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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

Modulation of autophagy as new approach in mesenchymal stem cell-basedtherapy

Jelena Jakovljevica, C. Randall Harrellb, Crissy Fellabaumb, Aleksandar Arsenijevica,Nemanja Jovicica, Vladislav Volarevica,⁎

aUniversity of Kragujevac Serbia, Faculty of Medical Sciences, Department of Microbiology and immunology, Center for Molecular Medicine and Stem Cell Research, 69Svetozar Markovic Street, 34000, Kragujevac, Serbiab Regenerative Processing Plant, LLC, 34176 US Highway 19 N Palm Harbor, Palm Harbor, Florida, United States

A R T I C L E I N F O

Keywords:AutophagyMesenchymal stem cellsTherapyApproach

A B S T R A C T

Due to their trophic and immunoregulatory characteristics mesenchymal stem cells (MSCs) have tremendouspotential for use in a variety of clinical applications. Challenges in MSCs’ clinical applications include lowsurvival of transplanted cells and low grafting efficiency requiring use of a high number of MSCs to achievetherapeutic benefits. Accordingly, new approaches are urgently needed in order to overcome these limitations.Recent evidence indicates that modulation of autophagy in MSCs prior to their transplantation enhances survivaland viability of engrafted MSCs and promotes their pro-angiogenic and immunomodulatory characteristics.Here, we review the current literature describing mechanisms by which modulation of autophagy strengthenspro-angiogenic and immunosuppressive characteristics of MSCs in animal models of multiple sclerosis, osteo-porosis, diabetic limb ischemia, myocardial infarction, acute graft-versus-host disease, kidney and liver diseases.Obtained results suggest that modulation of autophagy in MSCs may represent a new therapeutic approach thatcould enhance efficacy of MSCs in the treatment of ischemic and autoimmune diseases.

1. Introduction

Mesenchymal stem cells (also known as multipotent mesenchymalstromal cells, MSCs) were first discovered in 1960′s by Friedenstein andcolleagues as plastic adherent, fibroblast-like, bone marrow (BM)-de-rived cells [1]. Several other groups [2–4] revealed potential of thesecells to differentiate into a number of mesenchymal cell types includingosteoblasts, chondrocytes and adipocytes and called them “mesench-ymal stem cells” in reference to their high self-renewing properties andability to regulate normal turnover and maintenance of adult me-senchymal tissues [5]. During the last 50 years, MSCs were most usuallyisolated from BM, umbilical cord blood (UCB) and adipose tissue (AT)but have also been obtained from skeletal muscle, synovium, bloodvessel walls, dental pulp, amniotic fluid, liver and lungs [6], demon-strating that MSCs reside in almost all postnatal organs and tissues[6,7].

Despite their tissue localization, all MSCs have similar morpholo-gical and functional characteristics including multi-lineage differ-entiation capacity and potential for immunomodulation. MSCs havefibroblast-like morphology and display a variety of cell surface antigensthat may vary depending on the isolation and expansion methods

(Table 1) [6]. In an effort to better characterize human MSCs, the In-ternational Society for Cellular Therapy (ISCT) has proposed followingcriteria: (a) cells must adhere to plastic in standard culture conditionsusing tissue culture flasks; (b) cells must be able to differentiate intoadipocytes, osteoblasts, and chondrocytes under standard in vitro dif-ferentiating conditions; (c) more than 95% of the cell population mustexpress CD105, CD73 and CD90 but must lack expression of CD45,CD34, CD14 or CD11b, CD79a or CD19 and HLA class II [8]. However,it should be noted that several research groups suggested that CD34should not be considered as a negative marker for MSCs. They identi-fied CD34+ sub-populations of BM-MSCs and AT-MSCs [9,10] andindicated that CD34 is expressed on tissue-resident MSCs while atte-nuated or diminished expression of CD34 on MSCs could be the con-sequence of their long-term propagation in vitro [11].

