Multipotent mesenchymal stromal cells in articular diseases

Best Practice & Research Clinical RheumatologyVol. 22, No. 2, pp. 269–284, 2008

doi:10.1016/j.berh.2008.01.005
available online at http://www.sciencedirect.com
5

Multipotent mesenchymal stromal cells

in articular diseases

Christian Jorgensen*Inserm, Montpellier, France

Universite Montpellier 1, UFR de Medecine, Montpellier, France

Service d’immuno-Rhumatologie, Hopital Lapeyronie, Montpellier, France

Farida Djouad a

Carine Bouffi

Dominique Mrugala

Daniele NoelInserm, U844, Montpellier, France

Universite Montpellier 1, UFR de Medecine, Montpellier, France

Although cartilage defects are common features of osteoarthritis and rheumatoid arthritis,current treatments can rarely restore the full function of native cartilage. Recent studieshave provided new perspectives for cartilage engineering using multipotent mesenchymalstromal cells (MSC). Moreover, MSC have been used as immunosuppressant agents in auto-immune diseases and have tested successfully in animal models of arthritis. However, the se-quential events occurring during chondrogenesis must be fully understood before we canreproduce the complex molecular events that lead to MSC differentiation and long-termmaintenance of cartilage characteristics in the context of chronic joint inflammation. Thischapter focuses on the potential of MSC to repair cartilage, with an emphasis on the factors

* Corresponding author. Inserm U844, CHU Saint Eloi, Batiment INM, 80 avenue Augustin Fliche,

Montpellier F-34091, France.

E-mail address: [email protected] (C. Jorgensen)a Current address: Cartilage Biology and Orthopedics Branch, National Institutes for Health, Bethesda,

Maryland, USA.

1521-6942/$ - see front matter ª 2008 Published by Elsevier Ltd.

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270 C. Jorgensen et al

that are known to be required in inducing chondrogenesis and on their immunosuppressivepotential.

Key words: characterization; immunosuppression; mesenchymal stem cell.

POTENTIAL OF MESENCHYMAL STROMAL CELLSIN ARTICULAR DISEASES

In recent years, adult multipotent mesenchymal stromal cells or mesenchymal stem cells(MSC) have generated a great deal of interest as a potential source of cell-based thera-peutic strategies in bone and joint diseases. MSC are multipotent, non-haematopoieticprogenitors found mainly in the bone marrow (BM). They can be isolated as a result oftheir adherence to tissue culture plastic and have a triple potential for osteogenesis,adipogenesis and chondrogenesis.1 MSC readily generate single-cell-derived colonies,which can be highly expanded and differentiated into a variety of cell types, includingchondrocytes.2 Despite growing experience and knowledge concerning human MSCand their use in cell-based strategies, the molecular mechanisms that govern their chon-dral differentiation are not well understood and several reports have attempted todescribe the genes differentially expressed through the process.3 Although many ofthe molecular players involved in chondrogenic differentiation have been identified dur-ing limb development in the embryo, a comprehensive understanding of the mechanismsgoverning the steps of chondrogenesis in adult stem cells has not been achieved, partic-ularly in respect to the growth factors driving the process, among which bone morpho-genetic protein (BMP), Hedgehog (Hh) or transforming growth factor (TGF)-b familymembers have already been identified. As well as their cartilage repair potential, MSChave anti-inflammatory properties that make them suitable for cell therapy in osteoartic-ular diseases.4

CHARACTERIZATION OF MULTIPOTENT MESENCHYMALSTROMAL CELLS

The International Society for Cellular Therapy (ISCT) recently proposed a consensus forMSC characterization.5 The MSC is defined according to three criteria: (1) its property ofadherence to plastic; (2) its phenotype (CD14� or CD11b�, CD19� or CD79a�,CD34�, CD45�, HLA-DR�, CD73þ, CD90þ, CD105þ); and (3) its capacity to be dif-ferentiated into three lineages: chondrocyte, osteoblast and adipocyte.

MSC were first characterized by their clonogenic potential, which was determinedby the capacity to form fibroblast colony-forming units (CFU-F). In the BM, the fre-quency of CFU-F is in the range of one cell in 104–105 mononuclear cells,6,7 but thepercentage of mononuclear cells in the BM able to form CFU-F decreases with theage of the donor.8 Moreover, as for the majority of adult stem cells, cell division isaccompanied by telomere shortening, which reaches – with age – a critical size beyondwhich anomalies of cell division occur.

