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Contents lists available at ScienceDirect

Seminars in Cell & Developmental Biology

journa l homepage: www.e lsev ier .com/ locate /semcdb

eview

SC exosome as a cell-free MSC therapy for cartilage regeneration:mplications for osteoarthritis treatment

ei Seong Toh a,b,∗, Ruenn Chai Lai d, James Hoi Po Hui b,c, Sai Kiang Lim d,e,∗∗

Faculty of Dentistry, National University of Singapore, SingaporeTissue Engineering Program, Life Sciences Institute National University of Singapore, SingaporeCartilage Repair Program, Therapeutic Tissue Engineering Laboratory, Department of Orthopaedic Surgery, National University Health System, Nationalniversity of Singapore, SingaporeInstitute of Medical Biology, Agency for Science, Technology and Research, SingaporeDepartment of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

r t i c l e i n f o

rticle history:eceived 26 September 2016eceived in revised form7 November 2016ccepted 18 November 2016vailable online xxx

eywords:

a b s t r a c t

Mesenchymal stem cell (MSC) therapies have demonstrated efficacy in cartilage repair in animal and clin-ical studies. The efficacy of MSC-based therapies which was previously predicated on the chondrogenicpotential of MSC is increasingly attributed to the paracrine secretion, particularly exosomes. Exosomesare thought to function primarily as intercellular communication vehicles to transfer bioactive lipids,nucleic acids (mRNAs and microRNAs) and proteins between cells to elicit biological responses in recip-ient cells. For MSC exosomes, many of these biological responses translated to a therapeutic outcomein injured or diseased cells. Here, we review the current understanding of MSC exosomes, discuss the

artilageesenchymal stem/stromal cells

hondrocytesxosomesxtracellular vesiclesissue engineering

possible mechanisms of action in cartilage repair within the context of the widely reported immunomod-ulatory and regenerative potency of MSC exosomes, and provide new perspectives for development ofan off-the-shelf and cell-free MSC therapy for treatment of cartilage injuries and osteoarthritis.

© 2016 Elsevier Ltd. All rights reserved.

issue regeneration

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. MSCs in cartilage repair and regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. MSC secretome. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .004. Therapeutic MSC exosomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .005. Potential mechanisms underlying MSC exosomes in cartilage regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5.1. Bioenergetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.2. Cell number . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005.3. Immunomodulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

6. MSC exosomal miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: W.S. Toh, et al., MSC exosome as a costeoarthritis treatment, Semin Cell Dev Biol (2016), http://dx.doi.org

7. MSC exosomes: the next generation therapeutics for OA. . . . . . . . . . . . . . . . . .

8. Challenges for exosome-based therapy for OA . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Faculty of Dentistry, National University of Singapore,1 Lower Kent Ridge Road, 119083, Singapore.∗∗ Corresponding author at: Institute of Medical Biology (IMB), Agency for Sci-nce and Technology (A*STAR) 8A Biomedical Grove, #05-16 Immunos, 138648,ingapore.

E-mail addresses: dentohws@nus.edu.sg (W.S. Toh),aikiang.lim@imb.a-star.edu.sg (S.K. Lim).

ttp://dx.doi.org/10.1016/j.semcdb.2016.11.008084-9521/© 2016 Elsevier Ltd. All rights reserved.

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Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00Competing interests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. Introduction

Osteoarthritis (OA) is the most common form of chronic jointisease affecting 250 million people worldwide. It is presently the

astest growing major health condition. With an aging populationnd expanding obesity epidemic, OA incidence is anticipated toe the fourth leading cause of disability by the year 2020 [1–3].he pathology of OA is complex involving multiple tissues androcesses. It is characterized by the degradation of the articu-

ar cartilage, degeneration of menisci and ligaments, thickeningf the subchondral bone, and formation of osteophytes [4]. How-ver, it is often a sequela initiated by articular cartilage injuriesaused primarily by sports and recreational activities, [5] and aging6,7]. Articular cartilage lesions often result in potentially cripplingymptoms such as activity-related pain, swelling and impairedobility, and are a major risk factor for development of OA [4,8].

xacerbation of such lesions into full-thickness lesions expose jux-aposed joint bones causing direct bone-to-bone rubbing during

ovement to further exacerbate injury and progression to OA.There is currently no cure for OA and most treatments for

A are essentially symptomatic therapies to manage pain, stiff-ess and swelling. Since articular cartilage damage is central toA pathology, restoring the integrity and function of the artic-lar cartilage is critical in halting or reversing the progressionascade to OA where the final treatment option is prostheticeplacement. However, repair and regeneration of damaged artic-lar cartilage has been challenging as cartilage generally have poorealing capacity. There are presently no pharmacologic treatmentso repair and regenerate damaged articular cartilage. Both tra-itional and investigative new pharmacologic treatments for OAuch as acetaminophen, non-steroidal anti-inflammatory drugs,nd opioids, and sprifermin/recombinant human fibroblast growthactor-18 [9] Tanezumab [10] relieve pain and swelling but showittle to no ability in repair of damaged cartilage and restoration ofartilage homeostasis.

