Mfn1/2), facilitates the metabolic transition to glycolysis through the activation of the Ras-Raf and HIF-1α signaling pathways at the early stage of iPSC reprogramming [156]. Furthermore, the quality of mitochondrial population is maintained by the process of autophagy and mitophagy to remove the damaged mitochondria during differentiation. The up-regulation of mitochondrial respiration in the differentiation of stem cells may lead to an increase in the ROS levels, which can cause oxidative damage within mitochondria. Thus, the activation of autophagy for elimination of defective mitochondria at the early stage of stem cell differentiation might be important for the quality control of mitochondria. It was reported that the mitochondrial clearance occurs during the differentiation of erythrocytes [157, 158]. The cleaved type II light chain 3 (LC3-phosphatidylethanolamine conjugate, LC3-II) protein, a marker for active autophagosomes, has been shown to accumulate at the early stage of osteogenic differentiation of MSCs, indicating activation of autophagy upon stimulation of differentiation [96]. Autophagy inhibitors also down-regulate the osteogenic differentiation of MSCs [96]. Recently, Song et al. [159] showed that inhibition of the Notch signaling by (N-[N-(3,5-difluor-ophenacetyl-L-alanyl)]-Sphenylglycine t-butylester, DAPT), a γ-secretase inhibitor, promoted the adipogenic 20
differentiation of MSCs through the activation of autophagy. The treatment of autophagy
inhibitors
could
further
reduce
the
DAPT-induced
adipogenic
differentiation on MSCs. Nonetheless, the detailed characteristics and regulation of mitophagy during the differentiation of MSCs remain to be investigated.
6. Stem cells as vehicles for mitochondrial transfer It has been suggested that mitochondrial transfer can rescue the stressed cells [15, 160-163], reprogram the differentiated cells [164] and restore the loss of mitochondrial function in recipient cells [165] (Table 2). However, the mechanisms and conditions of mitochondrial transfer between cells remain to be clarified. In particular, different cell types, such as vascular smooth muscle cells [166], endothelial cells [167], endothelial progenitor cells [168, 169] and MSCs [160, 162, 164, 165, 170-172] and human Wharton’s jelly MSCs [163] have been suggested to be able to rescue mitochondrial function through tunneling nanotubes (TNTs). Wharton’s jelly MSCs are derived from umbilical cords from subjects undergoing full-term delivery. The umbilical cord is processed following the primary culture procedure. The isolated cells from the umbilical cord are finally resuspended in culture media comprised of high-glucose DMEM, supplemented with fetal bovine serum, glutamine, non-essential amino acids and fibroblast growth factors. Several in vivo studies also demonstrated the capacity of mitochondrial transfer via TNTs and Miro1, which is a Rho GTPase that mediates mitochondrial movement along the TNTs [161, 165, 169, 173] (Table 3). Intercellular mitochondrial transfer using stem cells as vehicles is thus promising for development of strategies in the treatment of mitochondria-associated pathologies and provides a new approach to treat diseases caused by mitochondrial dysfunction. 6.1. Mitochondrial transfer by different types of stem cells in vitro 21
In 2006, Spees et al. [160] proposed and demonstrated a mitochondria-based approach to treat mitochondrial diseases. By co-culturing the mtDNA-deficient cells with BM-MSCs, BM-MSCs effectively rescued the respiratory function by transferring their mitochondria to the mtDNA-deficient cells. The capacity of mitochondrial transfer was recently reviewed by Plotnikov et al. [174]. Furthermore, in the "metabolic state hypothesis" proposed by Dr. Prigione, it was suggested that mitochondrial state and cellular metabolism are different among stem cells at different developmental stages [175]. Notably, MSCs have been demonstrated to promote adult cardiomyocytes reprogramming back to a progenitor-like state through partial cell fusion and mitochondrial transfer [164]. Deprivation of mtDNA in the MSCs dramatically decreased the somatic reprogramming efficiency rather than cell fusion, suggesting that mitochondrial transfer from MSCs to adult cardiomyocytes helps them convert to the progenitor state through metabolic reprogramming. Until now, most studies have focused on the mitochondrial transfer of MSCs. We summarize recent findings of mitochondrial transfer by different types of stem cells in the rescue of damaged cells in vitro (Table 2).
6.2. Mitochondrial transfer as the therapeutic effects in vivo It has been shown that in a mouse model of induced acute lung injury, the therapeutic effects of MSCs are associated with mitochondrial transfer to alveolar epithelial cells and thereby allowed the mouse to recover from lung injury [15]. Furthermore, MSCs without the ability to form gap junctions or without functional mitochondria cannot transfer the organelles via cell-cell connections [16], suggesting that mitochondrial transfer may also account for the therapeutic effects of the MSCs that are rich in mitochondria. Furthermore, Li et al. [165] demonstrated that the efficiency of mitochondrial transfer of iPSCs-MSCs was higher than that from 22
BM-MSCs to rescue the cigarette smoke-induced mitochondrial damage. The improvement of fibrosis was also greater in the rats inoculated with the iPSC-MSCs than those inoculated with the BM-MSCs [165]. Furthermore, mitochondrial transfer was made possible through the tunneling nanotubes formed by MSCs, which was suggested by the observation that inhibition of the formation of tunneling nanotube blocked mitochondrial transfer [165]. We summarize the in vivo therapeutic effects of different types of stem cells through mitochondrial transfer (Table 3).
6.3. Mechanisms of mitochondrial transfer Some probable mechanisms of mitochondrial transfer have been proposed. It has been demonstrated that mitochondrial transfer does not go through endocytosis and vesicle trafficking [160]. Mitochondrial transfer by BM-derived stromal cells and epithelial cells has been shown to be mediated by actin-based tube structure named tunneling nanotubes and by connexin 43 gap junctions [15, 167]. Another mitochondrial Rho-GTPase 1 (Miro1) has been shown to regulate the movement of mitochondria [161, 176-178]. I was told that the mitochondrial acceptor cells treated with rotenone, a respiratory chain inhibitor, received more mitochondria from donor cells [161]. Co-culturing the rotenone-treated acceptor cells with MSCs reversed the ATP levels and activities of Complexes I and IV, and decreased mitochondrial ROS production and cytochrome c release [161]. Therefore, impairment of respiratory function may initiate the retrograde signaling triggered by ROS, Ca2+, AMP/ATP and NAD+/NADH ratio, for the acceptor cells to receive mitochondria from donor cells. Furthermore, when the mitochondrial donor MSCs were treated with rotenone, mitochondrial transfer was attenuated through reduction of the expression level of Miro1 [161]. MSCs, which are efficient mitochondrial donors, express high levels of Miro1 compared with the poorer mitochondrial donors, lung epithelial cells and 23
fibroblasts [161]. The transfer of mitochondria from the MSCs with low level of Miro1 to injured ECs is reduced in comparison with that from the MSCs with higher Miro1. This reduction is not due to a decrease in nanotube formation, but to the decrease in Miro1-mediated mitochondrial motility through the nanotubes [161]. Recently, a novel fluorescent-activated cell sorting-based protocol “MitoCeption” was developed to directly quantify the transferred mitochondria from mitochondria donor cells to the acceptor cells [179]. This MitoCeption technique might be useful for the study of how mitochondria from MSCs behave when they are transferred to the cells deficient of mitochondria or to terminally-differentiated cells. Furthermore, this technique may be used to investigate whether transfer of mitochondria from young cells to aged cells can result in rejuvenation of the recipient cells [179].
7. Conclusion The roles of protein kinases and sirtuins in the regulation of mitochondrial biogenetic function and lineage determination of MSCs have received increasing attention in recent years. Clarification of the metabolic reprogramming and the putative interplay of PTMs in this process may open up a new path for improvement of MSCs differentiation. Our unpublished data indicated a significant increase in the succinylation and malonylation levels of mitochondrial proteins in contrast to the increase of protein acetylation catalyzed by Sirt3 during adipogenic differentiation of ad-hMSCs. This might be a result of the down-regulation of Sirt5 at the late stage of adipogenesis of adipose-derived MSCs. It is worth mentioning that Sirt5 is the first and the only sirtuin that has been shown to be capable of catalyzing protein desuccinylation and demalonylation in mammalian cells. Interestingly, it has been demonstrated that Sirt3 and Sirt5 regulate the activities of mitochondrial proteins by a coordinated manner. They not only share the same protein targets but also act on 24
overlapping lysine residues.
Therefore, it is probable that there is a high degree of
overlap between succinylation and acetylation at specific lysine residues of the target proteins [61]. These studies have expanded the landscape of lysine acylation of proteins and deepened our understanding of the biological functions of protein deacylation catalyzed by Sirt3 and Sirt5, respectively. It is worth clarification as to whether these two mitochondrial sirtuins act synergistically or in a competitive manner, and how they discriminatively modulate deacetylation, desuccinylation and demalonylation of lysine residues on mitochondrial proteins. In addition, while the signaling pathways involved in mitochondrial transfer in the acceptor and donor cells remain unclear, identification of the controlling proteins in the transfer of mitochondria may improve future applications of mitochondrial transfer. Given the considerable therapeutic potential of MSCs and mitochondrial transfer, future investigation of the mechanisms underlying the regulation of oxidative metabolism and dynamics of mitochondria in MSCs may ultimately facilitate the development of effective stem cell therapies for treatment of mitochondrial diseases (Fig. 4).