MSC are stromal cells that play a role in haematopoiesis through their adhesion/in-teraction with the haematopoietic stem cells (HSC) and via the secretion of cytokinesand growth factors that are necessary to HSC differentiation.9 Indeed, MSC secretea number of growth factors, such as stem cell factor (SCF), interleukin (IL)-6 and lym-phocyte inhibitory factor (LIF), which are active on the most primitive haematopoieticprecursors [granulocyte macrophage-colony stimulating factor (GM-CSF), G-CSF and

Multipotent mesenchymal stromal cells in articular diseases 271

M-CSF, which act on the haematopoietic progenitors], and thrombopoietin, which actson the more mature cells. They also produce negative regulators of haematopoiesis,such as IL-8, macrophage inflammatory protein (MIP)-1a, transforming growth factor(TGF)-b and cytokines that induce the synthesis of others cytokines by the macro-phages [in particular, the proinflammatory cytokines IL-1 and tumour necrosis factor(TNF)-a]. These different cytokines often display multiple roles. They can act at var-ious levels during haematopoiesis, being at the same time negative regulators orgrowth factors (TGF-b, MIP-1a) according to the targeted cells, and regulating thehaematopoietic cells and the stromal cells (M-CSF, IL-6, TGF-b, IL-1, TNF-a) to con-trol their proliferation. Other cytokines, such as basic fibroblast growth factor (bFGF),which is produced by the stromal cells, are essentially mesenchymal growth factors.Indeed, bFGF was shown to sustain the proliferation of MSC while maintaining theirprimitive phenotype, although a similar role on haematopoiesis was also described.10

MSC also produce adhesion molecules, which are mediators involved in the controlof haematopoiesis by the stroma: these molecules can be extracellular or associatedwith the cell membrane. The adhesion molecules associated with the membrane areof various types. They can be: (1) cytokines, present either directly in the membrane(e.g. the transmembrane SCF isoform) or associated with membrane molecules, suchIL-3 and GM-CSF, which bind to heparin sulphate; (2) membrane molecules belongingto the integrin family (a1b1, a5b1) or the immunoglobulin superfamily (ICAM-1,VCAM-1, HCA); (3) CD44, the ligand of hyaluronic acid; or (4) other molecules ofthe extracellular matrix (ECM). In addition, the stromal cells synthesize and assemblemany molecules of the ECM: fibronectins, laminins, collagens, tenascins and glycosami-noglycans. Thus, molecules of the ECM are part of the architecture of the adherentlayer allowing the anchoring of HSC and the source of a great number of cytokines(SCF, IL-3, GM-CSF, M-CSF, TGF-b, bFGF, MIP-1a).

The capacity of MSC to differentiate into bone, cartilage and fat has been widelystudied and characterized. Although the MSC are defined by their capacity to be dif-ferentiated towards these three cell lineages, they display a broader differentiationpotential. Thus, the MSC are also described for their potential to differentiate intomyocytes, tendinocytes, ligamentocytes,11 cardiomyocytes,12 neuronal cells13,14 andother cell types.15

PRESENCE OF MARROW STROMAL CELLS IN THE JOINT

Previous studies have reported that MSC can be isolated from the synovial membraneby the same protocol as for synovial fibroblast cultivation, suggesting that MSC corre-spond to a subset of the adherent synovial cell population.16 MSC have been shown tobe present in the synovial tissue of normal and osteoarthritis patients, where they rep-resent up to 5% of the synovial cells. These synovium-derived mesenchymal progeni-tors display the trilineage differentiation potential and share the same phenotype asBM-derived MSC. Fluorescent-activated cell-sorting analysis revealed that both popu-lations were negative for CD14, CD34 and CD45 expression, and that both displayedequal levels of CD44, CD73, CD90 and CD105, a phenotype currently known to becharacteristic of BM-derived MSC.

Like BM-derived MSC, synovium-derived cells were shown to suppress the T-cellresponse in a mixed lymphocyte reaction, and to express the enzyme indoleamine2,3-dioxygenase (IDO) activity, which is a possible mediator of this suppressive activity.Transcription profiles revealed discrimination between the MSC-like cells from the


synovial membrane and the BM-MSC by 46 of 268 genes.2 In particular, activin A wasshown to be one major upregulated factor, secreted to high levels by BM-MSC. MSCsfrom BM, synovium, periosteum, adipose and muscle have been compared for theirproperties for yield, expansion and multipotentiality. The colony number per nucle-ated cell derived from synovium was 100-fold higher than for cells derived fromBM. With regard to expansion potential, synovium-derived cells exhibited the highestefficiency in colony formation, fold increase and growth kinetics. The in-vitro chondro-genic assay demonstrated that the pellets derived from synovium were heavier, be-cause of a greater production of cartilage matrix, than those from other tissues,indicating their superiority in chondrogenesis.