The intractability of articular cartilage to pharmacologic inter-ention for repair and regeneration is due primarily to it being

poorly vascularised, aneural and alymphatic load-bearing tis-ue supported by the underlying subchondral bone. Furthermore,igration of chondrocytes to the site of injury to participate in

artilage repair is hampered by its microenvironment of densextracellular matrix (ECM) [11]. To circumvent these intrinsicimiting factors, microfacture and drilling, and autologous chon-rocyte implantation (ACI) were introduced [5,12]. A rationale

or microfacture and drilling is to mobilise and stimulate MSCsn the bone marrow to participate in cartilage repair. However,hese techniques often yield inferior fibrocartilage repair [13] thatre prone to degeneration. ACI which was first reported to repairrticular cartilage lesions in 1994 [14] is generally more effectivehan microfracture [15] or osteochondral grafts [16]. However, a

eta-analysis of six trials involving 431 participants concludedhat there was insufficient evidence on the use of ACI to treatull-thickness articular cartilage defects in the knee [17]. Never-heless, another meta-analysis of 153 young patients with earlysteoarthritis observed that 92% of patients were functioning well

years post-operation [18]. The challenge in ACI is associated with

Please cite this article in press as: W.S. Toh, et al., MSC exosome as a costeoarthritis treatment, Semin Cell Dev Biol (2016), http://dx.doi.org

he use of autologous tissue where there is limited tissue availabil-ty, donor site morbidity and loss or modification of chondrocytehenotype after ex vivo expansion. In addition, ACI is also associatedith inferior fibrocartilage formation and high cost [12].

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

In recent years, stem cells particularly mesenchymalstem/stromal cells (MSCs) have emerged as an alternativecell type to circumvent some of the drawbacks of ACI. MSCs aremultipotent progenitor cells that possess self-renewal capabilityand can differentiate into multiple lineages including osteoblasts,adipocytes and chondrocytes [19]. Although MSCs have beenidentified and isolated in the cartilage [20], overlying synovium[21], and even synovial fluid [22], these endogenous MSCs arelimited in numbers, and effective mobilization of these cells tothe site of cartilage lesion to participate in tissue repair haveyet to be demonstrated. Hence, MSCs are routinely harvestedfrom autologous or allogenic tissues such as bone marrow andadipose tissues where they are expanded ex vivo to generate morecells for transplantation. The efficacy of autologous or allogenicMSCs in cartilage repair has been demonstrated in animal studies[23–26], and more recently in human clinical trials [27–32]. Theuse of MSCs to repair cartilage tissues was predicated on thehypothesis that these cells could differentiate into chondrocytesto replace the damaged tissue. In recent years, there is howeverincreasing evidence to suggest that MSCs secrete a wide rangeof trophic factors to modulate the injured tissue environmentand to orchestrate subsequent regenerative processes includingcell migration, proliferation, differentiation, and matrix synthesis[33,34]. In 2010, it was first reported that exosome was the activeagent in MSC secretion against myocardial ischemia reperfusion(I/R) injury [35]. Since then MSC exosomes were found to beefficacious against many disease targets of MSC. MSC exosomeswas recently reported to mediate cartilage repair and regeneration[36,37].

Here, we review the characteristics and properties of MSCs, anddiscuss the emerging role of MSC exosomes in cartilage repair andregeneration. In particular, we present novel perspectives for thedevelopment and implementation of MSC exosomes as an off-the-shelf and cell-free regenerative medicine approach for cartilagerepair.