Several differences between MSC populations of distinct anatomicorigin have been observed with respect to the success rate of isolating,clonality, proliferation and differentiation capacity [6]. For example, incontrast to BM-MSCs and AT-MSCs, UCB-MSCs have the highest rates ofcell proliferation and clonality and significantly lower expression ofp53, p21, and p16, well known markers of senescence. Under standardculture conditions, AT-MSCs showed the greatest ability to differentiate

https://doi.org/10.1016/j.biopha.2018.05.061Received 21 February 2018; Received in revised form 8 May 2018; Accepted 14 May 2018

⁎ Corresponding author.E-mail address: vladislav.volarevic@ana.unibe.ch (V. Volarevic).

Biomedicine & Pharmacotherapy 104 (2018) 404–410

0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.

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into adipocytes while UCB-MSCs and BM-MSCs differentiated readilyinto osteoblasts [12].

In recent years, the extent of MSC multipotency has been frequentlydiscussed, with many authors suggesting that MSCs have a differ-entiation potential broader than initially thought. Under certain in vitroconditions, MSCs have been shown to form neural cells, cardiomyo-cytes, hepatocytes, insulin-producing cells, epithelial cells demon-strating their endodermal and neuroectodermal differentiation poten-tial [13]. In vivo, differentiation capacity of engrafted MSCs is regulatedand limited by multiple extrinsic factors reflecting epigenetic adapta-tions that predispose MSCs to different cell fates depending on thetissue microenvironment [13,14].

In addition to their differentiation capacity, MSCs holds great po-tential in regenerative and transplantation medicine due to their im-munosuppressive and pro-angiogenic characteristics [15]. MSCs mod-ulate function of all immune cells affecting both innate and acquiredimmune response [15]. MSCs suppress proliferation of antigen primed Tcells by preventing their entry into the S phase of the cell cycle [16].Similarly, by mediating G0/G1 arrest, MSCs block the differentiation ofmonocytes into immature DCs [15] and suppress proliferation of B cells[17]. MSCs downregulate expression of co-stimulatory molecules onantigen presenting cells, significantly attenuating their ability to in-teract with T cells [15]. By promoting conversion of inflammatory (M1)in immunosuppressive (M2) phenotype, MSCs modulate phenotype andfunction of macrophages attenuating macrophage-driven inflammation[15,18]. MSCs significantly inhibit IL-2-stimulated proliferation ofresting natural killer (NK) cells and remarkably attenuate cytotoxicityof activated NK and natural killer T (NKT) cells [15,19].

Pro-angiogenic characteristics of MSCs are relied on their potentialto stimulate differentiation of endothelial progenitor cells (EPCs) and toenhance survival and proliferation of endothelial cells (ECs) [20]. Re-cently published data suggest that the pro-angiogenic effects of MSCsare mediated by transient, paracrine mechanisms comprising the se-cretion of MSC-derived soluble molecules: vascular endothelial growthfactor (VEGF) basic fibroblast growth factor (bFGF), platelet-derivedgrowth factor (PDGF), angiopoietin-1, hepatocyte growth factor (HGF)[21].

Due to their trophic and immunoregulatory characteristics me-senchymal stem cells (MSCs) have tremendous potential for use in avariety of clinical applications. Nevertheless, there are still severalobstacles that should be addressed for successful MSC-based therapy.Challenges in MSC’s clinical applications include low survival oftransplanted cells, limited targeting capabilities, and low grafting effi-ciency and potency, which often requires use of a high number of cellsto achieve therapeutic benefits [14]. Accordingly, new strategies andapproaches are urgently needed in order to overcome these limitations.Appearing evidence indicates that autophagy plays a consistent role inthe modulation of cell proliferation, differentiation and stemness in a

wide variety of cell types, including MSC [22]. In line with these ob-servations, herewith we present current knowledge and future per-spectives about the modulation of autophagy in MSCs as potentiallynew approach for improving efficency of MSC-based therapy.

2. Autophagy: a dynamic recycling system that produces newenergy for cell survival

Autophagy is the highly conserved fundamental cell biologicalpathway in which cytoplasmic materials are delivered to and degradedin the lysosome. Basal levels of autophagy are important in maintainingcellular homeostasis by elimination of damaged organelles, proteinaggregates and turnover of long-lived proteins. The purpose of autop-hagy is not the simple elimination of materials, but instead, autophagyserves as a dynamic recycling system that produces new energy forcellular renovation and homeostasis. In response to stress signals suchas starvation, hypoxia, reactive oxygen species, pathogen infection orirradiation, autophagy is rapidly elevated and acts primarily as a sur-vival mechanism by recycling cytoplasmic materials for energy pro-duction [23].