REGENERATIVE POTENTIAL OF MULTIPOTENT MESENCHYMALSTROMAL CELLS

Tissue engineering based on cell and gene therapy is one of the most promisingapproaches to the repair of articular cartilage in arthritis. This strategy involves theuse of different cell types that can act as chondroprogenitor cells and/or gene deliveryvehicles that produce a therapeutic protein. MSC offer new potential for cartilage re-pair, as they can be induced to differentiate towards various lineages, and, in particular,towards chondrocytes. The fate of these cells within the tissues is determined by spe-cific cell–cell and cell–matrix interactions, which are controlled by extracellular signal-ling molecules through the interaction with their respective receptors and intracellularevents that control gene transcription in a cell-specific manner. Various differentiationfactors, such as BMP, FGF and Wnt molecules have been shown to be required, butnot specific, for chondrogenesis. Actually, these factors promote both cartilage andbone formation in vivo; the exact molecular pathways governing each specific lineageare still under investigation.

The use of MSC is an attractive alternative to the transplantation of mature chon-drocytes. MSC have the potential of differentiating towards a chondrocytic phenotypefollowing in-vitro culture in a three-dimension system and serum-free medium. Theyare accessible from many tissues, including BM, adipose tissue, synovium, deciduousteeth, umbilical cord blood and blood vessels17–20; but the MSC isolated from BMare the most commonly studied. Although implantation of unmodified MSC hasbeen reported to repair full-thickness cartilage defects in rabbits21, delivery of uncom-mitted MSC to cartilaginous lesions does not yield to a reproducible and satisfactoryregenerated tissue but – most of the time – to fibrocartilage formation. One possibleexplanation for this is the insufficient local stimulation of implanted cells by factorsnecessary to drive their in-situ differentiation or local inflammation.22

Many studies have reported the use of various scaffolds, mainly based on the use ofhyaluronic acid, polylactic acid and/or polyglycolic acid, to improve the quality of theneotissue. These might help the cells inside the defect provide a chondroinductivematrix and mimic the natural tissue geometry. However, biodegradable scaffoldssuch as fibrin might not fulfil the biomechanical requirements for the joint resurfacingof the knee. Indeed, Shao et al have shown that whereas MSC seeded in a fibrin gluematrix were able to form a cartilage-like neotissue at 3 months, by 6 months prom-inent fissures and splits had appeared on the cartilage surface, in combination withpoor integration.23

As a result of their poor resistance to mechanical stress, natural materials, includingagarose, alginate, gelatin and collagen derivatives, are inferior to synthetic and hybrid


materials and their clinical usefulness is severely limited.24 The biomechanical qualitiesand the biodegradability of synthetic biomaterials are more easily modified than naturalpolymers. Thus, Li and co-workers have developed a nanofibrous scaffold (NFS) based onthe synthetic biodegradable polymer poly-caprolactone (PCL), and have examined itsability to support the chondrogenesis of MSC in vitro. This unique form has a microstruc-ture that is similar to the native fibrillar matrix of collagens, and chondrogenesis wasfound to be superior to that seen in pelleted cell culture. In addition, Li et al reportedzonal morphologies within the neocartilage similar to that observed in the native type.25

Gao et al used a two-phase composite material composed of injectable calciumphosphate (ICP) and a hyaluronan derivative loaded with MSC to attempt the repairof osteochondral defects.26 By 12 weeks, the zonal features of the repair tissue be-came distinct; chondrocytes were arranged in a columnar array, which integratedwith surrounding native cartilage and the new bone tissue that formed within theICP. Thus, the titanium implants that provide the biomechanical support for the hydro-static compression in the cartilage were relevant for the differentiation of cells intohyaline-like cartilage.27 Frosch et al also reported that round titanium implants seededwith MSC and inserted into an osteochondral defect resulted in a satisfactory regen-eration of the subchondral bone layer, after 6 months, in 50% of cases.27 This highlightsthe need to improve the scaffolds and the disadvantage of using a material such as ti-tanium, which does not fulfil one of the requisite criteria for a tissue engineering com-posite: resorbability.