2. MSCs in cartilage repair and regeneration

MSCs have been successfully isolated from several adult tissuesincluding the bone marrow, adipose tissue, synovium, and periph-eral blood. The wide use of MSCs in clinical trials is largely attributedto their ex vivo expansion capacity, easy accessibility and isola-tion from several adult tissues [19]. To date, MSCs including bonemarrow and adipose tissue-derived MSCs have been intensivelyinvestigated as a cell-based therapy or in combination with scaf-fold in a tissue engineering approach to treat cartilage lesions andOA in both animal [23,25,38] and human studies [27,31,39–42].Although many of these studies used autologous MSCs where therisk of immune rejection is minimal, allogenic MSCs have also beenfound to be equally safe and therapeutically efficacious in human[43]. MSC transplantation also offers additional advantages includ-ing minimal donor site morbidity from bone marrow aspirationunlike the surgical harvesting of cartilage for isolation of chondro-cytes in ACI [27,39], and the ease of minimally-invasive injectionsof bone marrow concentrate or expanded cells.

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3. MSC secretome

Increasingly, both animal and human studies present evidencethat MSCs mediate tissue repair via secretion of trophic factors

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33]. The trophic effects of MSCs on chondrocytes were reportedy Wu and colleagues in co-culture studies. They observed thatuman bone marrow MSCs promote proliferation and ECM synthe-is of chondrocytes through its secretion, and MSCs from differentources including bone marrow, adipose tissue and synovial mem-rane exert similar trophic effects, irrespective of the tissue originnd culture conditions [44,45]. However, the trophic factors medi-ting the paracrine effects of MSCs are not well-characterizedn these studies. With new advances in proteomics, many ofhese trophic factors are now being identified and they spanned aroad spectrum of trophic factors that include growth factors andytokines, as well as ECM molecules [33,46].

MSCs are often considered as ‘environmentally-responsive’ cellsnd they are known to secrete bioactive factors and signals inesponse to the local microenvironmental cues [47]. Notably, sev-ral studies reported that the MSC secretome changes significantlyhen the cells were exposed to inflammatory conditions present

n tissue injuries and diseases [48]. For instance, it has been shownhat under pro-inflammatory condition in the presence of TNF-�,uman adipose tissue MSCs produced higher levels of IL-6, IL-

and monocyte chemotactic protein (MCP-1) that are involvedn the migration of monocytes in response to inflammation [48].hese observations have led to much interest in understandingnd influencing the profile of MSC secretion using approachesuch as biophysical (bioreactor), physiological (hypoxia, growthactors/cytokines, cell-cell interactions), pharmacological (chemi-als and small molecules), and genetic manipulations (reviewed in34]). The increasing evidence for the therapeutic potency of MSCecretion has also provided much impetus in identifying the activeherapeutic agent in MSC secretome.

. Therapeutic MSC exosomes

For many years, efforts to identify the active therapeutic factorn the MSC secretome have focused on growth factors, cytokinesnd chemokines [33]. Despite many promising candidate factors,o single factor could sufficiently account for the efficacy of MSCs.

n 2009, Bruno et al. attributed the ameliorating effect of MSC secre-ion on glycerol-induced acute kidney injury to microvesicles [49].hese microvesicles had a size range of 80 nm to 1 �m. In contrast,ai and colleagues attributed the cardioprotection of MSC secre-ion to exosomes, a class of membrane vesicles with a diameter of0–100 nm, an endosomal origin, and possess exosome-associatedroteins such as Alix, tumor susceptibility 101 (TSG101) andetraspanins (CD9, CD63 and CD81)[35]. Both microvesicles andxosomes are extracellular vesicles (EVs). EVs are generally hypoth-sized to be intercellular communication vehicles and functiono transfer lipids, nucleic acids (mRNAs and miRNAs) and pro-eins between cells to elicit biological responses in recipient cellshat are reflective of the cargo contents [50]. The major differ-nces between exosomes and microvesicles are that exosomesre released through the fusion of multivesicular bodies withhe plasma membranes and have a diameter of 30–150 nm while

icrovesicles are shed from the plasma membrane and have diam-ters of 100–1000 nm. Of the two, exosomes are presently moremportant as evidenced by the exponentially increasing numberf exosome-related publications over that of microvesicle in theecent years [51].