The process of autophagy is tightly controlled through the co-ordinated activity of diverse regulatory components. To date, over 30autophagy related (Atg) proteins have been identified and character-ized, most of which exhibit marked homology between yeast andmammalian genomes [24]. There are roughly three classes of autop-hagy: macroautophagy, microautophagy, and chaperone-mediated au-tophagy (CMA). While each is morphologically distinct, all three havein common the delivery of cargo to the lysosome for degradation andrecycling enabling energy production for cell survival.

2.1. Macroautophagy

Macroautophagy involves several highly conserved steps: initiation,vesicle nucleation (formation of a cup-shaped double-membranestructure-isolation membrane), vesicle elongation, fusion and de-gradation [25]. Initiation step involves the assembly of ULK proteincomplex consisting of ULK1, Atg13, FIP200 and Atg101 which is as-sociated with and suppressed by mTOR complex 1 (mTORC1) thatphosphorylates and inactivates ULK1/2 and Atg13. Rapamycin or cel-lular stress such as starvation, induce dissociation of mTORC1 fromULK protein complex, resulting with its activation and induction ofmacroautophagy [26]. Nucleation of isolation membranes requires theformation of a large protein complex, known as Beclin 1/Class IIIphosphatidylinositol-3-kinase (PI3K) complex, coordinated by the in-teractions of several proteins including Beclin 1, UV irradiation re-sistance-associated tumor suppressor gene (UVRAG), Atg14, B-cellleukemia/lymphoma-2 (Bcl-2), p150, ambra1, endophilin B1, and Va-cuolar protein sorting 34 (Vps34), which activates PI3K to producephosphatidylinositol-3-phosphat [27]. After nucleation, several Atgproteins (assembled into two ubiquitin-like conjugation systems, At-g12–Atg5–Atg16 L and Atg8 (LC3)–phosphatidylethanolamine (PE)) arerecruited to the membrane of the pre-autophagosomes to promote itselongation and expansion. As a final step, the expanding vesicle matureand close to form a completed autophagosome, which traffics to andfuses with an endosome and/or lysosome, becoming an autophagoly-sosome (autolysosome), the structure where materials or organellestethered by autophagy receptor proteins are digested by lysosomalenzymes [28]. Termination of macroautophagy can be achievedthrough reactivation of mTOR (an essential component of the mTORC1complex) by nutrients generated during this process. This is an exampleof feedback mechanism that inhibits excessive activation of autophagyduring periods of starvation. Reactivated mTOR creates protolysosomaltubules or vesicles and these structures extrude from the autolysosomesand mature into functional lysosomes, thereby providing the full com-plement of the autophagy machinery [25,29].

Table 1Cell surface antigens expressed on MSCs.

Cell surfaceantigens

Description

CD105 endoglin, also identified as SH2, a component of the receptorcomplex of transforming growth factor- beta (TGF-β) involvedin proliferation, differentiation and migration

CD73 SH3/4, ectoenzyme that regulates the purinergic signalingthrough the hydrolysis of adenosine triphosphate (ATP)

CD44 hyaluronan receptor involved in migrationCD90 Thy-1, regulates differentiation of MSCsstromal antigen

1involved in MSC migration

CD166 vascular cell adhesion moleculeCD54/CD102 intracellular adhesion moleculeCD49b Integrin alpha-2, involved in adhesion and osteogenic

differentiation of MSCs

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2.2. Microautophagy

As a fundamental biological phenomenon, microautophagy refers toa process by which cytoplasmic contents enter the lysosome through aninvagination or deformation of the lysosomal membrane. With itsconstitutive characteristics, microautophagy of soluble substrates canbe induced by N-starvation or rapamycin via the regulatory signalingcomplexes. The maintenance of organellar size, membrane homeostasis,and cell survival under N-restriction are the main functions of micro-autophagy. For this purpose, microautophagy cooperates with macro-autophagy, CMA, and other forms of self-eating in a coordinated andcomplementary mode [30].