The above data indicate that scaffolds that permit anchoring, support cell differen-tiation and maintain a mature phenotype have the promise, together with the use ofstem cells, of obtaining the regeneration of a fully functional tissue. However, thedevelopment of scaffolds must also take into account the fact that the chondrogenicpotential of MSC is favoured by hypoxia and is not only dependent on hydrostaticpressure but also on the cell density inside the matrix and the presence of growthfactors.28

CHONDROGENESIS OF MULTIPOTENT MESENCHYMALSTROMAL CELLS: A COMPLEX PATHWAY

Many lines of evidence have shown the role of various transcription factors in the in-duction of various steps of chondrogenesis. Importantly, the Sox proteins – and in par-ticular Sox-9 – are necessary for chondrogenesis. Sox-9, Sox-5 and Sox-6 aremembers of the Sox family of transcription factors, which are characterized bya high-mobility-group (HMG)-box DNA-binding domain.29 Several genetics ap-proaches in mice have described how Sox-9 positively regulates proliferation and neg-atively regulates chondrocyte hypertrophy.30 Moreover, mice lacking Sox-9 displaya severe generalized chondrodysplasia similar to that seen in Sox-5, Sox-6 double-null mutant mice.30 Recently, Ikeda et al showed that these Sox family members arenot only necessary for chondrogenesis but that, in combination, the Sox trio (Sox-5, Sox-6 and Sox-9) is sufficient for the process.31 Despite its importance for chondro-genesis, the mechanisms by which Sox-9 regulates cartilage-specific transcription arepoorly understood. However, a recent work shows that peroxisome proliferator-activated receptor g coactivator 1 a (PGC-1a) and CREB-binding protein (CBP/p300) act as coactivators for Sox-9 to regulate chondrogenesis.32,33

The Bapx1 transcription factor is another important molecule in chondrogenesis.Bapx1 mediates Sonic Hedgehog (Shh) signalling to induce chondrogenic differentiation


in the sclerotome.34 The signalling mediated by Shh targets Pax1 and Pax9, which in turnactivate Bapx1 in sclerotomal cells.35

The Twist subfamily includes transcription factors, such as Twist, Scleraxis, Dermo-1, Paraxis and HAND2, which function as transcriptional enhancers36–38 or repres-sors.39,40 Scleraxis was previously shown to be expressed in developing chondrogeniccell lineages during embryogenesis. Scleraxis can transactivate the expression of aggre-can by binding to its high-affinity binding site in the promoter.41 Paraxis expressionprecedes that of Scleraxis in the region of the somite fated to form the axial skeletonand tendons, and can direct transcription from an E-box found in the Scleraxis pro-moter. Moreover, in the absence of Paraxis, Pax-1 is no longer expressed in thesomites and presomitic mesoderm. These results suggest that Paraxis acts upstreamScleraxis and regulates early events during chondrogenesis by positively directing tran-scription of sclerotome-specific genes.42

Along with the specific transcription factors, a number of growth factors acting asinductive signals for chondrogenesis have also been identified. Apart from the mem-bers of the insulin growth factors (IGF) and Hh families (for review, see Ref. 43),we will focus on the genes that are the most studied in animal models and, in partic-ular, on the genes belonging to the TGF-b, Wnt and FGF families. The development ofin-vitro systems of chondrogenesis has been important to the identification of factorsthat can promote chondrocyte differentiation of adult MSC and improve cartilage re-pair in vivo. In a defined medium containing dexamethasone and a differentiation factorsuch as BMP, chondrogenesis is induced when MSC are cultured as aggregates. In thissystem, the aggregates synthesize an extracellular matrix characteristic of cartilage,containing proteoglycans and type II collagen.44 The evaluation of the chondrogenicpotential of several TGF-b family members, including TGF-b1, TGF-b2 and TGF-b3,has also been reported. A similar cellular content was observed over 3 weeks, withTGF-b2 and TGF-b3 producing significantly more proteoglycans and collagen type IIthan TGF-b1.45 TGF-b1 is involved in the early stage of the process stimulating chon-drogenesis via transition from an initial N-cadherin-contributing stage to a subsequentfibronectin-contributing stage during the process of chondrogenesis in MSC.46 TGF-b2was shown to enhance in-vitro proliferation and redifferentiation of chondrocytes andto participate in adult and embryonic growth and development.47 Recently, Jin and co-workers reported that TGF-b3 upregulates the expression of Wnt5a, and that Wnt5a,at least in part, mediates the chondrostimulatory effect of TGF-b3 by modulatingPKC-a and p38-mitogen-activated protein kinase (MAPK) activity in chick wing-budmesenchymal cells.48 Furthermore, they showed that the protein levels of cell adhe-sion molecules, such as fibronectin and integrin a5, were consistently increased inthe presence of TGF-b3 and Wnt5a. These results indicate that upregulation ofWnt5a signalling by TGF-b3 promotes expression of cell adhesion molecules throughthe activation of p38 MAPK in early stage during chondrogenesis.

