Cell Transplantation, Vol. 24, pp. 133–149, 2015 Printed in the USA. All rights reserved. Copyright © 2015 Cognizant Comm. Corp.

0963-6897/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368913X675728 E-ISSN 1555-3892 www.cognizantcommunication.com

Review Recent Advances in Mesenchymal Stem Cell Immunomodulation: The Role of Microvesicles Alessandra Fierabracci,* Andrea Del Fattore,* Rosa Luciano,* Marta Muraca,† Anna Teti,‡ and Maurizio Muraca§ *Research Laboratories, Bambino Gesù Children’s Hospital, Rome, Italy †School of Medicine, University “Sapienza”, Rome, Italy ‡Department of Biotechnological and Applied Clinical Sciences, University of L’Aquila, L’Aquila, Italy §Department of Women's and Children's Health, University of Padua, Padua, Italy

Mesenchymal stem cells are the most widely used cell phenotype for therapeutic applications, the main reasons being their well-established abilities to promote regeneration of injured tissues and to modulate immune responses. Efficacy was reported in the treatment of several animal models of inflammatory and autoimmune diseases and, in clinical settings, for the management of disorders such as GVHD, systemic lupus erythematosus, multiple sclerosis, and inflammatory bowel disease. The effects of mesenchymal stem cells are believed to be largely mediated by paracrine signals, and several secreted molecules have been identified as contributors to the net biological effect. Recently, it has been recognized that bioactive molecules can be shuttled from cell to cell packed in microvesicles, tiny portions of cytoplasm surrounded by a membrane. Coding and noncoding RNAs are also carried in such microvesicles, transferring relevant biological activity to target cells. Several reports indicate that the regenerative effect of mesenchymal stem cells can be reproduced by microvesicles isolated from their culture medium. More recent evidence suggests that the immunomodulatory effects of mesenchymal stem cells are also at least partially mediated by secreted microvesicles. These findings allow better understanding of the mechanisms involved in cell-to-cell interaction and may have interesting implications for the development of novel therapeutic tools in place of the parent cells. Key words: Microvesicles; Exosomes; Mesenchymal stem cells; Immunity

INTRODUCTION Bone marrow is a complex organ where different resident lineages of hematopoietic and stromal cells support hematopoiesis (50). Mesenchymal stem (or stromal) cells (MSCs) represent a subpopulation of undifferentiated cells with the hallmark properties of self-renewal and differentiation. This population, first described in the 1960s as bone-forming cells (59), is one of the most widely investigated with still unexplored properties and potential for clinical application. The proper definition of MSCs has been extremely challenging. Initially identified as a subgroup of cells with osteogenic potential, they were subsequently recognized as fundamental players in the process of hematopoiesis (57). Researchers were attracted by their ability of differentiating toward the mesodermal lineage in adipogenic, chondrogenic, and osteogenic cells

emphasizing their potential in tissue regeneration, although extensive debate persists regarding their multipotential capacity in vitro (57). A particular challenge has been the absence of specific markers to define this plastic-adherent population with fibroblast-like morphology, despite the large number of proposed determinants (84), that is, cell surface expression of cluster of differentiation 105 (CD105; endoglin, SH2), CD73 (ecto-5¢-nucleotidase), and CD90 (thymocyate antigen 1; Thy1) and the absence of hematopoietic markers CD45, CD19 or CD79, CD14 or CD11b, and HLA (human leucocyte antigen)-DR. The International Society for Cellular Therapy has established the minimal criteria for defining multipotent MSCs (49). Of note, MSCs could also be derived from adipose tissue (102,188), placental tissue (80), skin (148), dental pulp (64), umbilical cord blood (54), amniotic fluid (120), umbilical cord

Received July 11, 2013; final acceptance November 20, 2013. Online prepub date: November 21, 2013. Address correspondence to Alessandra Fierabracci, Ph.D., Research Laboratories, Children’s Hospital Bambino Gesù, Piazza Sant’Onofrio 4, 00165 Rome, Italy. Tel: +39 06 6859 2656; Fax: +39 06 6859 2904; E-mail: [email protected]