2.3. Chaperone-mediated autophagy

Unlike macro and microautophagy, which can both nonspecificallyengulf bulk cytoplasm, CMA is a uniquely selective form of autophagyby which specific cytosolic proteins are transported one-by-one acrossthe lysosomal membrane for degradation. CMA degrades a wide rangeof substrate proteins, including certain glycolytic enzymes, transcrip-tion factors and their inhibitors, calcium and lipid binding proteins,proteasome subunits, and proteins involved in vesicular trafficking[31].

3. The importance of autophagy in regulation of stemness anddifferetiation potential of stem cells

Autophagy plays an important role in regulation of self-renewal andpluripotency of embryonic stem cells (ESCs) and induced pluripotentstem cells (iPSCs). ESCs are pluripotent stem cells derived from theinner cell mass of a blastocyst, while iPSCs are pluripotent stem cellsthat are reprogrammed from adult cells [32]. ATG3-dependent autop-hagy is of crucial importance for the maintenance of pluripotency. AfterAtg3 deletion, removal of damaged mitochondria were compromised,leading to weakened self-renewal and pluripotency of ESCs and iPSCs[33]. In order to maintain multi-lineage differentiation capacity, ESCsand iPSCs exhibit a high autophagic flux driven by transcription factorForkhead box protein O1 (Foxo1). Inhibition of Atg3 or Foxo1 sig-nificantly compromised self-renewal, pluripotency, and differentiationcapacity of ESCs and iPSCs, indicating the importance of autophagy forfunctional characteristics of pluripotent stem cells [34].

Similarly, autophagy regulates stemness and differentiation capa-city of MSCs [22,35,36]. The housekeeping level of autophagy in MSCsmaintains their stemness while deletion of autophagy-related genesinduce genomic instability and telomere changes in MSCs acceleratingtheir senescence [35]. The exact relationship between autophagy andsenescence of MSCs had not been clarified [37]. Some researchers haveproposed a direct, positive relation between autophagy and senescence,while others imply an inverse relationship [38,39]. Extensive activationof autophagy in MSCs, manifested by upregulation of Beclin-1, Atg5,Atg7, and increased LC3-II conversion, resulted with their prematuresenescence manifested by enlarged and flat morphology and reducedproliferation capacity [40]. On contrary, inhibition of autophagy by 3-methyladenine (3-MA) prevented cellular degeneration and maintainedstemness of MSCs which were cultured under hyperglycemic stresfullconditions [40]. These, on first sight opposite findings, indicate thatautophagy is a stress adaptation response which must be carefullycontrolled. It has been shown that reduced autophagy induces senes-cence [37], however an excess of autophagy may be harmful. At thehousekeeping level, autophagy is required to prevent senescence ofMSCs while excessive autophagic activation abbreviates lifespan andstem cell characteristics of MSCs. There is controversy between au-tophagy inhibition and augmentation for stemness and differentiationpotential of MSCs. Having in mind that suitable cellular stress responseis important for maintaining homeostasis, it seems that autophagy re-presents a way to protect transplanted MSCs from external and internal

stressors and, accordingly, could be regulated by the microenvironmentin which MSCs were engrafted [37]. In line with these observations arefindings recently reported by Capasso and coworkers [37] who inducedsenescence in MSCs by irradiation, doxorubicin, peroxide hydrogentreatments and replicative exhaustion and demonstrated that in all se-nescent forms of MSCs (with the exception of high irradiation dose andreplicative senescence), the autophagy flux was notably reduced, sug-gesting that autophagy counteracts deteriorative processes, and its de-cline triggers senescence [37].

Activation of autophagy in MSCs slightly increased their prolifera-tion and self-renewal under normal culture conditions, while inhibitionof autophagy resulted in a lower survival rate of MSCs [41], indicatingthat stem cell characteristics of MSCs may be controlled through theactivation of autophagy pathway. In line with these observations, au-tophagy interferes with differentiation potential of MSCs by regulatingcommitment towards the adipogenic and osteoblastic lineages [22].Since significant number of undegraded autophagic vacuoles were ob-served in undifferentiated MSCs, it seems that undifferentiated MSCsare in the state of arrested autophagy. Induction of autophagy sup-pressed adipogenic and promoted osteogenic differentiation of MSCs(Fig. 3) [22,36]. Rapamycin treatment attenuates capacity of MSCs foradipogenic differentiation by suppressing activity of mTOR whichpromotes adipogenesis of white adipocytes, brown adipocytes, andmuscle satellite cells [42]. Opposite to adipogenic differentiation, eitherearly mTOR inhibiton or late activation of Akt induced differentiationof dental pulp-derived MSCs towards osteoblast lineage [22]. Similarly,upregulation of autophagy-related genes, induced by special AT-richbinding protein 2 (SATB2), markedly enhanced osteogenic differentia-tion of BM-MSCs in vitro and promoted regeneration of bone defects inBM-MSC-treated animals in vivo [43].