Early in the limb, BMP signalling is known to play a crucial role in the formation ofmesenchymal condensations and the formation of the joints. In-vitro and in-vivo stud-ies have shown that BMP signalling is required both for the formation of precartila-ginous condensations and for the differentiation of precursors into chondrocytes.49

BMP-2, BMP-9 and BMP-13 have been described to serve as potent anabolic factorsfor juvenile cartilage, which contains chondroprogenitors, but not for adult cartilage;BMP-7 has been demonstrated to have a strong anabolic activity in both young andadult cartilage.50 Adenoviral-BMP-2 infected MSC aggregates showed more intensestaining for proteoglycans and collagen type II than adenoviral TGF-b1 aggregates.51

BMP-2 is known to induce the formation not only of new cartilage but also of bone


tissue, thus demonstrating a true capability of BMP-2 to induce both chondrogenicand osteogenic pathways in appropriate precursor cells under specific microenviron-mental conditions.8,52,53 Knippenberg and co-workers pushed further, and demon-strated that, at day 4, a short treatment of MSC with low-concentration (10 ng/ml) of BMP-2, but not BMP-7, stimulated runx-2 and osteopontin gene expression,and that at day 14 BMP-7 downregulated expression of these genes and stimulatedaggrecan gene expression.54 Therefore, MSC triggered with BMP-2 or BMP-7 in spe-cific conditions may provide a feasible tool for both bone and cartilage tissue engi-neering. BMP-4 and BMP-6 have also been shown to promote collagen type IIproduction and to assist in differentiation.16,55 A recent study underlines the roleof BMP-14 in the delay of cellular recruitment and chondrocyte differentiation inthe early stages fracture repair in the absence of BMP-14. The authors supportthe hypothesis that BMP-14 deficiency leads to a delay in fracture healing and empha-size the importance of more closely examining the role of BMP-14 in normal fracturehealing, and the mechanism by which it works.56

The role of the members of the FGF family in skeletal development is poorly de-fined, probably because many genes have essential functions in other tissues duringembryogenesis and functional redundancy of some members has been observed.However, it has been clearly described that the congenital absence of either FGF-18or FGF-R3 results in a similar expansion of the growth plate in fetal mice.57 FGF-2seems to play a dual role in chondrogenesis. On the one hand, FGF-2 has been shownto enhance TGF-b1-induced periosteal chondrogenesis;58 more recently, Davidsonand co-workers have reported that FGF-18 signals through FGFR3 to promote chon-drogenesis.59 On the other hand, FGF-2 might induce the proliferation of MSC priorto chondrogenesis,60,61 inhibit the inductive effect of TGF-b3 on murine MSC culturedin micromass62 and block the synergetic effect of Shh and BMP-2 on transfected pre-chondrogenic cells.58

Members of the Wnt family are important regulators of several developmentalprocesses including skeletogenesis. After the binding of Wnt to the Frizzled familyof receptors and the LRP5/6 family of coreceptors, the canonical Wnt signalling path-way will stabilize the b-catenin that translocates to the nucleus and interacts withmembers of the TCF/LEF families to activate target genes. Whereas inactivation ofb-catenin causes ectopic formation of chondrocytes at the expense of osteoblastformation, the canonical Wnt pathway leads to enhanced ossification and suppressionof chondrocytes due to the transcriptional downregulation of Sox-9.63,64 Indeed,Church et al have shown that Wnt4 blocks the initiation of chondrogenesis and accel-erates terminal chondrocyte differentiation in vitro. By contrast, Wnt5a and Wnt5bpromote early chondrogenesis in vitro while inhibiting terminal differentiation invivo.65 Whereas it has been clearly demonstrated that Wnt7a blocks chondrogene-sis,66,67 the exact role of Wnt3a is more controversial. Indeed, Wnt3a has the capacityto enhance BMP-2-mediated chondrogenesis of mesenchymal micromass cultures viathe regulation of N-cadherin-mediated adhesion, the inhibition of GSK-3b kinase activ-ity and the nuclear signalling of b-catenin and LEF-1.68 More recently, another studyhas demonstrated that Wnt3a inhibits chondrogenesis by stabilizing cell–celladhesion, leading to the dedifferentiation of chondrocytes by activating the b-cate-nin–TCF/LEF transcriptional complex and the c-Jun/AP-1 pathway.69 This discrepancycould be explained in part by the differences in the experimental systems particularlyin the choice of cells. These studies illustrate that the Wnt/b-catenin signalling playsan essential role in MSC in controlling the osteoblastic and chondrocyticdifferentiation.