Exosomes are secreted by many cell types, such as B and T lym-hocytes, dendritic cells, mast cells, platelets and tumor cells andre found in most bodily fluids such as blood, urine, cerebrospinal

Please cite this article in press as: W.S. Toh, et al., MSC exosome as a costeoarthritis treatment, Semin Cell Dev Biol (2016), http://dx.doi.org

iquid, breast milk and saliva [50]. Diseased cells also use exosomess vehicles to disseminate injurious signals [52,53], and have beenecently implicated in development of OA [54]. Kato et al., 2014ound that exosomes from IL-1� stimulated synovial fibroblasts

PRESSmental Biology xxx (2016) xxx–xxx 3

to induce OA-like changes with increased expression of catabolicmarkers including a disintegrin and metalloproteinase with throm-bospondin motifs (ADAMTS)-5 and metalloproteinase (MMP)-13in chondrocytes and matrix degradation with proteoglycan releasefrom cartilage explants [54].

In recent years, exosomes have been increasingly reported asthe principal therapeutic agent in MSC secretion that underpinsthe regenerative and immunomodulatory capabilities of MSCs intissue repair (reviewed in [55]). To date, exosomes have been iso-lated from MSCs derived from adipose tissue, bone marrow, fetaltissues, umbilical cord, ESC-, and iPSC [35,56–61]. Although it hasbeen reported that the RNA cargo in MSC exosome varies accordingto the tissue sources of MSC exosomes [62], it was also reported thatthe therapeutic efficacy of MSC exosomes is not dependent on thetissue sources of the MSCs [63]. This is consistent with the observa-tion that the trophic effects of MSCs derived from different tissuessuch as bone marrow, adipose tissue and synovial membrane havesimilar effects on chondrogenesis [44,45]. We however did observethat tissue source of MSC has an impact on exosome yield which isinversely correlated to the developmental age of the tissue [63].

Detailed analyses performed by mass spectrometry, antibodyarray and microarray further revealed that MSC exosomes carrya complex cargo of nucleic acids, proteins and lipids, with >850unique gene products (www.exocarta.org) [64] and >150 miR-NAs [65]. The exosomal proteins and microRNAs are functionallycomplex, and are implicated in many diverse biochemical and cel-lular processes such as communication, structure and mechanics,inflammation, exosome biogenesis, tissue repair and regenera-tion, and metabolism. This wide distribution of biological activitiesconfers on MSC exosomes the potential to elicit diverse cellularresponses and interact with many cell types [66].

Although the role of each of these individual gene products, i.e.mRNAs, miRNAs, proteins and lipids in the therapeutic activity ofexosome has not yet been determined, the combined functionalcomplexity of the cargo provides a rationale for the wide rangingtherapeutic efficacies that have been reported of MSC exosomes[67,68]. To date, human MSC exosomes have been reported to pro-tect against myocardial I/R injury [35], attenuate limb ischemia[69], enhance wound healing [70,71], ameliorate graft-versus-host-disease (GVHD) [72], reduce renal injury [58], promote hepaticregeneration [73], inhibit pulmonary hypertension [74], alleviateretinal injury[75], and more recently improve cartilage [36] andbone regeneration [56].

5. Potential mechanisms underlying MSC exosomes incartilage regeneration

We had recently reported that human MSC exosomes promotecartilage regeneration in an immunocompetent rat osteochondraldefect model [36]. In that study, MSC exosomes accelerated neotis-sue filling and enhanced matrix synthesis of type II collagen andsulphated glycosaminoglycan (s-GAG). By the end of 12 weeks,exosome-treated rats displayed complete restoration of cartilageand subchondral bone with characteristic features including a hya-line cartilage with good surface regularity, complete bonding toadjacent cartilage, and an ECM deposition that closely resemblethat of age-matched native control. In contrast, there were onlyfibrous repair in contralateral control defects treated with the salinevehicle.

Like the other therapeutic efficacies reported for MSC exo-somes such as wound healing and cardioprotection, the mechanism

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underlying cartilage regeneration by MSC exosomes has also notbeen elucidated. While the cargo of MSC exosome is sufficientlylarge and diverse to support different mechanistic hypothesis foreach of the therapeutic efficacies reported for MSC exosome, it

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s difficult to rationalize the purpose of the diverse cargo in MSCxosomes. We have previously proposed that MSCs being stromalupport cells function primarily to maintain a homeostatic tissueicroenvironment for optimal tissue function. As such, they must

e able to sense changes in the microenvironment and respond in aimely and effective manner to restore homeostasis. MSC exosomeseing intercellular communication vehicles are eminently suitedo transmit MSC response in a timely and effective manner forhis purpose [67]. The bi-lipid membrane of MSC exosomes confershe versatility to interact with multiple cell types both intracellu-arly through endocytosis or membrane fusion, and extracellularlyhrough membrane receptor-ligand interactions. In addition, MSCxosomes are rich in ECM proteins and enzymes that could modu-ate and restore homeostasis to the ECM. Most importantly, we havereviously proposed that MSC exosomes have an intrinsic capac-