Therapeutic potential of autophagy-dependent increased differ-entiation of MSCs towards osteoblast lineage has been evaluated inglucocorticoid-induced osteoporosis (GIOP), the most common type ofsecondary osteoporosis [44]. Oral glucocorticoids reduce the pro-liferation and increase the apoptosis of osteoblasts, prolong the survivalof osteoclasts and enhance bone resorption, resulted with the re-markably increased risk of bone fractures in patients that receive glu-cocorticoid therapy [45]. Due to their differentiation capacity, MSCsrepresent key cellular source for bone repair and regeneration in GIOPpatients [44]. Autophagy has an important role in maintenance of bonetissue homeostasis in GIOP due to its effects on survival of transplantedMSCs [44]. Autophagy, induced by glucocorticoid therapy, protectedengrafted MSCs from starvation-induced apoptosis, while 3-MA-in-duced inhibition of autophagy reduced proliferation and increasedapoptosis of MSCs resulting with the reduction in bone mass [44]. Sinceautophagy, at the same time protects engrafted MSCs from apoptosisand promote their differentiation towards osteoblasts, induction ofautophagy has huge potential to become new strategy aimed to increasetherapeutic effects of MSCs in the treatment of GIOP patients.

Modulation of autophagy is important for differentiation of MSCsinto myocytes, hepatocyte and neuronal cells (Fig. 3). Induction ofautophagy in tonsil-derived MSCs (T-MSCs) is required for their dif-ferentiation into myoblasts and skeletal myocytes [46]. Similarly, ac-tivation of autophagy in T-MSCs enhanced their differentiating intohepatocyte-like cells which was followed by the attenuation of carbontetrachloride (CCl4)-induced liver fibrosis [47]. Importantly, inhibitionof autophagy in T-MSCs completely diminished these effects, con-firming that modulation of autophagy may regulate differentiation andtherapeutic potential of MSCs [48]. In line with these findings, induc-tion of autophagy has been reported to promote differentiation of MSCsinto neuron-like cells enabling enhanced neural regeneration afterMSC-based therapy [49,50].

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4. Augmentation of autophagy as new approach for attenuatingapoptosis of transplanted MSCs

Since autophagy normally provides a survival effect for cells understress, several lines of evidence indicate that it plays a crucial role forthe increased survival of transplanted MSCs [22,51–53]. MSCs culturedin serum-free medium survive prolonged serum deprivation utilizingautophagy to recycle macromolecules and synthesize anti-apoptoticfactors [51]. Autophagy has an important role in protecting MSCs fromreactive oxygen species (ROS)-induced injury after irradiation [52].During gamma radiation, high-energy photons bombard the irradiatedcell causing electron displacement and generation of ROS, which aid inbreaking chemical bonds within DNA in an indirect manner [53]. Inorder to maintain their stemness, MSCs actively reduce a deteriorationprocess by establishing low ROS environments [22,52]. Starvation orrapamycin-induced autophagy can significantly reduce ROS accumu-lation-associated DNA damage, while inhibition of autophagy leads toenhanced ROS accumulation and increased DNA damage, ultimatelyresulting with genome rearrangements and significant decrease in via-bility of irradiated MSCs [52].