Acknowledgement Part of this review article was prepared on the basis of our studies supported by grants from the Ministry of Science and Technology (MOST) of Taiwan Government (MOST103-2314-B-715-001-MY2, MOST104-2314-B-715-003-MY3 and MOST103 -2321-B-715-001), and intramural research grants from Mackay Medical College (MMC1012A10,
MMC1012B13,
RD1010061,
RD1020021,
RD1020088,
MMC1031B05, RD1030006, RD1030098, RD1040109) and from Mackay Memorial Hospital (MMH-MM-10304, MMH-MM- 10405, MMH-MM- 10505). 25
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structure-mediated mitochondrial transfer, Microvasc. Res., 92 (2014) 10-18. [174] E.Y. Plotnikov, V.A. Babenko, D.N. Silachev, L.D. Zorova, T.G. Khryapenkova, E.S. Savchenko, I.B. Pevzner, D.B. Zorov, Intercellular transfer of mitochondria, Biochemistry (Mosc.), 80 (2015) 542-548. [175] A. Prigione, J. Adjaye, Modulation of mitochondrial biogenesis and bioenergetic metabolism upon in vitro and in vivo differentiation of human ES and iPS cells, Int. J. Dev. Biol., 54 (2010) 1729-1741. [176] S. Fransson, A. Ruusala, P. Aspenstrom, The atypical Rho GTPases Miro-1 and Miro-2 have essential roles in mitochondrial trafficking, Biochem. Biophys. Res. Commun., 344 (2006) 500-510. [177] M. Saotome, D. Safiulina, G. Szabadkai, S. Das, A. Fransson, P. Aspenstrom, R. Rizzuto, G. Hajnoczky, Bidirectional Ca2+-dependent control of mitochondrial dynamics by the Miro GTPase, Proc. Natl. Acad. Sci. USA, 105 (2008) 20728-20733. [178] A.F. Macaskill, J.E. Rinholm, A.E. Twelvetrees, I.L. Arancibia-Carcamo, J. Muir, A. Fransson, P. Aspenstrom, D. Attwell, J.T. Kittler, Miro1 is a calcium sensor for glutamate receptor-dependent localization of mitochondria at synapses, Neuron, 61 (2009) 541-555. [179] A. Caicedo, V. Fritz, J.M. Brondello, M. Ayala, I. Dennemont, N. Abdellaoui, F. de Fraipont, A. Moisan, C.A. Prouteau, H. Boukhaddaoui, C. Jorgensen, M.L. Vignais, MitoCeption as a new tool to assess the effects of mesenchymal stem/stromal cell mitochondria on cancer cell metabolism and function, Sci. Rep., 5 (2015) 9073.
45
Figure 1. Alterations of metabolic profile during adipogenic differentiation of MSCs. (A) Mitochondrial respiration was analyzed in MSCs undergoing adipogenic differentiation for 7 days by using Seahorse XF24e extracellular fluid analyzer. Overall mitochondrial oxygen consumption rates (OCR) of differentiated adipocytes and MSCs were measured after injection of 2 µg/ml oligomycin A, 2 µM CCCP, and 5 µM antimycin A, respectively. (B) The metabolic profile (aerobic metabolism and glycolytic flux) in adipocytes and MSCs. Basal OCR and extracellular acidification rate (ECAR) were measured concurrently and compared.
Figure 2. Upregulation of Sirt3 is involved in the mitochondrial biogenesis during adipogenic differentiation of human MSCs. (A) Increase in lipid accumulation during adipogenic differentiation of MSCs. MSCs were incubated in the adipogenic induction medium for up to 4 weeks. Lipids in the cells were visualized by Oil Red O staining after adipogenic differentiation for 7, 14, 21, and 28 days, respectively. The cells were photographed under a light microscope. Scare bar: 100 μm. (B) The quantification of lipid content was measured by the absorbance at 510 nm with an ELISA reader. (C) MSCs were incubated in the adipogenic induction medium for up to 2 week. The protein levels of mitochondrial Sirt3 and Sirt5 in MSCs during adipogenic differentiation were analyzed by Western blot. α-Tubulin was used as the internal control. (D) MSCs were infected with lentivirus for two days and then induced to undergo adipogenic differentiation for 1 week. The protein level of Sirt3 was analyzed by Western blot. α-Tubulin was used as the internal control. Sirt3: sirtuin 3; Sirt5: sirtuin 5; α-Tub: α-tubulin. The data shown here are from three independent experiments. (E-I) MSCs were infected with lentivirus for two days and then induced to undergo adipogenic differentiation for 7 days. The mRNA expression level of adipogenic marker genes including PPARand adiponectin were measured by a TaqMan-based real-time PCR technique (E). The data were normalized with the 18S rRNA gene as an internal control. (F) The amount of human adiponectin in cell culture supernatant was measured by the adiponectin (human) ELISA Kit (Adipogen AG). (G) The mRNA expression level of PGC-1α was measured in adipocytes differentiated form MSCs with or without Sirt3 knockdown. (H) After adipogenic induction for 7 days, the protein levels of mitochondrial respiratory enzymes were analyzed in adipocytes with or without Sirt3 knockdown by Western blot. (I) Quantification of the protein expression levels. NDUFA9: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 9; mtTFA: Mitochondrial transcription factor A. The data shown here are means ± standard deviation of the results from three independent experiments. *: p < 0.05.
(A)
(B)
OCR (pmole/min/104cells)
shSirt3 D7
(Mitochondrial Respiration)
shLuc D7
Aerobic metabolism shLuc D7
Anaerobic glycolysis
shSirt3 D7
(Glycolysis)
Figure 3. Impairment of mitochondrial respiration and metabolic shift in adipocytes differentiated from MSCs with Sirt3-knockdown. MSCs were infected with lentivirus for two days and then induced to undergo adipogenic differentiation for 7 days. (A) Mitochondrial respiration was analyzed by using the Seahorse XF24 extracellular fluid analyzer. Overall mitochondrial oxygen consumption rates (OCR) of Sirt3- and luciferase-knockdown adipocytes were measured after injection of 2 µg/ml oligomycin A, 2 µM CCCP, and 5 µM antimycin A, respectively. (B) The metabolic profile (aerobic metabolism and glycolytic flux) in adipocytes with luciferase- and Sirt3-knockdown. Basal OCR and extracellular acidification rate (ECAR) were measured concurrently and compared.
Figure 4. Regulation of mitochondrial metabolism in differentiation of MSCs and mitochondrial transfer in stem cells. During the adipogenic differentiation of MSCs, mitochondrial biogenesis is activated by PGC-1 and Sirt3. ROS generated in the differentiation process are removed by the antioxidant system that is up-regulated by an increase in the expression of FOXO3a and MnSOD. The adipogenic differentiation is attenuated by hypoxia, aging and oxidative stress. Furthermore, when co-culturing MSCs with cells harboring abnormal mitochondria, the healthy mitochondria will be transferred from MSCs to rescue the cells with abnormal mitochondria.
Table 1. Roles of different sirtuins in mitochondrial metabolism and differentiation of MSCs
Sirt1
Epigenetic
Expression
Lineage
Metabolic
Oxidative
Expression
regulation
level change
determination
targets
response
level change in aging
Yes
Increased Increased Decreased Decreased
Osteogenesis Chondrogenesis Adipogenesis Myogenesis
Mitochondrial biogenesis,
FOXO1,
Decreased
[105, 106]
PGC-1
References
PGC-1, MnSOD, Catalase
Sirt2
N.A.
Declined
Adipogenesis
N.A.
FOXO1, FOXO3a
Decreased
[119-121]
Sirt3
N.A.
Increased
Unknown
Numerous
FOXO3a,
Decreased
[132, 147]
metabolic PGC-1, pathways, PDH, MnSOD SDHA Sirt5
N.A.
Decreased
Unknown
PDH
N.A.
N.A.
[138]
Sirt6
Yes
Increased Decreased
Osteogenesis Adipogenesis
N.A.
N.A.
Constant
[148, 149]
Sirt7
Yes
Increased
Adipogenesis
Fatty acid metabolism
N.A.
Decreased
[150, 151]
FOXO: forkhead box O; N.A.: not available; PGC-1: proliferator activated receptor gamma coactivator-1α; PDH: pyruvate dehydrogenase; SDHA: succinate dehydrogenase complex, subunit A; Sirt: sirtuin.
Table 2. Mitochondrial transfer by different types of stem cells or differentiated cells in vitro Donor cells
Acceptor cells
Treatments or defects
Effects
Route
References
EPCs
Cardiomyocytes
None
N.A.
TNTs
[168]
BM-MSCs
Lung adenocarcinoma mitochondrial DNA Mitochondrial 0 A549 cell line (mtDNA)-depleted ρ cells respiratory function rescued
Cellular contact
[160]
MSCs
Cardiomyocytes
None
N.A.
TNTs
[170]
EPCs
HUVECs
Adriamycin-induced damages
The number of cells receiving mitochondria increased two-fold
TNTs
[169]
MSCs
RTCs
None
N.A.