ty to restore homeostasis because of its enzyme-rich protein cargo64,76–78]. Enzyme-mediated reactions are catalytic rather thantoichiometric, and enzymatic activity increases in proportion tohe loss of homeostatic equilibrium between the enzyme substratend product. Hence, loss of homeostasis during injury and diseaseill activate enzyme-based exosomes to restore homeostasis to

acilitate tissue function in repair and regeneration. Upon resolu-ion of the injury, homeostasis is restored and exosome enzymectivities would also cease. Hence, exosome-based therapeuticsould be highly responsive to and yet limited by the disease pre-ipitating microenvironment [78].

As MSCs from different tissue sources produce exosomes withimilar therapeutic activity but at a different yield per cell [63,79],he key exosome enzymes mediating the homeostatic activity areikely to be conserved across all MSC exosomes. Indeed, all MSC exo-omes are characterized by a common set of proteins and RNAs. Wead previously suggested that exosomes from other cell sourcesay have similar therapeutic activity as all exosomes shared an

volutionary conserved set of proteins and RNA [80]. However, exo-omes from other cell types many not be as ideal as MSC exosomesecause MSCs are easily available and expansible from a wide vari-ty of tissues, not immunologically reactive and have a strongocumented clinical safety record. In addition, they are prolific pro-ucer of exosomes [80]. We previously noted that the evolutionaryonserved set of proteins in exosomes includes many housekeep-ng enzymes that could modulate homeostasis in bioenergetics, cellumber and immunomodulation [67]. Here we propose that MSCxosomes facilitate endogenous cartilage repair and regenerationy restoring homeostasis in these areas (Fig. 1).

.1. Bioenergetics

Mitochondrial dysfunction and damage which are integral tohe aging process have been associated with the pathology ofA. OA chondrocytes are found to have reduced mitochondrialiogenesis [81] and decreased mitochondrial electron transporthain (ETC) proteins [82]. As the ETC is critical for ATP produc-ion by oxidative phosphorylation in the mitochondria, reducedTC activity and mitochondria number would lead to inefficientTP production and a loss of homeostasis in bioenergetics. This

oss provides a mechanistic rationale for the compromised cellularctivities frequently observed in OA such as increased oxida-ive stress, defective chondrocyte matrix biosynthesis and growthactor responses, increased cytokine-induced chondrocyte inflam-

ation and matrix catabolism, cartilage matrix calcification, andncreased chondrocyte apoptosis [83–85]. In such a scenario, theapacity of OA chondrocyte to repair would be severely compro-

Please cite this article in press as: W.S. Toh, et al., MSC exosome as a costeoarthritis treatment, Semin Cell Dev Biol (2016), http://dx.doi.org

ised and restoring bioenergetic homeostasis in OA tissues woulde fundamental to the initiation of repair and regeneration activ-

ties in OA chondrocytes. MSC exosomes carry a cargo rich inctive glycolytic ATP-generating enzymes such as phosphoglucok-

Fig. 1. Proposed MSC exosome mechanism of action in cartilage repair and regen-eration. MSC exosomes have the biochemical potential to restore homeostasis inbioenergetics, cell number and immunomodulation.

inase and pyruvate kinase, and ATP-generating enzymes such asadenylate kinase and nucleoside-diphosphate kinase. We have pre-viously proposed that these glycolytic and ATP generating enzymesfacilitate the increase in ATP level observed in exosome-treatedreperfused ischemic myocardium [76,86,87]. Similarly, it is plau-sible that these same enzymes could compensate for the reducedmitochondrial ATP production in OA chondrocytes. Although ATPsynthesis per glucose molecule in glycolysis is inefficient in com-parison to mitochondrial oxidative phosphorylation, the cellularcapacity to increase glycolytic flux by a factor of 10–100 readilycompensate for this inefficiency. In addition, glycolysis producesmetabolic intermediates for anabolic processes and help restoreredox potential (reviewed in [88]) that would be needed to facilitaterepair of OA cartilage.