Interestingly, ROS induces autophagy in MSCs, manifested by anincreased LC3-II expression and decreased p62 expression, and sig-nificantly affects interplay between autophagy and apoptosis [54]. Bcl-2 has a crucial role in maintaining signaling crosstalk between thesetwo cell death pathways. Bcl-2 binds to Beclin 1 and affects formationof Beclin 1/Vsp34 complex [55]. Mitogen-activated protein kinases(MAPKs) such as Jun N-terminal kinases (JNK) are involved in the in-duction of autophagy through Bcl-2 phosphorylation and disruption ofthe Bcl-2/Beclin1 complex. Since ROS activates JNK, JNK-mediatedBcl-2 degradation results in Beclin1-mediated autophagy activation inROS-treated MSCs (Fig. 1) [54]. Dissociation of Beclin-1 from Bcl-2 bypro-apoptotic BH3 proteins (such as Bad) and Beclin-1 phosphorylationby DAP kinase (DAPK) represent other ways for induction of autophagy[56,57]. Furthermore, DAPK triggers autophagy by phosphorylation ofprotein kinase D (PKD), which then phosphorylates and activates Vps34[57]. Although ROS-activated autophagy is cytoprotective againstapoptosis, apoptosis is overwhelming, and autophagy is unable tooverride apoptosis without extensive induction. Accordingly, augmen-tation of autophagy in MSCs prior to transplantation is needed to re-duce apoptosis and to prolong survival of engrafted MSCs. The efficacyof this approach has been demonstrated in the MSC-based therapy ofmyocardial infarction (MI) and diabetic limb ischemia [58].

4.1. Autophagy: a novel therapeutic approach for MSC-based treatment ofMI

MSCs have been widely used in cell-based therapy for myocardialinfarction (MI) [58]. After MI, the ischemic environment seriously af-fects the survival of transplanted MSCs. Precisely, engrafted MSCs un-dergone an acute death in one week after transplantation in the hypoxicmicroenvironment of infarcted heart [58]. Since MSCs are in vitro cul-tured at a pO2 level of 142mmHg or 20% O2, which is much higherthan that of the in vivo environment, transplanted MSCs are rapidly lostafter engraftment due to the hypoxic stress-induced apoptosis [59].Preconditioning by brief hypoxia prior to cell administration allowsMSCs to better adapt to the lower pO2 tissue environment and promotestheir survival by inducing autophagy [60–62]. Hypoxia-inducible factor1 (HIF-1), which functions as a master regulator of adaptive responsesto hypoxia, regulates the autophagy when MSCs are cultured underhypoxic conditions [60,61]. Hypoxia promotes proliferation of MSCs,through the activation of autophagy related mitogenic neuropeptideApelin [61]. Induction of autophagy significantly attenuates hypoxia-induced apoptosis of MSC enhancing their survival in hypoxic micro-environment [62]. Survival of MSCs post transplantation in damagedmyocardium is enhanced by drugs like atorvastatin, which activatesautophagy and helps survival of MSCs [62]. Similarly, rapamycin-in-duced activation of AMPK/mTOR signaling pathway attenuates, while3-MA promotes hypoxia-induced apoptosis of MSCs confriming that,under hypoxic conditions, the apoptosis of MSCs may be regulated byautophagy [63]. Accordingly, activation of autophagy in MSCs prior totheir transplantation in iscemic myocardium may be useful approach toenhance survival and therapeutic effects of MSCs in the treatment ofMI.

4.2. Autophagy as new strategy for enhanced efficacy of MSC-basedtherapy of diabetic limb ischemia

Transplantation of MSCs hold great promise as an alternative re-vascularization therapy for the treatement of diabetic lower limbischemia by virtue of their proangiogenic properties [64]. However,only few clinical studies have managed to demonstrate a signifcantimprovement in limb salvage after MSC trearment due to the reducedsurvival rate and viability of MSCs after engraftment in ischemic mi-croenvironment of diabetic limbs [65]. Since autophagy regulatesapoptosis of MSCs under hypoxic conditions, hypoxic pretreatment ofMSCs prior to transplantation has become a primary method to improvesurvival and therapeutic potential of MSCs in the treatment of diabetic

Fig. 1. An interplay between autophagy and apoptosis. The pro-death role of autophagy is complicated due to the extensive interference between different signalingpathways. Bcl-2 has been identified as a crucial molecule in maintaining signaling crosstalk among autophagy and apoptosis. When nutrients are sufficient, Beclin-1and Bax/Bak binds with Bcl-2 preventing initiation of autophagy and apoptosis respectively. During stress conditions, several mechanisms mediate the disruption ofthis interaction to allow induction of autophagy and apoptosis. Some of these mechanisms include JNK-mediated phosphorylation of Bcl-2, Beclin-1 phosphorylationby DAPK, phosphorylation and activation of Vps34 by PKD (upper panel) and competitive pro-apoptotic BH3-only protein interactions with Bcl-2 (lower panel).