TNTs
[171]
MSCs
Cardiomyocytes
None
Converting to the progenitor state
TNTs
[164]
BM-MSCs
Lung epithelium cells
Endotoxin-induced damages
Mitochondrial respiratory function rescued
Connexin 43-gap [15] junction and microvesicles
VSMCs
MSCs
EtBr treatment
Proliferation
TNTs
[166]
ECs
Cancer cells
Breast cancer cell line MCF7
Chemoresistance
TNTs
[167]
BM-MSCs
Airway epithelial cells CS-exposed
Preservation of ATP
TNTs
[165]
levels iPSCs-MSCs
Airway epithelial cells CS-exposed
Preservation of ATP levels
TNTs
[165]
MSCs
Epithelial cells
Epithelial injury
Rescued
Miro1
[161]
MSCs
Osteosarcoma cells
EtBr treatment or short-term Mitochondrial R6G treatment function rescued
TNTs or cellular [162] contact
MSCs
Cybrid cells
mtDNA mutations (A3243G No effects mutation or 4,977 bp deletion)
TNTs or cellular [162] contact
Wharton's jelly-derived MSCs
ρ0 cells
mtDNA-depleted
Mitochondrial function rescued
TNTs
[163]
MSCs
Rat cortical neurons
Post-stroke
Neuroprotective effects
Miro1
[172]
BM: bone marrow; CS: cigarette smoke; ECs: endothelial cells; EPCs: endothelial progenitor cells; EtBr: ethidium bromide; HUVECs: human umbilical vein endothelial cells; iPSCs: induced pluripotent stem cells; MSCs: mesenchymal stem cells; N.A.: not available; RTCs: renal tubular cells; R6G: rhodamine 6G, a highly fluorescent rhodamine family dye; TNTs: tunneling nanotubes; VSMCs: vascular smooth muscle cells.
Table 3. Mitochondrial transfer as an in vivo therapeutic tool Donor cells
Acceptor cells
Treatments or defects
Effects
Route
References
EPCs
ECs
Adriamycin-associated nephropathy
Reduced inflammation, apoptosis and increased vascular density
TNTs
[169]
BM-MSCs
Airway epithelial cells CS-induced lung damage
Reduced alveolar destruction and lung fibrosis
TNTs
[165]
iPSC-MSCs
Airway epithelial cells CS-induced lung damage
Reduced alveolar destruction and lung fibrosis
TNTs
[165]
MSCs
Epithelial cells
Allergen-induced asthma
Reversed mitochondrial dysfunction
Miro1
[161]
MSCs
ECs
Ischemia-reperfusion
Rescued endothelial function
TNTs
[173]
BM: bone marrow; CS: cigarette smoke; ECs: endothelial cells; EPCs: endothelial progenitor cells; iPSCs: induced pluripotent stem cells; MSCs: mesenchymal stem cells; TNTs: tunneling nanotubes
S1084-9521(16)30053-2 http://dx.doi.org/doi:10.1016/j.semcdb.2016.02.011 YSCDB 1967
To appear in:
Seminars in Cell & Developmental Biology
Received date: Revised date: Accepted date:
31-12-2015 3-2-2016 5-2-2016
Please cite this article as: Hsu Yi-Chao, Wu Yu-Ting, Yu Ting-Hsien, Wei YauHuei.Mitochondria in mesenchymal stem cell biology and cell therapy: From cellular differentiation to mitochondrial transfer.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2016.02.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Mitochondria in mesenchymal stem cell biology and cell therapy: from cellular differentiation to mitochondrial transfer
Yi-Chao Hsu1#, Yu-Ting Wu1,2#, Ting-Hsien Yu1,2, and Yau-Huei Wei1,2
1
Institute of Biomedical Sciences, Mackay Medical College, New Taipei City 252
2
Institute of Biochemistry and Molecular Biology, National Yang-Ming University,
Taipei, Taiwan 112
*Corresponding Author: Dr. Yau-Huei Wei, Institute of Biomedical Sciences, Mackay Medical College, New Taipei City, Taiwan 252 Tel: 886-2-26360303 ext. 1100, Fax: 886-2-26361134 E-mail: [email protected]
#
These two authors made equal contributions to this work
1
Highlights 1. An integrated view on different protein kinases and sirtuins in MSCs is provided. 2. The regulation of mitochondrial biogenesis and metabolism in MSCs are summarized. 3. Recent advances in mitochondrial dynamics and transfer are discussed.
2
Abstract Mesenchymal stem cells (MSCs) are characterized to have the capacity of self-renewal and the potential to differentiate into mesoderm, ectoderm-like and endoderm-like cells. MSCs hold great promise for cell therapies due to their multipotency in vitro and therapeutic advantage of hypo-immunogenicity and lower tumorigenicity. Moreover, it has been shown that MSCs can serve as a vehicle to transfer mitochondria into cells after cell transplantation. Mitochondria produce most of the energy through oxidative phosphorylation in differentiated cells. It has been increasingly clear that the switch of energy supply from glycolysis to aerobic metabolism is essential for successful differentiation of MSCs. Post-translational modifications of proteins have been established to regulate mitochondrial function and metabolic shift during MSCs differentiation. In this article, we review and provide an integrated view on the roles of different protein kinases and sirtuins in the maintenance and differentiation of MSCs. Importantly, we provide evidence to suggest that alteration in the expression of Sirt3 and Sirt5 and relative changes in the acylation levels of mitochondrial proteins might be involved in the activation of mitochondrial function and adipogenic differentiation of adipose-derived MSCs. We summarize their roles in the regulation of mitochondrial biogenesis and metabolism, oxidative responses and differentiation of MSCs. On the other hand, we discuss recent advances in the study of mitochondrial dynamics and mitochondrial transfer as well as their roles in the differentiation and therapeutic application of MSCs to improve cell function in vitro and in animal models. Accumulating evidence has substantiated that the therapeutic potential of MSCs is conferred not only by cell replacement and paracrine effects but also by transferring mitochondria into injured tissues or cells to modulate the cellular metabolism in situ. Therefore, elucidation of the underlying 3
mechanisms in the regulation of mitochondrial metabolism of MSCs may ultimately improve therapeutic outcomes of stem cell therapy in the future.
Abbreviations: AMPK, AMP-activated protein kinase; BM, bone marrow; CAT, catalase;
Complex
I,
NADH-coenzyme
Q
oxidoreductase;
Complex
II,
succinate-coenzyme Q oxidoreductase; Complex III, ubiquinol-cytochrome c oxidoreductase; COX, cytochrome c oxidase; Complex V, FoF1ATPase; DAPT, N-[N-(3,5-difluor-ophenacetyl-L-alanyl)]-S-phenylglycine t-butylester; ETC, electron transport chain; ESCs, embryonic stem cells; MSCs, mesenchymal stem cells; FOXO3a, forkhead box O 3a; GPx, glutathione peroxidase; HIF-1α, hypoxia inducible factor-1α; HLA-DR, human leukocyte antigen - antigen D Related; H2O2, hydrogen peroxide; LC3, type II light chain 3; mTOR, rapamycin serine/threonine kinase; Miro1, mitochondrial Rho-GTPase 1; Mfn, mitofusin; mtTFA, mitochondrial transcription factor A; NRFs, nuclear respiratory factors; OPA1, optic atrophy protein 1; OXPHOS, oxidative phosphorylation; O2-., superoxide anion; p70S6K, p70 S6 kinase; PGC-1α, proliferator activated receptor gamma coactivator-1α; PSCs, pluripotent stem cells; PKC, protein kinase C; PDH, pyruvate dehydrogenase; PDHC, pyruvate dehydrogenase complex; PDK, PDH kinase; PSCs, pluripotent stem cells; PTM, post-translational modification; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen
species;
SOD,
superoxide
dismutase;
SREBP-1,
sterol
regulatory
element-binding protein 1; Sirt, sirtuins; TCA cycle, tricarboxylic acid cycle; TSC1/2, tuberous sclerosis complex1/2; TNTs, tunneling nanotubes.
Keywords: mitochondrial metabolism, mitochondrial transfer, mesenchymal stem cells, sirtuins. 4
1. Introduction Stem cells are capable of self-renewal and have the potential of differentiating into multiple cell lineages. In light of the rapid progress in stem cell research in the last decade, stem cell therapy has hold great promise in regenerative medicine. The role of mitochondria in the maintenance and differentiation of stem cells has attracted increasing attention. Mesenchymal stem cells (MSCs) are adherent fibroblast-like multipotent cells, which can differentiate into mesoderm cell lineages such as osteoblasts, chondrocytes, myoblasts and adipocytes [1]. Recently, it was demonstrated that MSCs could differentiate into cells in different germ layers, such as ectodermal neuronal-like cells as well as endodermal hepatocyte-like cells [2, 3]. MSCs were first derived from bone marrow (BM) [1], and were identified and characterized by Friedenstein [4]. MSCs have been isolated from the adipose tissue [4], umbilical cord blood [5, 6], amniotic fluid [7], placenta [8-10] and dental pulp [11]. The Mesenchymal and Tissue Stem Cell Committee of International Society for Cellular Therapy has defined human MSCs based on the following four criteria: (1) plastic-adherent and fibroblast-like cells; (2) the ability of osteogenic, chondrogenic and adipogenic differentiation; (3) simultaneous expression of CD105, CD73, CD90 and CD105; (4) lack of expression of CD11b, CD14, CD19, CD34, CD45, CD79a and human leukocyte antigen-DR [12]. MSCs are full of potential for cell therapies due to their multipotency and the therapeutic advantage of hypo-immunogenicity [13], low tumorigenicity [14] and the capacity of mitochondrial transfer [15, 16]. During tissue injury, MSCs can migrate preferentially to the injured sites in the organs such as the heart [17], brain [18, 19], skeletal muscle [20] and kidney [21]. The therapeutic potential of MSCs have been demonstrated in different diseases such as myocardial infarction [22-26], acute renal failure [27], pulmonary hypertension [28-32], sepsis [33, 34], pulmonary fibrosis [28, 35], emphysema [36, 37], diabetes 5
[38-41], asthma [42-45], bone defects [46], graft-versus-host disease [47], ischemic stroke [48] and hepatic failure [49]. In light of the potential of applications of MSCs in regenerative medicine and cell therapy, the roles of mitochondrial metabolism and dynamics in the maintenance and differentiation of MSCs have received increasing attention from clinicians and life scientists [50]. The switch of energy source from glycolysis to aerobic metabolism is a hallmark of the differentiation of MSCs, which is referred to as the metabolic shift. Accumulating evidence has revealed that the metabolic shift and upregulation of mitochondrial function is essential for successful differentiation of MSCs [51]. In this review, we synthesize and discuss recent progress in the studies of metabolism and dynamics of mitochondria in the differentiation and therapeutic application of MSCs.