ANTI-INFLAMMATORY POTENTIAL OF MULTIPOTENTMESENCHYMAL STROMAL CELLS IN ARTHRITIS

MSC have immunosuppressive effect, as demonstrated in various inflammatory diseasesincluding graft versus host (GVH) disease and autoimmune encephalitis. MSC have beenshown to inhibit the lymphocyte proliferation induced by allogenic antigens in mixed lym-phocyte reaction (MLR), mitogens (phytohaemagglutinin or concavalin A) or anti-CD3and anti-CD28 antibodies. MSC suppress the immune function of the various subpopu-lations of T lymphocytes: CD3þ, CD4þ and CD8þ. These cells express the major his-tocompatibility complex (MHC) class I molecules; class II molecules can be induced onlyafter stimulation by IFN-g.2,70,71 The upregulation of MHC class II molecules by IFN-gdoes not elicit a proliferative response72, but these data are controversial.73,74 However,the immunosuppressive effect is independent of the presence of these molecules as MSCthat are devoid of or expressing both class I and class II antigens can inhibit the activationof T lymphocytes.72,75 Several studies have shown than either human or murine MSC cansuppress the lymphocyte proliferative response to allogenic or xenogenic antigens.4,76

MSC modulate the function of the major immune cell populations when stimulated bya mitogenic signal.77 The inhibitory effect of MSC on B lymphocytes was recently shownto occur through an arrest in the G0/G1 phase of the cell cycle, and not through the in-duction of apoptosis.78 Both CD8þ cytotoxic T lymphocytes (CTL) and natural killer(NK) cells are effector cells that display cytotoxic activities important for eliminatingtransformed or infected cells. CTL are activated on interaction with peptides expressedon MHC class I molecules. MSC are not sensitive to CTL-mediated lysis and are able toinhibit CTL cytotoxicity in a dose-dependent manner when present at CTL priming.79,80

NK cells are constitutively cytotoxic against cells that lack MHC class I molecules orwhen these molecules are not recognized by the killer immunoglobulin-like receptor(KIR) on NK cells. Although MSC were reported to be unable to activate NK cells,80,81

they inhibit IFN-g production by IL-2 stimulated NK cells82 and are lysed by IL-2-acti-vated NK cells.82–84 This suppressive effect of MSC is dose dependent, decreasingwith decreasing quantities of MSC in the MLR but a weak concentration of MSC hasbeen shown to have a stimulating effect on T-cell proliferation.72,83,85 The suppressionof the immune response is mediated by soluble factors after MSC activation by culturein presence of immune cells. IL-1b secreted by CD14þ cells and/or IFN-g producedby activated T lymphocytes or NK cells has been found to activate MSC.85–87 The iden-tities of the soluble factors produced by the activated MSC, as well as the mechanisms bywhich these cells act, are still the object of controversy.

A number of possible mechanisms of MSC-mediated immune suppression havebeen evaluated, including effects on dendritic cells (DC) and T regulatory cells, aswell as through prostaglandin E2 (PGE2), IDO, nitric oxide (NO) and cytokine expres-sion. Among the soluble factors known to have immunosuppressive functions, TGF-b and hepatocyte growth factor (HGF) are the most studied. Although contradictoryresults were reported in the literature – probably related to the various types of re-sponding cells and mitogens used – it seems that a single role for TGF-b has been ex-cluded, although it could act in synergy with HGF.88 The involvement of otherscytokines, such as IL-10 or IL-6, was also described. The secretion of IL-6 by MSCis upregulated after culture in a MLR and the addition of neutralizing antibodies restorepartially the lymphocyte proliferation.88–90 PGE2 plays a role in many immune func-tions, including the activation of B lymphocytes and the induction of regulatory T cells.MSC express both cyclo-oxygenase isoforms, COX-1 and COX-2, which are


responsible for the synthesis of PGE2. PGE2 inhibition by indometacin partially re-stored the proliferation of T cells in presence of MSC from human or murine origin,suggesting a probable but not essential role for this molecule. The IDO activity, in-duced by IFN-g, catalyses the conversion of tryptophan to kynurenine, resulting, onthe one hand, in the depletion of tryptophan (which is essential for lymphocyte pro-liferation) in the extracellular medium and, on the other hand, in the accumulation ofkynurenine breakdown products, which are toxic for the cells. A first study showedthat IDO is expressed by the MSC and is functional after activation by IFN-g.89,91