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complications. Liu and colleagues found that hypoxic pretreatment ofMSCs up-regulated autophagy through AMPK/mTOR signalingpathway, remarkably increased viability of engrafted MSCs and notablyenhanced pro-angiogenic characteristics and therapeutic effects ofMSCs in the repair of diabetic lower limb ischemia [66].

5. Modulation of autophagy as new approach for enhancement ofimmunosuppressive characteristics of MSCs

Several lines of evidence showed that modulation of autophagy inMSCs can provide a novel strategy to improve MSCs-based therapeuticeffects in the treatment of immune cell-mediated diseases.

Results from a recently published study [67] demonstrated thatautophagy enhances MSC-dependent inhibition of CD4+T cells byincreasing production of immunosuppressive TGF-β1. Pre-treatmentwith rapamycin remarkably strengthened the capacity of MSCs to se-crete TGF-β1 and suppress proliferation of CD4+T lymphocytes,whereas use of 3-MA significantly attenuated TGF-β1-dependent in-hibition of T cells by MSCs. Addition of recombinant TGF-β1 managedto recover immunosuppressive capacity of 3-MA-pretreated MSCs,whereas anti-TGF-β1 antibody mediated blockade of TGF-β1 com-pletely abbrogated immunosuppressive potential of rapamycin-pre-treated MSCs [67]. It is well known that MSC-derived TGF-β1 sup-presses activation of Jak-Stat signaling pathway in T cells, causing theG1 cell cycle arrest while inhibition of TGF-β partially alleviate MSC-mediated attenuation of lymphocyte expansion [6]. Accordingly, in-duction of autophagy may be used to increase production of TGF-β1and several other immunosuppressive factors in MSCs significantlyenhancing their therapeutic efficacy in the treatment of immune cell-mediated diseases. This approach was confirmed in MSC-based therapyof acute Graft-versus-host disease (aGVHD), a lethal complication inallogeneic bone marrow transplantation (BMT) recipients (Fig. 2). Thebeneficial effects of rapamycin-induced activation of autophagy inMSCs resulted with increased production of immunosuppressive factors(TGF-β1, IL-10 and IDO) in MSCs while clinical manifestations ofaGVHD were significantly reduced in the mice that received rapamycin-treated MSCs compared with animals that received rapamycin-un-treated MSCs [68]. Through the production of IL-10, MSCs affect ma-turation of DCs and inhibit activation of T cells while MSC-derived IDOis critical molecular switch that stimulates immunosuppressive prop-erties of T regulatory cells (Tregs) and simultaneously blocks re-pro-gramming of Tregs into IL-17 producing effector Th17 cells [6]. In linewith these findings, increased production of IL-10 and IDO in rapa-mycin-treated MSCs corresponded with increased expansion of Tregs

and reduced presence of inflammatory Th17 cells in mice that receivedrapamycin-treated MSCs [68], indicating that activation of autophagyin MSCs prior to their transplantation signifcantly increased their im-munosuppressive effects against CD4+T cells.

Interestingly, completely opposite findings were reported by Dangand colleagues [69] who investigated the effects of autophagy-relatedmodulation in MSC-based therapy of experimental autoimmune en-cephalomyelitis (EAE), animal model of multiple sclerosis. They de-monstrated that inhibition, and not activation, of autophagy promotedMSC-mediated suppression of CD4+T cell driven inflammation incentral nervous system (CNS) [69]. Inflammatory cytokines (TNF-α andIFN-γ), produced by myelin specific CD4+T cells, activated autophagyin transplanted MSCs by inducing expression of Beclin 1. Inhibition ofautophagy, by knocking down Beclin 1, managed to significantly im-prove therapeutic effects of MSCs in EAE by preventing activation ofautoreactive CD4+T cells, but had no influence on transdifferentiationbetween Tregs and Th17 cells or on cytokine profile of effector T cells[69]. Mechanistically, inhibition of autophagy increased generation ofROS and activation of MAPK1/3 in MSCs, resulting with increasedproduction of immunosuppressive PGE2 which, through the down-regulation of IL-2 receptor and janus kinase (JAK)-3, attenuated re-sponsiveness of T cells to mitogen IL-2 [6].