2. Mitochondrial biogenesis and upregulation of antioxidant enzymes occur concurrently in the differentiation of MSCs Mitochondria produce the majority of ATP through respiration and oxidative phosphorylation (OXPHOS). The electron transport chain (ETC) is composed of NADH-coenzyme
Q
oxidoreductase
(Complex
I),
succinate-coenzyme
Q
oxidoreductase (Complex II), ubiquinol-cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (COX, Complex IV). The electron flow through the ETC establishes a proton gradient across the inner membrane, which is utilized to drive ATP synthesis by the FoF1ATPase (Complex V) [52]. Upregulation of mitochondrial biogenesis [53] and aerobic metabolism [51] are the hallmarks of the differentiation of MSCs. Mitochondrial biogenesis is regulated by extracellular stimuli such as cold exposure, fasting, exercise, oxidative stress and cell differentiation. It is established that proliferator activated receptor gamma coactivator-1α (PGC-1α) regulates 6
mitochondrial biogenesis by increasing the expression of mitochondrial biogenesisassociated genes, such as nuclear respiratory factors (NRFs) [54]. NRF-1 and NRF-2 are transcription factors, which regulate nuclear DNA-encoded proteins involved in mitochondrial respiration and OXPHOS. In addition, NRF-1 can activate the expression of mitochondrial transcription factor A (mtTFA), which binds to the D-loop of mtDNA and coordinates with DNA polymerase γ to replicate mtDNA [55]. Many studies have revealed that defects in mitochondrial biogenesis or OXPHOS are involved in the pathophysiology of mitochondrial disease, metabolic syndrome, heart disease, and type 2 diabetes [56]. We have demonstrated that upon osteogenic induction of MSCs, proteins involved in mitochondrial biogenesis are all increased, which include mtTFA, DNA polymerase γ and PGC-1α, and some of the enzymes of TCA cycle and protein subunits of respiratory enzymes [53]. Furthermore, it was reported that osteogenic differentiation of MSCs is accompanied by cristae development and increase in the expression levels of the constituent subunits and activity of respiratory enzyme complexes [57, 58]. Moreover, it was found that the respiration rate, mitochondrial membrane potential and intracellular ATP content were also increased, indicating an upregulation of oxidative metabolism in mitochondria during the differentiation of MSCs [53]. ATP synthesis is coupled with respiration in normal mitochondria, which also produce a certain amount of reactive oxygen species (ROS) as byproducts. During respiration of mitochondria, the electrons may leak out from Complex I and Complex III to react with O2 to generate the superoxide anion (O2-.) [59], which can be converted to hydrogen peroxide (H2O2) by superoxide dismutase (SOD) and further decomposed to O2 and H2O by catalase (CAT) or glutathione peroxidase (GPx). Excess ROS can cause DNA damage, lipid peroxidation and oxidative modification of proteins, which are detrimental to cell function [60]. Accumulated evidence has 7
substantiated the notion that oxidative stress can lead to a wide spectrum of human diseases such as cardiovascular disease, neurological disorders, diabetes and cancers [61]. Therefore, the intracellular levels of ROS should be effectively controlled by the antioxidant defense system, including SOD, CAT and GPx. MSCs express significant levels of active forms of CAT, GPx and SOD, which confer the differentiating cells with the ability to cope with ROS-mediated oxidative stress and avoid damage [62]. It was reported that the mitochondrial ROS generated from Complex III during respiration is also involved in the regulation of adipogenic differentiation [63]. We found that MnSOD, CAT, GPx and glutathione reductase are up-regulated during adipogenic differentiation of human mesenchymal stem cells [64]. Furthermore, we found that forkhead box O 3a (FOXO3a), an upstream factor that regulates MnSOD transcription, is involved in the upregulation of antioxidant enzymes at the early stage of adipogenic differentiation of MSCs [64]. It has been shown that adipogenic differentiation is associated with the up-regulation of FOXO1 to counteract the increase in the ROS production, which has a pivotal role in maintaining cellular redox homeostasis and regulate the expression of antioxidant enzymes (e.g., MnSOD and CAT) [65]. Moreover, it was reported that activation of FOXO1 is involved in osteogenic differentiation [66].
3. Metabolic switch during the differentiation of MSCs In a previous study, we demonstrated that oxidative metabolism is activated by induction of the expression of respiratory enzymes and increase of the OXPHOS of mitochondria during osteogenic differentiation of MSCs [53]. In addition, glycolysis is reduced during osteogenic differentiation of MSCs as revealed by a dramatic decrease in the production of lactate [67]. These findings suggest a metabolic switch upon the osteogenic differentiation of MSCs. A similar change in metabolic profile 8
was also observed during adipogenic differentiation. A remarkable increase in mitochondrial respiration and decrease in glycolytic flux were observed in adipocytes differentiated form MSCs (Fig. 1). In particular, the metabolic state of stem cells may depend on their niche. Most differentiated cells are located around the blood vessels for ample supply of oxygen to support oxidative metabolism. By contrast, the stem cells or transplanted MSCs in the injured tissues are usually exposed to low oxygen tension or hypoxic stress that disturbs aerobic metabolism. Under such conditions, cells utilize anaerobic glycolysis to supply the majority of energy needed for the execution of cellular functions. It was demonstrated that hypoxia inducible factor-1α (HIF-1α) can suppress oxidative metabolism through inhibition of pyruvate dehydrogenase (PDH) by PDH kinase (PDK) and activate the expression of glycolytic enzymes [68]. We further showed that hypoxia could increase HIF-1α expression and inhibit osteogenic differentiation by blocking the metabolic switch of MSCs [69, 70]. In addition, it was observed that adipogenic differentiation of MSCs was inhibited by HIF-1α and enhanced by knockdown of HIF-1α under both normoxia and hypoxia [71].
4. Post-translational protein modifications in the regulation of mitochondrial function and metabolic shift during MSCs differentiation Although human tissue-derived MSCs hold great promise for cell-based regenerative therapy of various diseases, it remains an open question as to how the differentiation capacity of MSCs can be maintained in vivo. Epigenetic mechanisms regulating gene expressions are important elements that affect differentiation and function of stem cells. In addition to the study on epigenetic and transcriptomic characteristics, systemic proteomic approaches have been applied to identify and quantify functional alterations and post-translational modifications (PTMs) of proteins in the maintenance 9
of stemness of MSCs [72-74]. In recent years, PTMs of certain target proteins have been demonstrated to play important roles in the regulation of multipotency and differentiation of MSCs. Emerging evidence shows that PTMs such as phosphorylation, glycosylation and acetylation play critical roles in the regulation of protein and enzyme functions, signaling networks and cell fates in human pluripotent stem cells (PSCs) [74-76].