In response to antigenic stimulation, the na€ıve T lymphocytes are activated by a firstsignal (interaction between the T cell receptor and the MHC molecules) and a secondsignal of co-stimulation (interaction between CD28 and B7 molecules). In the absenceof a co-stimulatory signal, the T lymphocytes become anergic, i.e. they cannot proliferateor secrete IL-2 in response to antigenic stimulation. Anergy can, however, be abrogatedby addition of IL-2. MSC that lack expression of the co-stimulatory molecules (CD40,CD80, CD86) could render T lymphocytes anergic.92,93 However, Glennie and collabo-rators showed that removal of MSC restored IFN-g production, but not T lymphocyteproliferation, despite IL-2 addition. They propose that MSC mediate T lymphocyte arrestin the G0/G1 phase of the cell cycle.77 A possible mechanism explaining the inhibition oflymphocyte proliferation is the induction of cellular death by apoptosis. Thus, Plumas andcollaborators showed that MSC inhibit cell proliferation by inducing apoptosis of acti-vated T lymphocytes.94 Under their conditions, the apoptosis was related to IDO activ-ity. NO, produced by nitric oxide synthase (NOS), is known to inhibit T-cell proliferationthrough Stat5 phosphorylation.95 MSC were recently showed to possess inducible NOS(iNOS) activity.96 The induction of iNOS was readily detected in MSC but not in T cells,and a specific inhibitor of NOS restored the T-cell proliferation. Furthermore, MSC fromiNOS�/�mice had a reduced ability to suppress T-cell proliferation, suggesting that NOproduced by MSC is one mediator of the immune suppression.

Regulatory T cells play a crucial role in the suppression of immune responses, par-ticularly in the autoimmune pathologies. MSC increase the number of CD4þ/CD25þregulatory T cells in an MLR.81,97 By contrast, the depletion in CD4þ/CD25þ cellsbefore antigenic stimulation has been shown not to affect MSC-induced suppression.98

The divergences between these data suggest that MSC can contribute to the expan-sion of regulatory T cells without inducing a new regulatory cell population from naiveT cells.

Finally, MSC have been found to inhibit the generation of mature DC from the periph-eral monocytes or the progenitor cells from BM, and can induce the reversion of DC phe-notype towards a less mature stage by decreasing the expression of MHC class II andco-stimulatory molecules (CD40, CD80, CD86) 99. These DC display a reduced capacityto stimulate the lymphocyte proliferation in a MLR and, under these conditions, a de-creased production of proinflammatory cytokines IFN-g, TNF-a, IL-2 is also observed.One suppressive mechanism of MSC would thus be to direct the maturation of DC to-wards a suppressor phenotype that results in an attenuated or regulatory T-cellresponse.

IN-VIVO IMMUNE SUPPRESSION: THERAPEUTIC EFFECTOF MSC IN ARTHRITIS

The use of MSC as immunosuppressant agents in autoimmune diseases has been pro-posed and successfully tested in animal models. Bartholomew and collaborators were


among the first to show that intravenous injection of MSC prolongs the survival of anallogenic skin graft in baboons.100 Later, our team showed that, on the one hand,allogenic MSC are not rejected by the host immune system after implantation in animmunocompetent mouse and, on the other hand, that systemic injection of MSC al-lows the proliferation of allogenic tumour cells according to a mechanism probablyclose to allograft.4 More recently, Zappia and collaborators reported the therapeuticefficacy of MSC in the murine model of multiple sclerosis, the experimental autoim-mune encephalomyelitis (EAE).93 In this model, MSC decrease the clinical signs asso-ciated to demyelinization (ataxia, paralysis of one or more members) when they areinjected before or at the onset of the disease. However, no therapeutic effect is ob-served when the injection occurs after disease stabilization.

Injection of allogeneic MSC has been proposed as therapy for collagen-induced ar-thritis (CIA), a mouse model for human rheumatoid arthritis. DBA/1 mice were immu-nized with type II collagen in Freund’s complete adjuvant, and some of the animalsreceived an intraperitoneal injection of allogeneic MSC. A single injection of MSC pre-vented the occurrence of damage to bone and cartilage. MSC induced the hypores-ponsiveness of T lymphocytes, as evidenced by a reduction in active proliferationand modulated the expression of inflammatory cytokines. Moreover, the serum con-centration of TNF-a was significantly decreased.101 However, two recent studies havemoderated these results. In a murine model of allogenic HSC transplantation, injectionof MSC originating from the receiver improves the long-term allograft, whereas theinjection of MSC from the donor caused a significant increase of cell rejection.102 Sim-ilarly, in a mouse model of GVH disease, the injection of donor MSC had no beneficialeffect on the incidence or severity of the GVH disease.103 In our hands, in the CIAmodel, intra-articular injections of the C3HT101/2 murine MSC line did not protectfrom arthritis. Both the clinical and the immunological findings suggested that MSCwere associated with accentuation of the Th1 response. In-vitro experiments showedthat the addition of TNF-a was sufficient to reverse the immunosuppressive effect ofMSC on T cell proliferation, and this observation was associated with an increase in IL-6 secretion.104 This suggests that environmental parameters, in particular those re-lated to inflammation, might influence the immunosuppressive properties of MSC,and that any clinical application of MSC in articular inflammatory diseases should beassociated with anti-TNF-a therapy.