Due to their immunomodulatory characteristics MSCs are con-sidered as new therapeutic agents in the cell-based therapy of acutekidney injury (AKI) [70]. It was recently demonstrated that activationof autophagy in renal tubular epithelial cells, induced by human um-bilical cord MSC-derived exosomes (hucMSC-Ex), significantly reducedcisplatin-induced nephrotoxicity and AKI [71]. Wang and colleaguesidentified a hucMSC-ex-derived protein 14-3-3ζ that was transportedinto renal tubular cells where interacted with ATG16 L and promoted itslocalization to the outer surface of the phagophore [71].

Immunosuppressive properties of MSCs are mainly responsible fortheir beneficent effects in the treatment of immune cell-mediated acuteand chronic liver diseases [47]. Inhibition of autophagy in MSCs viadown-regulation of ATG7 increased the viability of MSCs and conse-quently enhanced their therapeutic effects [41]. Furthermore, whenautophagy is inhibited in MSCs, their potential for liver regenerationwas significantly improved, mainly due to increased secretion of im-munomodulatory and hepato-protective cytokines IL-6 and IL-10 [72].

6. Conclusions

The implications of autophagy in modulation of stemness, apop-tosis, viability and survival of MSCs should be used as new therapeutic

Fig. 2. Modulation of autophagy as a novel strategy for enhancement of immunosuppressive characteristics of MSCs. Induction of autophagy may be used to increasetherapeutic efficacy of MSCs in the treatment of immune cell-mediated diseases, such as aGVHD. Through increased production of several immunosuppressive factors(TGF-β1, IL-10 and IDO), rapamycin-treated MSCs affect maturation of DCs and inhibit activation of T cells, stimulate immunosuppressive properties of Tregs andsimultaneously blocks re-programming of Tregs into IL-17 producing effector Th17 cells. The beneficial effects of rapamycin-induced activation of autophagy in MSCsresulted with significantly reduced clinical manifestations of aGVHD in the mice that received rapamycin-treated MSCs compared with animals that receivedrapamycin-untreated MSCs.

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approach of MSCs priming before MSC-based therapy of degenerativeand autoimmune diseases.

It should be emphasized that in most of published studies autophagywas modulated in MSCs in vitro, prior to their transplantation in ex-perimental animals and that observed effects of autophagy on differ-entiation and immunomodulatory properties of MSCs may not be ne-cessarily confirmed in vivo. Accordingly, there is a growing need amongthe stem cell community to monitor and modulate autophagy in alreadyengrafted MSCs. For this purpose, MSCs derived from transgenic micethat systemically express green fluorescent protein (GFP) fused to LC3,a mammalian homologue of yeast Atg8 (Aut7/Apg8), which serves as amarker protein for autophagosomes [73], could be used. These animalscould also be used to determine the effects of autophagy on stem cellcharacteristics of resident, endogenous MSCs at various time pointsafter tissue injury or during regeneration. For this purpose, MSCs couldbe isolated from specific tissues based on the expression of surfacemarkers: CD49a, CD63, CD73, CD105, CD106, CD140b, CD271, TNAP,Hsp90-beta, as well as orphan antigens defined by antibodies STRO-1,W3D5, W5C5. Additionally, placenta-derived MSCs could be pre-ferentially isolated using antibodies against CD349, SSEA-4, and TRA-1–81, which contrasts with antibodies usually used for isolation ofBM–MSCs (CD271 or TNAP), AT-MSCs and AF-MSCs (CD117) [74–76].

Importantly, opposite findings regarding the consequences of au-tophagy activation and inhibition for MSC-dependent modulation ofhost immune response suggests that great caution has to be taken andfurther animal studies should be conducted before considering thetransplantation of autophagy-modulated MSCs in patients.

Funding

This work was supported by Serbian Ministry of Science(ON175069, ON175103) and Faculty of Medical Sciences University ofKragujevac (JP02/09).

Declaration of interest

The authors declare no potential conflicts of interest.

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