4.1 Role of protein kinase in MSCs differentiation A recent study on phospho-proteome of human embryonic stem cells (ESCs) revealed that dynamic change in protein phosphorylation events occurs in the early stage of differentiation [77]. It has been shown that activation of multiple kinases is associated with different signaling pathways during stem cell differentiation. Accumulating evidence suggests that the evolutionarily conserved protein kinases act as major effectors of stem cell homeostasis and lineage differentiation via regulation of metabolism. The mammalian target of rapamycin serine/threonine kinase (mTOR) has emerged as a cellular sensor of energy status that can regulate metabolic processes, protein synthesis and cell proliferation [78]. It contains two distinct multi-protein complexes, mTORC1 (rapamycin-sensitive complex) and mTORC2. mTORC1 has a significant impact on the maintenance of metabolic homeostasis via the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway [79]. Activation of mTORC1 phosphorylates its downstream targets to up-regulate the expression levels of genes involved in glycolysis and lipid synthesis. It has been demonstrated that overexpression of mTORC1 can induce the differentiation of MSCs into adipocytes, osteoblasts and neuronal-like cells [80-82]. In adipocytes, mTOR is critical for lipid metabolism and serves as a sensor of nutrient availability to regulate the expression of PPAR. mTORC1 signaling promotes fatty acids and triacylglycerol synthesis via the 10
activation of the transcriptional factor SREBP-1 and induction of the expression of lipogenic genes [83]. It has been reported that mTORC1 signaling promotes adipogenic differentiation by activation of PPAR [84]. It has been demonstrated that activation of mTORC1 is involved in insulin-stimulated adipogenesis through PI3K/AKT-mediated inhibition of the mTOR inhibitory proteins and tuberous sclerosis complex1/2 (TSC1/2) [80, 84]. Moreover, it has been reported that transcriptional activation of CREB by p70 S6 kinase (p70S6K), the downstream target of mTOR, is involved in adipogenic differentiation of BM-derived MSCs (BM-MSCs) [80]. Inhibition of mTORC with rapamycin or shRNA silencing has been shown to reduce the expression levels of adipogenic markers and lipid accumulation in 3T3-L1 preadipocytes [85]. Negative effect of rapamycin on adipogenesis was also observed in human pre-adipocytes, human MSCs [86] and murine MSCs [78]. mTOR-mediated signaling pathways are also involved in the proliferation and differentiation of osteoblasts. Unlike the effect on adipogenesis, the effect of mTOR on osteogenic differentiation is controversial. Emerging data indicate that PI3K/mTOR signaling is critical for the maintenance of ESCs pluripotency via suppressing the induction of differentiation-related transcription factors [87, 88]. Although inhibitory effects of rapamycin on osteogenic gene expression and ALP activity were observed in a murine model, most studies support that the disruption of mTOR contributes to osteogenesis of ESCs and MSCs [82, 89]. The above-mentioned observations strongly suggest that mTOR plays a critical role in the regulation of stem cell differentiation. In recent years, numerous studies suggest that AMPK-mediated signaling pathways favor osteogenic differentiation and play a role in the lineage commitment of MSCs. AMPK is a master regulator of cellular metabolism and energy homeostasis. It can sense cellular AMP/ATP ratio and regulate metabolic activity and maintain cell survival under insufficient oxygen supply. Moreover, AMPK regulates mitochondrial 11
biogenesis through increasing the expression level or phosphorylation of PGC-1 [90]. Indeed, AMPK activation was shown to increase the expression of PGC-1α and mtTFA in MSCs [91]. One of our previous studies also showed that PGC-1 and mitochondrial biogenesis are up-regulated during osteogenic differentiation of human MSCs [53]. Interestingly, crosstalk of AMPK with Wnt-β-catenin signaling, a master pathway that determines the differentiation of BM-MSCs into osteoblasts and adipocytes, has also been demonstrated to contribute to osetogenesis of MSCs [92, 93]. It was found that the expression and phosphorylation levels of AMPK were increased during osteogenic differentiation of MSCs [94]. It has been shown that inhibition of AMPK signaling by its inhibitor, compound C, or knockdown by shRNA suppress osteogenesis of MSCs and increase the lipid droplet formation upon osteogenic induction. Conversely [95], activation of AMPK with metformin promotes osteogenesis and inhibits adipogenesis of MSCs [95]. It has been established that AMPK activation inhibits mTORC1 by phosphorylation of TSC1/2 [96]. Coordination of AMPK and AKT/mTOR signaling modulate osteogenic differentiation of MSCs. AMPK has been shown to induce autophagy through inhibition of mTOR at the early stage of osteogenesis in MSCs. Protein kinase C (PKC) is a family of serine/threonine protein kinases that is known to be involved in the regulation of glucose metabolism and mitochondrial biogenesis. Several studies have indicated that specific PKC isoforms are associated with embryonic bone formation. PKCλ/ι was reported to be involved in the upregulation of the biogenesis and function of mitochondria in stem cell differentiation [97]. Under differentiation condition, loss of PKCλ/ι impairs mitochondrial function and induces a metabolic shift to glycolysis through the HIF-1α-mediated inhibition of PDH by PDK, which increases the pluripotency of mouse ESCs [97]. In this process, stabilization of HIF-1α increases the expression of pluripotency genes while repressing the expression of PGC-1α. Interestingly, similar to the role of AMPK in MSCs 12
differentiation, PKC has a positive effect on osteogenesis. The expression and phosphorylation levels of PKC were found to increase in osteogenic differentiation of BM-MSCs [98].
4.2 Inhibitory regulation of Sirt1 and Sirt2 on adipogenic differentiation of MSCs Numerous studies have substantiated the importance of protein deacetylation catalyzed by sirtuins in the control of the function and differentiation capacity of stem cells [75, 76, 99]. Mammalian sirtuins belong to a conserved family of proteins with NAD+-dependent deacylase activities, which catalyze lysine deacylation by coupling with NAD+ hydrolysis and ADP-ribosylation [100, 101]. Seven sirtuins (Sirt1-Sirt7) are involved in the regulation of longevity, energy homeostasis, stress response and cell development in mammals. It has been thought that these enzymes affect stem cell differentiation through deacetylation of histone and non-histone proteins associated with the regulations of gene expression, cytoskeleton remodeling and metabolic reprogramming during the differentiation process [72-75]. Here, we summarize the roles of different sirtuins in the regulation of MSCs differentiation (Table 1). Among all the sirtuins, Sirt1 is the most widely studied sirtuin that is involved in epigenetic regulation of the expression of tissue-specific genes through histones deacetylation during differentiation of MSCs [102-104]. Up-regulation of Sirt1 was found to promote osteogenic differentiation of MSCs by silencing the inhibitory gene of bone formation [102, 105, 106]. In addition to acting on histones, deacetylation of transcription factors, PPAR-γ, p53, NFκB and FOXO1 has also been suggested to be involved in Sirt1-mediated regulation of lineage determination of MSCs [103]. Simic et al. [104] showed that the MSCs obtained from MSCs-specific Sirt1 knockout mice exhibited a reduction in the efficiency of osteogenic and chondrogenic differentiation, but no such difference was found in adipogenesis. Accumulated evidence suggests a 13
potential role of Sirt1 in fate decision of MSCs based on the findings that Sirt1 activation favors osteogenic differentiation while inhibits adipogenic differentiation [104, 107]. In contrast to its positive regulation of osteogenesis, Sirt1 negatively regulates adipogenic differentiation. Down-regulation of Sirt1 was observed during adipogenic induction of mouse MSCs cell lines [108-110]. We also found that the protein expression level of Sirt1 was decreased during adipogenic differentiation of adipose-derived human MSCs. However, the mechanism by which Sirt1 inhibits adipogenesis has remained unclear. Inhibition of Sirt1 by nicotinamide stimulates adipogenesis through increase of the number of adipocytes and expression of adipocyte markers. Sirt1 was shown to reduce fat and delay adipogenesis through inhibiting PPARγ signaling and stimulating lipolysis in 3T3-L1 preadipocytes [111]. It has been reported that Sirt1 regulates lipolysis and fat mobilization through the deacetylation and activation of FOXO1 transcription factor in adipose tissues [112]. Recent studies revealed that the deacetylation of FOXO1 at the forkhead DNA binding domain by Sirt1 could increase its transcriptional activity, and in turn inhibit PPARγ expression, which might contribute to the inhibitory effect of FOXO1 on adipogenic differentiation at the early stage [113, 114]. Therefore, inactivation of Sirt1 during adipogenic induction of MSCs could maintain the gene expression of PPARγ and induce adipogenic gene expression. This is in line with the observation that resveratrol blocked adipocyte development and increased the expression of osteogenic markers in MSCs [107]. Furthermore, miR-146b repressed the transcription of Sirt1 and increased adipogenic differentiation of 3T3-L1 preadipocytes, which was mediated by inhibition of the Sirt1-FOXO1 cascade [99]. The above-mentioned findings suggest that Sirt1 may serve as a key regulator to determine the adipogenic and osteogenic differentiation, respectively, of MSCs. Like 14
Sirt1, the Sirt2 protein level was also decreased at the early stage of adipogenic differentiation of MSCs [115]. Sirt2 localizes to cytosol and acts on the deacetyltion of -tubulin and regulates microtubule stability to control the cell morphology change during differentiation [116, 117]. Acetylated α-tubulin was demonstrated to facilitate the transport of mitochondria on the microtubules [118]. Acetylation of α-tubulin is increased during adipogenesis, and impairment in adipogenic differentiation was observed in the preadipocytes expressing an acetylation-resistant α-tubulin mutant in the mouse [116]. Moreover, it was shown that hyperacetylation of α-tubulin can initiate cytoskeleton remodeling by activating microtubule-severing protein, katanin, which in turn facilitates the expansion of lipid droplets and controls morphological transition towards mature adipocytes [117]. Decrease of Sirt2 expression promotes the differentiation of 3T3L1 preadipocytes via enhanced acetylation and phosphorylation of FOXO1, which promotes its cytoplasmic translocation and inactivation [119]. It was shown that FOXO1 deacetylation by Sirt2 suppressed adipogenic differentiation of MSCs through down-regulation of PPARγ [120, 121]. On the other hand, regulation of GSK3β by Sirt2 was reported to be required for early cell lineage commitment of mouse ESCs [121]. However, whether inactivation of Sirt2 is associated with lineage determination between adipogenesis and osteogenesis remains to be elucidated.