5.2. Cell number

In OA, the injured/diseased cartilage is often further aggra-vated by inflammation resulting in cell death, matrix degradationand finally loss of structure and function [89,90]. Cells die mainlythrough the process of apoptosis, induced as a result of inflamma-tion, oxidative stress and mitochondrial dysfunction present in OA[7]. To restore the cell number, tissue structure and function, thedepleted cells would have to be replaced to a homeostatic steadystate level and this is best evidenced by the importance of mes-enchymal cell proliferation in initiating a chondrogenic reparativeresponse followed by matrix deposition and tissue formation atthe defect site [91,92]. Consistent with this, we observed that dur-ing MSC exosome treatment, neotissue formation and subsequenthyaline cartilage regeneration at 12 weeks in the rat osteochon-dral defect model was preceded by enhanced cellular proliferationas evidenced by the presence of proliferative cell nuclear antigen(PCNA) at 2 weeks [37].

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We have previously proposed that in injured tissues, MSCexosome induces proliferation through adenosine-mediated phos-phorylation of the survival kinases, ERK1/2 and AKT. When tissuesare injured during tissue trauma (e.g. shear or mechanical stress

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93,94], chemotherapy [95] or hypoxia [96], they released dangerignals such as ATP (reviewed [97,98]). Extracellular ATP stimulatesmmunogenic cells to remove injured and dying cells [95,99]. How-ver, sustained injury could lead to excessive ATP signal-inducedell death and a “bystander effect” on healthy neighbouring cellseading to a net loss in cell number and compromised tissue func-ion. Therefore, to repair tissue after damaged tissues are removed,xtracellular ATP death signal must be eased and cell prolifera-ion initiated. Extracellular ATP and ADP with short half-lives of1 s [100] and <3.2 min [101] respectively, are rapidly erased byydrolysis to AMP which is then degraded to adenosine, a potentctivator of survival kinases through adenosine receptors [102]. Theydrolysis of extracellular AMP to adenosine is catalysed by CD73he only known extracellular ecto 5′nucleotidase and also a highlyegulated enzyme [103]. Hence, the pro-death signal presented byhe highly unstable extracellular ATP could be readily converted todenosine a pro-survival signal by CD73. CD73 is a hallmark fea-ure of MSC exosomes and most exosomes as well. In the presencef AMP, MSC exosomes activate phosphorylation of survival kinasesRK and AKT through theophylline-sensitive adenosine receptors87]. The significance of this observation is that MSC exosome CD73an restore pro-death ATP and pro-survival adensone signalling ton equilibrium state [87]. Hence, MSC exosome through CD73 couldonvert a death signal from injured tissues, ATP to a pro-survivalignal, adenosine to initiate cell proliferation for tissue repair andegeneration.

.3. Immunomodulation

The primary role of the immune system is traditionally vieweds an integral component of the body defence system against thexternal environment and pathogens. However, it is becoming evi-ent that the immune system is important in influencing tissueepair. In the case of cartilage injury, the rapid upregulation of pro-nflammatory cytokines including IL-1�, IL-6 and IL-8, and MMP-3ontributes to subsequent matrix degradation and joint damage90]. These pro-inflammatory cytokines and MMPs are mainly pro-uced by the synovium and immune cells such as the macrophages,nd contribute to the onset and development of OA [89]. Recently,t was reported that M1 polarised macrophages in OA synovium tis-ues inhibit chondrogenic differentiation of MSCs via IL-6 in vitro104] while M2 macrophage polarization support survival of car-ilage graft by production of anti-inflammatory IL-10 to suppressdverse inflammation [105]. Therefore, modulation of the pro-nflammatory environment in injured cartilage or OA is importantn regenerative cartilage therapy [106].

It is well-established that MSCs are immunomodulatory33,107] and this immunomodulation is mediated largely througharacrine secretion of trophic factors such as interferon (IFN)-�108,109], transforming growth factor (TGF)-�1 [110], hepatocyterowth factor (HGF) [111], heme oxygenase-1 [112], IL-6 [113]nd prostaglandin E2 (PGE2) [114]. However, the immunomod-latory activity of MSCs could not be sufficiently rationalised byny one of these secreted factors alone [108,110,115], suggestinghat the immunomodulatory activity of MSCs require the syner-ism of multiple factors. MSC exosome with a proteome of >200mmunomodulatory proteins is an ideal vehicle for this syner-ism [64,67,116]. Consistent with this, MSC exosomes could induceigh levels of anti-inflammatory IL-10 and TGF-�1, and attenu-ted levels of pro-inflammatory IL-1�, IL-6, TNF-� and IL-12P40 inHP-1 monocytes in vitro [72]. Most significantly, we observed that

Please cite this article in press as: W.S. Toh, et al., MSC exosome as a costeoarthritis treatment, Semin Cell Dev Biol (2016), http://dx.doi.org

SC exosomes induce Tregs in mice with active immune reactivitynduced by the grafting of allogenic skin but not in un-grafted con-rol mice, indicating MSC exosomes are immunomodulatory andot immunosuppressive [72].