ENGINEERED MESENCHYMAL STROMAL CELLS FOR CARTILAGEREPAIR

MSC therapeutic potential might be improved by combining gene transfer of growthfactors or immunosuppressive cytokines (such as IL-10) and cell transplantation, asshown in a rat model of partial-thickness lesions created in articular cartilage.MSC infected with recombinant adenoviral vectors expressing BMP-2 or IGF-1were able to repair the cartilage of hyaline morphology containing a type-II-colla-gen-positive and type-I-collagen-negative proteoglycans-rich matrix that restoredthe articular surface in most lesions.105 However, cells in excess were partially dis-located to the joint margins, leading to osteophyte formation when BMP-2-express-ing cells were used, whereas these adverse effects were not observed with IGF-1-expressing cells. Osteophyte formation might be prevented by the forced expressionof Sox-9, which promotes cell condensation and early differentiation, and blocks hy-pertrophy (for review, see Ref. 106). Other authors have shown that the implantation


of genetically modified MSC expressing BMP-7 or Shh in articular cartilage lesions sig-nificantly enhanced the quality of the repair tissue, resulting in a more hyaline-appear-ing cartilage, when compared with untransduced MSC.107 However, there wasa noticeable difference in the persistence of the cartilage phase between the groups.The subchondral compartment seemed to remodel with bone much faster in thegroup that received the BMP-7 gene. Together with the growth factor, the qualityof the repaired cartilage depends on the delivery scaffold. The most encouraging re-sults were found when BMP-7 was combined with a collagen matrix, whereas linkingBMP-7 to chitosan led to only partial healing of the articular surface.108 However,although hyaline-appearing cartilage was formed, bone was also obtained. These re-sults underline the necessity of identifying chondral-specific factors that could beused for tissue engineering.

CONCLUSION

In summary, MSC have a great potential for therapy in arthritis, as they combinecartilage repair and immunosuppressive functions. Cartilage engineering throughengineered MSC is a promising approach in different pathological situations to cir-cumvent current treatments, which rarely restore the full functions of the tissueto promote its return to a native normal state. However, to fulfil the criteriaof stem-cell-based repair, it is possible that far more sophisticated strategies willbe required to faithfully reproduce the complex molecular events of the chondro-genic process and the long-term maintenance of the articular cartilage phenotype.Several experimental approaches currently under investigation might prove useful,such as the local implantation of genetically modified MSC into cartilage defects.Regardless of the method, the challenge at the present time is the characterizationof candidate gene products that could guide specifically the chondrogenic processin vivo.

MSC exert their immunosuppressive effect by secreting soluble factors (cytokines,IDO, PGE2) after activation by cytokines such as IFN-g and IL-1b. On activation, MSCwould direct DC towards a suppressor phenotype responsible for the reduction in theT lymphocyte proliferation and probably associated with the generation of regulatoryT cells. Nevertheless, these mechanisms are only partially responsible for the immunemodulation of MSC, as these cells can act independently of the presence of DC. How-ever, a local inflammation mediated by TNF-a might shift the immunosuppressive ef-fect of the cells to a more aggressive status. The immunomodulatory potential ofMSC still needs to be confirmed in vivo in chronic inflammatory diseases and in partic-ular in arthritis.

Practice points

� Isolation, characterization and good manufacture practice (GMP) production ofhuman MSC are available.� The feasibility and tolerance of MSC injection in humans have been evaluated.� The immunosuppressive effect of MSC for the prevention of GVH disease is

currently under evaluation. This is a step towards the use of MSC in autoim-mune diseases, particularly rheumatoid arthritis.

Research agenda

� The differentiation potential of MSC is proven but it is necessary to identifya differentiation factor that is specific for chondrogenesis.� Maintenance of differentiated phenotype to be obtained over time after in-vivo

implantation.� A chondrosupportive scaffold, adapted to MSC implantation, is to be

developed.� Preclinical development and biomechanical testing in large animal models is

required.� The therapeutic potential of MSC in experimental arthritis is to be assessed.


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Multipotent mesenchymal stromal cells in articular diseases