4.3 Roles of Sirt3 and Sirt5 in mitochondrial biogenesis and oxidative metabolism during adipogenic differentiation of MSCs Mitochondrial dysfunction is one of the factors causing defects in adipogenesis and dysfunction of adipocytes, which culminate in insulin resistance and type 2 diabetes [122, 123]. In a previous study, we showed that activation of oxidative metabolism is required for adipogenic differentiation [124]. Increases in oxygen consumption and 15
activities of respiratory enzyme complexes were observed in the differentiation of adipocytes. Therefore, activation of mitochondrial function is crucial for the success of the differentiation of stem cells. Sirt3 is the major sirtuin in mitochondria and is preferentially expressed in the tissues of high metabolic demand [125]. It exhibits robust deacetylase activity and acts on numerous targets that are involved in oxidative metabolism. We and other investigators have shown that Sirt3-mediated protein deacetylation plays a crucial role in the regulation of energy homeostasis through the activation of mitochondrial enzymes involved in OXPHOS, TCA cycle, -oxidation of fatty acids, urea cycle and ketogenesis, respectively [126-129]. Recent studies revealed a decline of Sirt3 in diabetic rats and that the deficiency of Sirt3 may increase the risk of developing insulin resistance and obesity [130]. These findings suggest that Sirt3 is involved in the regulation of glucose metabolism of adipocytes. However, the roles of Sirt3 during adipogenesis of MSCs and in the function of mature adipocytes have remained poorly understood. In contrast to the suppressive effect of above-mentioned sirtuins on differentiation, Sirt3 activity was shown to be essential for brown adipogenic differentiation in vitro [131]. By using an adipogenic induction system of MSCs, we found a significant increase in the protein level of Sirt3 during adipogenic differentiation (Fig. 2A-2C). We demonstrated that the expression levels of differentiation markers and adiponectin secretion were decreased in adipocytes differentiated from Sirt3-knockdown MSCs, which suggest that Sirt3 is essential for adipogenesis of MSCs (Fig. 2D-2F). We also showed that adipocytes differentiated
from
Sirt3-knockdown
MSCs
displayed
impairment
in
the
mitochondrial biogenesis including the decrease of PGC-1 mtTFA and protein subunits of respiratory enzyme complexes (Fig. 2G-I) and respiratory dysfunction (Fig. 3A). It was also reported that up-regulation of Sirt3 could enhance myogenic differentiation by activation of mitochondrial function [132]. In this study, silencing 16
of Sirt3 was found to down-regulate PGC-1 and MnSOD, which led to abnormal differentiation of myoblasts. Likewise, increase in mtDNA content and expression of mitochondrial biogenesis-related transcription factors have been observed in neurons overexpressing Sirt3 [133, 134]. These findings together suggest that Sirt3 might be involved in the up-regulation of the mitochondrial biogenesis and respiratory function during adipogenic differentiation of MSCs. In addition to lysine acetylation, malonylation and succinylation catalyzed by Sirt5 have recently been suggested to regulate the activities of some mitochondrial proteins [135, 136]. These two types of acylation are achieved by using intermediary metabolites malonyl-CoA and succinyl-CoA as the substrate, which are generated by catabolism of fatty acids and amino acids. Although the deacetylase activity of Sirt5 is very low, it is the only sirtuin known to catalyze desuccinylation and demalonylation of mitochondrial proteins [135-137]. Recent studies of a mouse model revealed an association of Sirt5-catalyzed protein succinylation with energy metabolism [135, 137]. Like the role of Sirt3 in the regulation of lipid metabolism, loss of Sirt5 results in the impairment of -oxidation of fatty acids in liver and skeletal muscle of the Sirt5 knockout mice [137]. The protein expression level of Sirt5 was decreased during adipogenic differentiation and was dramatically reduced at the terminal stage of differentiation of MSCs [138] (Fig. 2C), whereas our unpublished data showed that the succinylation and malonylation levels of proteins in mitochondria were significantly increased in MSCs after adipogenic differentiation. Whether Sirt5 is involved in adipogenic differentiation of MSCs warrants further investigation. Sirt5 was recently shown to catalyze protein desuccinylation and down-regulate the activities of pyruvate dehydrogenase complex (PDHC) and succinate dehydrogenase [137], which resulted in subsequent impairment of aerobic metabolism in human embryonic kidney cells HEK293. Mitochondrial PDHC is the pivotal regulator of 17
oxidative metabolism in mammalian cells [139]. Its deficiency decreases the conversion of pyruvate to acetyl-CoA in mitochondria and impairs aerobic metabolism. Activation of PDHC [140] and down-regulation of glycolysis [51] are important for the maintenance of high aerobic metabolism during adipogenic differentiation. It was found that acetylation of PDH-E1 inhibited its activity by increasing its phosphorylation and thereby increasing the glycolytic flux [141]. Deacetylation of PDHC by Sirt3 could activate its enzyme activity and in turn enhance oxidative metabolism [142]. Actually, we observed that Sirt3 deficiency impaired mitochondrial respiration (Fig. 3A) and induced a metabolic shift to glycolysis (Fig. 3B) during adipogenic differentiation of MSCs. In contrast to the activation of PDHC by Sirt3, Sirt5 seems to negatively regulate the activity of this enzyme. It remains to be investigated whether a decline of Sirt5 is essential for the activation of PDHC in MSCs differentiation and how Sirt3 and Sirt5 regulate aerobic metabolism, in a reciprocal manner, during adipogenic differentiation of MSCs. In addition to Sirt1, a decline of mitochondrial Sirt3 was observed in the tissues of aged mice and in mammalian cells subject to oxidative stress [126]. Accumulated evidence has indicated that Sirt3 is likely associated with an extension of lifespan of humans by counteracting oxidative stress, though some studies are controversial. Sirt3 is considered a potential regulator of the ROS detoxification due to its up-regulation on the deacetylation and activation of MnSOD and isocitrate dehydrogenase 2 to enhance the antioxidant defense of target cells [143-145]. In myocardial tissues and cardiomyocytes, Sirt3-mediated activation of FOXO3a by deacetylation eliminates excess ROS through induction of the expression of antioxidant enzymes MnSOD and CAT [146]. Our unpublished data suggest that up-regulation of Sirt3 in MSCs contributes to the increase in the protein level of FOXO3a, and that Sirt3 deficiency caused an increase of mitochondrial ROS at the initial stages of adipogenic 18
differentiation. Sirt3 deficiency could lead to defects in mitochondrial respiration and accumulation of ROS, which in turn may affect the signaling pathways involved in adipogenic differentiation and function of maturate adipocytes. Recently, it was reported that overexpression of Sirt3 in human BM-MSCs improves cell resistance to oxidative stress-induced apoptosis through the up-regulation of MnSOD and CAT. Wang et al. [132] showed that the expression level of exogenous Sirt3 was decreased in BM-MSCs under severe oxidative stress induced by a high concentration of H2O2. They further demonstrated that the reduction of Sirt3 by H2O2 increased the cellular susceptibility of aged BM-MSCs to oxidative stress [132]. In consideration of the importance of Sirt3 in the mitochondrial function and antioxidant defense, we contended that oxidative stress-induced decline of Sirt3 may affect differentiation ability of aged MSCs. Brown et al. [147] showed that up-regulation of Sirt3 could improve the regenerative capacities of aged hemopoietic stem cells. Furthermore, other sirtuins, such as Sirt6 [148, 149] and Sirt7 [150, 151] have also been suggested to play important roles in the differentiation of MSCs. Thus, manipulation of sirtuins levels in MSCs by potential drugs or compounds (e.g., resveratrol) to increase mitochondrial function and antioxidant defense during adipogenic and osteogenic differentiation can be used to improve the quality of MSCs in advance for cell therapy in future clinical applications.
5. Mitochondrial dynamics Mitochondrial fusion, fission, biogenesis, degradation, movement within the cytoplasm and even intercellular transfer contribute to mitochondrial dynamics. Three GTPase proteins, including mitofusin-1 (Mfn1), mitofusin-2 (Mfn2), and optic atrophy protein 1 (OPA1), are involved in the regulation of mitochondrial fusion [152]. The fusion process enables the two-way exchange of mtDNA and proteins 19
between mitochondria and between mitochondria and the nucleus to maintain precise mtDNA transmission and promote overall mitochondrial functions. During mitosis, mitochondrial fission regulates the partitioning process of mitochondrial contents and further generates heterogeneity, and thereby removes damaged mitochondria [153]. Abnormal mitochondrial dynamics has been shown to cause neuromuscular disorders [154]. Furthermore, mitochondrial fission and fusion may also play important roles in the maintenance of pluripotency as inhibition of fission in mouse ESCs will result in the loss of colony morphology and pluripotency [155]. On the other hand, depletion of fusion proteins, mitofusins 1 and 2 (Mfn1/2), facilitates the metabolic transition to glycolysis through the activation of the Ras-Raf and HIF-1α signaling pathways at the early stage of iPSC reprogramming [156]. Furthermore, the quality of mitochondrial population is maintained by the process of autophagy and mitophagy to remove the damaged mitochondria during differentiation. The up-regulation of mitochondrial respiration in the differentiation of stem cells may lead to an increase in the ROS levels, which can cause oxidative damage within mitochondria. Thus, the activation of autophagy for elimination of defective mitochondria at the early stage of stem cell differentiation might be important for the quality control of mitochondria. It was reported that the mitochondrial clearance occurs during the differentiation of erythrocytes [157, 158]. The cleaved type II light chain 3 (LC3-phosphatidylethanolamine conjugate, LC3-II) protein, a marker for active autophagosomes, has been shown to accumulate at the early stage of osteogenic differentiation of MSCs, indicating activation of autophagy upon stimulation of differentiation [96]. Autophagy inhibitors also down-regulate the osteogenic differentiation of MSCs [96]. Recently, Song et al. [159] showed that inhibition of the Notch signaling by (N-[N-(3,5-difluor-ophenacetyl-L-alanyl)]-Sphenylglycine t-butylester, DAPT), a γ-secretase inhibitor, promoted the adipogenic 20
differentiation of MSCs through the activation of autophagy. The treatment of autophagy
inhibitors
could
further
reduce
the
DAPT-induced
adipogenic
differentiation on MSCs. Nonetheless, the detailed characteristics and regulation of mitophagy during the differentiation of MSCs remain to be investigated.