PRESSmental Biology xxx (2016) xxx–xxx 5

In other studies, MSC exosomes have been reported to mod-ulate the inflammatory response present in hypoxic pulmonaryhypertension [74], diabetic cutaneous wound [117], myocardial I/Rinjury [118], and retinal laser injury [75]. Notably, in the mousemodel of hypoxic pulmonary hypertension, MSC exosomes havebeen found to suppress influx of macrophages and induction ofpro-inflammatory monocyte chemoattractant protein-1 (MCP-1)to ameliorate the disease progression [74]. While the immunomod-ulatory role of MSC exosomes has yet to be addressed in OA, it wasrecently found that exosomes present in serum play a significantrole in protecting human OA cartilage from s-GAG loss in the pres-ence of pro-inflammatory IL-1� [119]. We therefore hypothesisethat MSC exosome could alleviate OA since a parallel early increasein M2 macrophages was observed during MSC exosome treatmentto promote cartilage regeneration in immunocompetent rats [37].

6. MSC exosomal miRNA

MSC exosome has a large cargo of about 150 miRNAs [65]. Manyare potent regulators of important signal transduction pathwayssuch as SMAD, AKT and ERK pathways. As such, these miRNAs arelikely to play key roles in mediating the efficacies of MSC exosomeagainst injuries and diseases including OA. For example, some of themiRNAs in the MSC exosomes such as miR-23b and miR-92a couldbe therapeutic against OA through their regulatory roles in pro-liferation and chondrogenesis while others such as miR-125b andmiR-320 could be therapeutic for their roles in modulating matrixsynthesis [120–127] (Table 1).

miR-92a could mediate the efficacy of MSC exosome in alle-viating OA by targeting noggin3 to upregulate proliferation ofchondrocyte and matrix synthesis through the PI3 K/AKT/mTORpathway [122,127] while exosomal miR-23b could exert its effectby inhibiting protein kinase A (PKA) signalling to induce chon-drogenic differentiation of human MSC [120]. On the otherhand, miR-125b and miR-320 could alleviate OA by attenuat-ing ECM breakdown by downregulating expression of ADAMTS-4(aggrecanase-1) and MMP-13, two ECM proteinases that are upreg-ulated in human OA chondrocytes [121,125].

7. MSC exosomes: the next generation therapeutics for OA

MSC therapy to treat OA is presently under clinical testing.Based on the large number of clinical trials using MSCs for a widerange of disease indications, it is evident that MSC-based thera-pies are generally safe. Although MSC has not been approved forOA, the widespread use of MSC to treat OA in pets and other ani-mals [128,129] provides a compelling rationale to use MSC therapyto treat OA in human. The discovery that MSC exosomes mediateMSC therapeutic activity could radically transform MSC therapy byeliminating many difficult issues associated with using living cellsas therapeutics. The use of viable cells carries inherent risks suchas occlusion in microvasculature leading pulmonary embolism anddeath [130], transformation of transplanted cells into inappropri-ate cell types or cancer, immune rejection, proarrhythmic sideeffects [131–133], ossifications and/or calcifications [134]. A peren-nial risk of cell-based treatment is that it is considered a permanenttreatment as the transplanted cells cannot be removed in eventof adverse activity or upon disease resolution. The manufacture ofcell-based therapeutics is also challenging as cell viability, potencyand transformation have to be monitored and maintained through-out the manufacturing process, storage, and delivery to patient.

ell-free MSC therapy for cartilage regeneration: Implications for/10.1016/j.semcdb.2016.11.008

Replacing cells with exosomes eliminates many of these chal-lenges such as occlusion of blood vessels and generation ofinappropriate cell types as exosomes are not viable and small.Unlike cell therapy, exosome treatment is not a permanent treat-

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6 W.S. Toh et al. / Seminars in Cell & Developmental Biology xxx (2016) xxx–xxx

Table 1MSC exosomal miRNAs involved in regulation of chondrogenesis and cartilage haemostasis.

microRNA Targets Effects Reference

miR-23b PKA Induce chondrogenic differentiation of human MSCs by inhibiting PKAsignalling