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perivascular cells (144), Wharton’s jelly (174), synovial tissue (45), and breast milk (70,125). MSCs IN REGENERATIVE MEDICINE Currently, 167 actively recruiting, MSC-based interventional clinical studies are registered at the NIH site (http://clinicaltrials.gov/). The present review will address more in detail the use of MSCs in the treatment of immune disorders and provide a concise overview of MSC-based cell therapy in tissue regeneration. For a more complete discussion of the latter topic, the reader is referred to excellent recent reviews [see, e.g., (84,139)]. However, the above distinction is largely artificial, since consolidated experimental evidence indicates that the therapeutic effects result from an interplay of different mechanisms including immunomodulation, protection from injury, and regenerative stimuli. Moreover, a better understanding of the interactions between administered MSCs and local microenvironments is a prerequisite for the development of more successful treatments. In immune disorders, the therapeutic efficacy of MSCs probably depends on the nature of different diseases because of the distinct inflammatory environments. Even for one specific disease, the variety of microenvironments in different tissues may result in different levels of efficacy. For instance, a recent trial on graft-versus-host disease (GVHD) showed significant effects of MSC treatment only on gastrointestinal and hepatic involvement (112). Clinical trials of MSCs in tissue regeneration have largely addressed heart and limb ischemia, bone defect and trauma repair, and neurodegenerative diseases. In a fundamental study, MSCs were administered after acute myocardial infarction in 53 patients, showing the safety of the procedure and providing preliminary efficacy data (68), including a significant reduction of ventricular arrhythmias and improvement in pulmonary function. Moreover, improved left ventricular function was achieved in a subset of patients receiving MSCs compared with the placebo group. These results provided the basis for further phase II–III clinical trials. Several issues are being addressed in designing present and forthcoming clinical trials using MSCs for heart repair, including timing of MSC administration, route of delivery, patient selection, and (last but not least) the efficacy index (37), since improvement in ventricular ejection fraction may not correlate with clinical outcome. MSCs need to be combined with a suitable scaffold in order to provide efficient bone regeneration, especially with large bone defects. Such a tissue-engineering approach provides abundant callus formation and good integration of the implants in long-term follow-up (133). A combination with scaffolds was also used in clinical trials for the treatment of osteoarthritis with MSCs, resulting in symptomatic relief and promotion of cartilage repair after intra-articular injection (3,44,74,82,173). Various MSC-based clinical studies have been carried out with patients suffering from

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neurological disorders. Preliminary encouraging results have been reported in stroke (73), multiple sclerosis, and amyotrophic lateral sclerosis (33,42,83). Chronic skin ulcers represent a common pathology where effective cell therapy is much waited for, due to insufficient results of current conventional treatments, also involving significant nurse care time. In August 2013, a press release announced the results of a randomized, controlled clinical trial in patients with chronic diabetic foot ulcers comparing the safety and effectiveness of a human cellular repair matrix including living MSCs (Grafix®) to standard of care. For the primary endpoint, 62% of patients receiving Grafix had complete wound closure compared to 21% (p < 0.0001) of patients who received conventional treatment. The interim analysis conducted on the first 97 enrolled patients met the prespecified stopping rules for proven efficacy as determined by the data monitoring committee (4). IMMUNOMODULATION BY MSCs In Vitro Studies MSCs are immunoprivileged due to the low expression of class II major histocompatibility complex (MHC II) and costimulatory molecules CD40, CD40L, CD80, CD86 on the cell surface, thus escaping T-cell recognition (24). They also interfere with different pathways both through cell–cell interaction and secretion of soluble factors. MSCs can alter the response of both the adaptive and the innate immune systems by suppressing T-cell and dendritic cell maturation, affecting B-cell activation and proliferation, inhibiting proliferation and cytotoxicity of natural killer (NK) cells, and inducing regulatory T-cells (Tregs) (84). MSCs were shown to modulate T-cell proliferation and activation (181,187) in different experimental protocols. Di Nicola et al. (47) found that both autologous and allogeneic bone marrow stromal cells (BMSCs) reduced T-cell proliferation in mixed lymphocyte reactions (MLRs) with allogeneic dendritic cells (DCs) or peripheral blood lymphocytes (PBLs). When human MSCs were added in cocultures with purified subpopulations of immune cells, the cytokine secretion profile of DCs, naive and effector T helper 1 (Th1), Th2, and NK cells was altered, resulting in a more anti-inflammatory phenotype, while increasing the proportion of Tregs (2). Furthermore, MSCs inhibited the division of stimulated T-cells avoiding their entry into the S phase of the cell cycle and inducing irreversible G0/ G1 phase arrest (62). Work by Beyth et al. (19) supports an immunoregulatory mechanism wherein MSCs inhibit T-cells indirectly by contact-dependent induction of regulatory antigen-presenting cells (APCs) with T-cell-suppressive properties. Additional studies suggest that T-cell inhibition is not antigen specific, but acting through HLA both on primary and secondary responses (90,181). There is increasing interest in the effect of MSCs on B lymphocytes, since these cells are increasingly recognized