6. Stem cells as vehicles for mitochondrial transfer It has been suggested that mitochondrial transfer can rescue the stressed cells [15, 160-163], reprogram the differentiated cells [164] and restore the loss of mitochondrial function in recipient cells [165] (Table 2). However, the mechanisms and conditions of mitochondrial transfer between cells remain to be clarified. In particular, different cell types, such as vascular smooth muscle cells [166], endothelial cells [167], endothelial progenitor cells [168, 169] and MSCs [160, 162, 164, 165, 170-172] and human Wharton’s jelly MSCs [163] have been suggested to be able to rescue mitochondrial function through tunneling nanotubes (TNTs). Wharton’s jelly MSCs are derived from umbilical cords from subjects undergoing full-term delivery. The umbilical cord is processed following the primary culture procedure. The isolated cells from the umbilical cord are finally resuspended in culture media comprised of high-glucose DMEM, supplemented with fetal bovine serum, glutamine, non-essential amino acids and fibroblast growth factors. Several in vivo studies also demonstrated the capacity of mitochondrial transfer via TNTs and Miro1, which is a Rho GTPase that mediates mitochondrial movement along the TNTs [161, 165, 169, 173] (Table 3). Intercellular mitochondrial transfer using stem cells as vehicles is thus promising for development of strategies in the treatment of mitochondria-associated pathologies and provides a new approach to treat diseases caused by mitochondrial dysfunction. 6.1. Mitochondrial transfer by different types of stem cells in vitro 21
In 2006, Spees et al. [160] proposed and demonstrated a mitochondria-based approach to treat mitochondrial diseases. By co-culturing the mtDNA-deficient cells with BM-MSCs, BM-MSCs effectively rescued the respiratory function by transferring their mitochondria to the mtDNA-deficient cells. The capacity of mitochondrial transfer was recently reviewed by Plotnikov et al. [174]. Furthermore, in the "metabolic state hypothesis" proposed by Dr. Prigione, it was suggested that mitochondrial state and cellular metabolism are different among stem cells at different developmental stages [175]. Notably, MSCs have been demonstrated to promote adult cardiomyocytes reprogramming back to a progenitor-like state through partial cell fusion and mitochondrial transfer [164]. Deprivation of mtDNA in the MSCs dramatically decreased the somatic reprogramming efficiency rather than cell fusion, suggesting that mitochondrial transfer from MSCs to adult cardiomyocytes helps them convert to the progenitor state through metabolic reprogramming. Until now, most studies have focused on the mitochondrial transfer of MSCs. We summarize recent findings of mitochondrial transfer by different types of stem cells in the rescue of damaged cells in vitro (Table 2).
6.2. Mitochondrial transfer as the therapeutic effects in vivo It has been shown that in a mouse model of induced acute lung injury, the therapeutic effects of MSCs are associated with mitochondrial transfer to alveolar epithelial cells and thereby allowed the mouse to recover from lung injury [15]. Furthermore, MSCs without the ability to form gap junctions or without functional mitochondria cannot transfer the organelles via cell-cell connections [16], suggesting that mitochondrial transfer may also account for the therapeutic effects of the MSCs that are rich in mitochondria. Furthermore, Li et al. [165] demonstrated that the efficiency of mitochondrial transfer of iPSCs-MSCs was higher than that from 22
BM-MSCs to rescue the cigarette smoke-induced mitochondrial damage. The improvement of fibrosis was also greater in the rats inoculated with the iPSC-MSCs than those inoculated with the BM-MSCs [165]. Furthermore, mitochondrial transfer was made possible through the tunneling nanotubes formed by MSCs, which was suggested by the observation that inhibition of the formation of tunneling nanotube blocked mitochondrial transfer [165]. We summarize the in vivo therapeutic effects of different types of stem cells through mitochondrial transfer (Table 3).
6.3. Mechanisms of mitochondrial transfer Some probable mechanisms of mitochondrial transfer have been proposed. It has been demonstrated that mitochondrial transfer does not go through endocytosis and vesicle trafficking [160]. Mitochondrial transfer by BM-derived stromal cells and epithelial cells has been shown to be mediated by actin-based tube structure named tunneling nanotubes and by connexin 43 gap junctions [15, 167]. Another mitochondrial Rho-GTPase 1 (Miro1) has been shown to regulate the movement of mitochondria [161, 176-178]. I was told that the mitochondrial acceptor cells treated with rotenone, a respiratory chain inhibitor, received more mitochondria from donor cells [161]. Co-culturing the rotenone-treated acceptor cells with MSCs reversed the ATP levels and activities of Complexes I and IV, and decreased mitochondrial ROS production and cytochrome c release [161]. Therefore, impairment of respiratory function may initiate the retrograde signaling triggered by ROS, Ca2+, AMP/ATP and NAD+/NADH ratio, for the acceptor cells to receive mitochondria from donor cells. Furthermore, when the mitochondrial donor MSCs were treated with rotenone, mitochondrial transfer was attenuated through reduction of the expression level of Miro1 [161]. MSCs, which are efficient mitochondrial donors, express high levels of Miro1 compared with the poorer mitochondrial donors, lung epithelial cells and 23
fibroblasts [161]. The transfer of mitochondria from the MSCs with low level of Miro1 to injured ECs is reduced in comparison with that from the MSCs with higher Miro1. This reduction is not due to a decrease in nanotube formation, but to the decrease in Miro1-mediated mitochondrial motility through the nanotubes [161]. Recently, a novel fluorescent-activated cell sorting-based protocol “MitoCeption” was developed to directly quantify the transferred mitochondria from mitochondria donor cells to the acceptor cells [179]. This MitoCeption technique might be useful for the study of how mitochondria from MSCs behave when they are transferred to the cells deficient of mitochondria or to terminally-differentiated cells. Furthermore, this technique may be used to investigate whether transfer of mitochondria from young cells to aged cells can result in rejuvenation of the recipient cells [179].
7. Conclusion The roles of protein kinases and sirtuins in the regulation of mitochondrial biogenetic function and lineage determination of MSCs have received increasing attention in recent years. Clarification of the metabolic reprogramming and the putative interplay of PTMs in this process may open up a new path for improvement of MSCs differentiation. Our unpublished data indicated a significant increase in the succinylation and malonylation levels of mitochondrial proteins in contrast to the increase of protein acetylation catalyzed by Sirt3 during adipogenic differentiation of ad-hMSCs. This might be a result of the down-regulation of Sirt5 at the late stage of adipogenesis of adipose-derived MSCs. It is worth mentioning that Sirt5 is the first and the only sirtuin that has been shown to be capable of catalyzing protein desuccinylation and demalonylation in mammalian cells. Interestingly, it has been demonstrated that Sirt3 and Sirt5 regulate the activities of mitochondrial proteins by a coordinated manner. They not only share the same protein targets but also act on 24
overlapping lysine residues.
Therefore, it is probable that there is a high degree of
overlap between succinylation and acetylation at specific lysine residues of the target proteins [61]. These studies have expanded the landscape of lysine acylation of proteins and deepened our understanding of the biological functions of protein deacylation catalyzed by Sirt3 and Sirt5, respectively. It is worth clarification as to whether these two mitochondrial sirtuins act synergistically or in a competitive manner, and how they discriminatively modulate deacetylation, desuccinylation and demalonylation of lysine residues on mitochondrial proteins. In addition, while the signaling pathways involved in mitochondrial transfer in the acceptor and donor cells remain unclear, identification of the controlling proteins in the transfer of mitochondria may improve future applications of mitochondrial transfer. Given the considerable therapeutic potential of MSCs and mitochondrial transfer, future investigation of the mechanisms underlying the regulation of oxidative metabolism and dynamics of mitochondria in MSCs may ultimately facilitate the development of effective stem cell therapies for treatment of mitochondrial diseases (Fig. 4).