[120]

miR-92a Noggin3 Targets noggin3 and activates PI3K/AKT/mTOR pathway to positively regulateproliferation and matrix synthesis of chondroprogenitors

[122,127]

miR-125b ADAMTS-4 miR-125b overexpression suppresses IL-1�-induced upregulation ofADAMTS-4 in human OA chondrocytes

[125]

miR-320 MMP-13 Downregulates MMP-13 expression in both ATDC5 cell model ofchondrogenesis and IL-1�-treated primary mouse chondrocytes

[121]

miR-145 Sox9 miR-145 inhibition upregulates Sox9 expression and promotes MSCchondrogenesis

[124]

miR-221 MDM2 Downregulates MDM2 to prevent slug protein degradation, that in turnnegatively regulates chondroprogenitor proliferation

[123]

miR-22 PPAR-�, BMP-7 miR-22 inhibition upregulates BMP-7 and PPAR-� expression, inhibits IL-1�resse

[126]

B PKA: p

mwtpapspm

8

efiSabsioteilcbe

9

amwlipiccemfaoat

expression and supp

MP-7: bone morphogenetic protein 7; MDM2: mouse double-minute 2 homolog;

ent and can be easily suspended when there are adverse effects orhen the disease resolves. Exosome production is more amenable

o process optimization. For example, the cell source for exosomeroduction could be selected or genetically manipulated to gener-te a high exosome-yielding clonal cell line with infinite expansionotential to ensure reproducible cost-effective production of exo-omes. All things considered, MSC exosome therapy for OA couldotentially be a safer, cheaper and a more effective treatmentodality than cell-based MSC therapy.

. Challenges for exosome-based therapy for OA

Translating MSC exosomes into an OA therapeutic carry sev-ral challenges that are unique to exosomes as they represent arst-in-class drug. The primary concern is its safety and toxicity.ince MSC which has been tested in more than 600 MSC clinical tri-ls and generally found to be safe, MSC exosome could reasonablye assumed to be as safe if not safer. Indeed, MSC exosome werehown to be generally well-tolerated and to have minimal risk ofmmunogenicity and toxicity [35,36,72]. However, in the contextf cartilage repair, there are still important questions regardingherapeutic efficacy, biosafety, kinetics and bio-distribution of MSCxosomes that needs to be answered in a large animal study. Fornstance, the bio-distribution and clearance of MSC exosomes fol-owing intra-articular injection in the joint environment (articularartilage, synovium and synovial fluid) need to be evaluated toetter establish the biosafety and therapeutic profile of the MSCxosome therapy.

. Conclusion

MSC exosome is now widely accepted as the principal ther-peutic agent present in the MSC secretion and is sufficient toediate the many reported therapeutic efficacies of MSC. Recently,e demonstrated that human MSC exosomes promote carti-

age regeneration in a full-thickness cartilage defect model inmmunocompetent adult rats [36]. This provides a compellingroof-of-principle that MSC exosomes could alleviate OA via repair-

ng and regenerating the damaged articular cartilage which isentral to the pathogenesis of OA. Although translation to clini-al trials would require validation of the safety and efficacy of MSCxosomes to repair and regenerate cartilage lesions in a large ani-al model, the study using a rat model rationalizes a paradigm shift

rom conventional cell-based MSC therapies to a more accessible

Please cite this article in press as: W.S. Toh, et al., MSC exosome as a costeoarthritis treatment, Semin Cell Dev Biol (2016), http://dx.doi.org

nd safer, off-the-shelf and cell-free MSC therapy. The mechanismf action underpinning MSC exosome-mediated cartilage regener-tion reflects the functional role of MSC as a stromal support cello optimize the viability and vitality of the tissues. Consistent with

s MMP-13 expression in OA chondrocytes

rotein kinase A; PPAR-�: Peroxisome proliferator-activated receptor alpha.

this, the proteome of MSC exosome is enriched in biochemicallyactive housekeeping enzymes that could restore homeostasis to themost essential activities in the cell and tissue microenvironment.Although the use of MSC exosomes to treat OA could be rationalisedby scientific evidences, its ultimate use in the clinic will require val-idation in the appropriate animal models and rigorous evaluationin properly controlled clinical trials.

Funding

This work was supported by in part by grants fromNational Medical Research Council Singapore (R221000080511)and National University of Singapore (R221000090112). SKL is sup-ported by core grants from BMSI, A*STAR.

Competing interests

The authors report that they have no conflicts of interest in theauthorship and publication of this article.

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