Acknowledgement Part of this review article was prepared on the basis of our studies supported by grants from the Ministry of Science and Technology (MOST) of Taiwan Government (MOST103-2314-B-715-001-MY2, MOST104-2314-B-715-003-MY3 and MOST103 -2321-B-715-001), and intramural research grants from Mackay Medical College (MMC1012A10,
MMC1012B13,
RD1010061,
RD1020021,
RD1020088,
MMC1031B05, RD1030006, RD1030098, RD1040109) and from Mackay Memorial Hospital (MMH-MM-10304, MMH-MM- 10405, MMH-MM- 10505). 25
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45
Figure 1. Alterations of metabolic profile during adipogenic differentiation of MSCs. (A) Mitochondrial respiration was analyzed in MSCs undergoing adipogenic differentiation for 7 days by using Seahorse XF24e extracellular fluid analyzer. Overall mitochondrial oxygen consumption rates (OCR) of differentiated adipocytes and MSCs were measured after injection of 2 µg/ml oligomycin A, 2 µM CCCP, and 5 µM antimycin A, respectively. (B) The metabolic profile (aerobic metabolism and glycolytic flux) in adipocytes and MSCs. Basal OCR and extracellular acidification rate (ECAR) were measured concurrently and compared.
Figure 2. Upregulation of Sirt3 is involved in the mitochondrial biogenesis during adipogenic differentiation of human MSCs. (A) Increase in lipid accumulation during adipogenic differentiation of MSCs. MSCs were incubated in the adipogenic induction medium for up to 4 weeks. Lipids in the cells were visualized by Oil Red O staining after adipogenic differentiation for 7, 14, 21, and 28 days, respectively. The cells were photographed under a light microscope. Scare bar: 100 μm. (B) The quantification of lipid content was measured by the absorbance at 510 nm with an ELISA reader. (C) MSCs were incubated in the adipogenic induction medium for up to 2 week. The protein levels of mitochondrial Sirt3 and Sirt5 in MSCs during adipogenic differentiation were analyzed by Western blot. α-Tubulin was used as the internal control. (D) MSCs were infected with lentivirus for two days and then induced to undergo adipogenic differentiation for 1 week. The protein level of Sirt3 was analyzed by Western blot. α-Tubulin was used as the internal control. Sirt3: sirtuin 3; Sirt5: sirtuin 5; α-Tub: α-tubulin. The data shown here are from three independent experiments. (E-I) MSCs were infected with lentivirus for two days and then induced to undergo adipogenic differentiation for 7 days. The mRNA expression level of adipogenic marker genes including PPARand adiponectin were measured by a TaqMan-based real-time PCR technique (E). The data were normalized with the 18S rRNA gene as an internal control. (F) The amount of human adiponectin in cell culture supernatant was measured by the adiponectin (human) ELISA Kit (Adipogen AG). (G) The mRNA expression level of PGC-1α was measured in adipocytes differentiated form MSCs with or without Sirt3 knockdown. (H) After adipogenic induction for 7 days, the protein levels of mitochondrial respiratory enzymes were analyzed in adipocytes with or without Sirt3 knockdown by Western blot. (I) Quantification of the protein expression levels. NDUFA9: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 9; mtTFA: Mitochondrial transcription factor A. The data shown here are means ± standard deviation of the results from three independent experiments. *: p < 0.05.
(A)
(B)
OCR (pmole/min/104cells)
shSirt3 D7
(Mitochondrial Respiration)
shLuc D7
Aerobic metabolism shLuc D7
Anaerobic glycolysis
shSirt3 D7
(Glycolysis)
Figure 3. Impairment of mitochondrial respiration and metabolic shift in adipocytes differentiated from MSCs with Sirt3-knockdown. MSCs were infected with lentivirus for two days and then induced to undergo adipogenic differentiation for 7 days. (A) Mitochondrial respiration was analyzed by using the Seahorse XF24 extracellular fluid analyzer. Overall mitochondrial oxygen consumption rates (OCR) of Sirt3- and luciferase-knockdown adipocytes were measured after injection of 2 µg/ml oligomycin A, 2 µM CCCP, and 5 µM antimycin A, respectively. (B) The metabolic profile (aerobic metabolism and glycolytic flux) in adipocytes with luciferase- and Sirt3-knockdown. Basal OCR and extracellular acidification rate (ECAR) were measured concurrently and compared.
Figure 4. Regulation of mitochondrial metabolism in differentiation of MSCs and mitochondrial transfer in stem cells. During the adipogenic differentiation of MSCs, mitochondrial biogenesis is activated by PGC-1 and Sirt3. ROS generated in the differentiation process are removed by the antioxidant system that is up-regulated by an increase in the expression of FOXO3a and MnSOD. The adipogenic differentiation is attenuated by hypoxia, aging and oxidative stress. Furthermore, when co-culturing MSCs with cells harboring abnormal mitochondria, the healthy mitochondria will be transferred from MSCs to rescue the cells with abnormal mitochondria.
Table 1. Roles of different sirtuins in mitochondrial metabolism and differentiation of MSCs
Sirt1
Epigenetic
Expression
Lineage
Metabolic
Oxidative
Expression
regulation
level change
determination
targets
response
level change in aging
Yes
Increased Increased Decreased Decreased
Osteogenesis Chondrogenesis Adipogenesis Myogenesis
Mitochondrial biogenesis,
FOXO1,
Decreased
[105, 106]
PGC-1
References
PGC-1, MnSOD, Catalase
Sirt2
N.A.
Declined
Adipogenesis
N.A.
FOXO1, FOXO3a
Decreased
[119-121]
Sirt3
N.A.
Increased
Unknown
Numerous
FOXO3a,
Decreased
[132, 147]
metabolic PGC-1, pathways, PDH, MnSOD SDHA Sirt5
N.A.
Decreased
Unknown
PDH
N.A.
N.A.
[138]
Sirt6
Yes
Increased Decreased
Osteogenesis Adipogenesis
N.A.
N.A.
Constant
[148, 149]
Sirt7
Yes
Increased
Adipogenesis
Fatty acid metabolism
N.A.
Decreased
[150, 151]
FOXO: forkhead box O; N.A.: not available; PGC-1: proliferator activated receptor gamma coactivator-1α; PDH: pyruvate dehydrogenase; SDHA: succinate dehydrogenase complex, subunit A; Sirt: sirtuin.
Table 2. Mitochondrial transfer by different types of stem cells or differentiated cells in vitro Donor cells
Acceptor cells
Treatments or defects
Effects
Route
References
EPCs
Cardiomyocytes
None
N.A.
TNTs
[168]
BM-MSCs
Lung adenocarcinoma mitochondrial DNA Mitochondrial 0 A549 cell line (mtDNA)-depleted ρ cells respiratory function rescued
Cellular contact
[160]
MSCs
Cardiomyocytes
None
N.A.
TNTs
[170]
EPCs
HUVECs
Adriamycin-induced damages
The number of cells receiving mitochondria increased two-fold
TNTs
[169]
MSCs
RTCs
None
N.A.
TNTs
[171]
MSCs
Cardiomyocytes
None
Converting to the progenitor state
TNTs
[164]
BM-MSCs
Lung epithelium cells
Endotoxin-induced damages
Mitochondrial respiratory function rescued
Connexin 43-gap [15] junction and microvesicles
VSMCs
MSCs
EtBr treatment
Proliferation
TNTs
[166]
ECs
Cancer cells
Breast cancer cell line MCF7
Chemoresistance
TNTs
[167]
BM-MSCs
Airway epithelial cells CS-exposed
Preservation of ATP
TNTs
[165]
levels iPSCs-MSCs
Airway epithelial cells CS-exposed
Preservation of ATP levels
TNTs
[165]
MSCs
Epithelial cells
Epithelial injury
Rescued
Miro1
[161]
MSCs
Osteosarcoma cells
EtBr treatment or short-term Mitochondrial R6G treatment function rescued
TNTs or cellular [162] contact
MSCs
Cybrid cells
mtDNA mutations (A3243G No effects mutation or 4,977 bp deletion)
TNTs or cellular [162] contact
Wharton's jelly-derived MSCs
ρ0 cells
mtDNA-depleted
Mitochondrial function rescued
TNTs
[163]
MSCs
Rat cortical neurons
Post-stroke
Neuroprotective effects
Miro1
[172]
BM: bone marrow; CS: cigarette smoke; ECs: endothelial cells; EPCs: endothelial progenitor cells; EtBr: ethidium bromide; HUVECs: human umbilical vein endothelial cells; iPSCs: induced pluripotent stem cells; MSCs: mesenchymal stem cells; N.A.: not available; RTCs: renal tubular cells; R6G: rhodamine 6G, a highly fluorescent rhodamine family dye; TNTs: tunneling nanotubes; VSMCs: vascular smooth muscle cells.
Table 3. Mitochondrial transfer as an in vivo therapeutic tool Donor cells
Acceptor cells
Treatments or defects
Effects
Route
References
EPCs
ECs
Adriamycin-associated nephropathy
Reduced inflammation, apoptosis and increased vascular density
TNTs
[169]
BM-MSCs
Airway epithelial cells CS-induced lung damage
Reduced alveolar destruction and lung fibrosis
TNTs
[165]
iPSC-MSCs
Airway epithelial cells CS-induced lung damage
Reduced alveolar destruction and lung fibrosis
TNTs
[165]
MSCs
Epithelial cells
Allergen-induced asthma
Reversed mitochondrial dysfunction
Miro1
[161]
MSCs
ECs
Ischemia-reperfusion
Rescued endothelial function
TNTs
[173]
BM: bone marrow; CS: cigarette smoke; ECs: endothelial cells; EPCs: endothelial progenitor cells; iPSCs: induced pluripotent stem cells; MSCs: mesenchymal stem cells; TNTs: tunneling nanotubes