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to play an important role in autoimmune disorders and in transplant rejection. The immunomodulatory effect of MSCs on B-cells is still controversial, and the mechanisms involved are unclear. The role of soluble factors is generally recognized, and it was even found to be more relevant in mediating the effect on B than on T lymphocytes (12). When cocultured with purified B-cells, MSCs were reported to have different and even opposite effects in different experimental conditions. MSCs inhibited Bcell proliferation and antibody production in vitro in the presence of cytosine–phosphate–guanosine (CpG), CD40L, anti-immunoglobulin, interleukin (IL)-2, and IL-4 (43), while they had no effect after B-cell stimulation by a combination of allogeneic T-cell-depleted peripheral blood mononuclear cells (PBMCs) and CpG (90). In the latter work, B-cells became susceptible to the suppressive activity of MSCs in the presence of exogenously added interferon-g (IFN-g). On the other hand, in the work of Traggiai et al. (164), MSCs promoted proliferation and differentiation into immunoglobulin-secreting cells of transitional and naive B-cells stimulated with CpG, in the absence of B-cell receptor triggering. Using bacterial/viral antigen stimulation of PBMCs, Rasmusson et al. (136) demonstrated MSC-dependent induction or inhibition of antibody secretion in B-cells depending on the strength of the baseline stimulus, inhibition being associated with a stronger stimulation. A perhaps more consistent MSC-mediated inhibition of B lymphocyte function was observed where T-helper effect on B-cell function is maintained such as in the work of Comoli et al. (41) or in PBMC cocultures (22,141). Collectively, these findings suggest that MSC-mediated inhibition of B lymphocyte function involves T-helper suppression rather than a direct effect on B lymphocytes, given also the observation that Ig production is not inhibited by MSCs when CD40 engagement is obtained by agonist anti-CD40L antibody rather than by the direct help of T-cells (41). MSCs induce inhibition of differentiation of monocytes to immature DCs (121,181). MSCs may also affect NK cell function by downregulating IFN-g secretion (2). Contrasting data were reported by Rasmusson et al. (137), suggesting that MSCs do not affect NK cell or cytotoxic T lymphocyte (CTL) lysis. Such discrepancies may result from the different experimental settings, since it seems that MSCs can inhibit T-cells in the early activation phase, but not in the effector phase of allograft rejection (137). Similarly, the contrasting results on NK cell lysis may be related to the timing of the experiment, since the late lytic phase of NK cells may be mediated by cell surface molecules that are not affected by MSCs. Of note, MSCs significantly inhibited IL2-stimulated proliferation on resting NK cells (149), while the proliferation of activated NK cells was only partially affected. Soluble mediators of MSC immunomodulatory effects have been identified in several studies. MSCs inhibit T-cell

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proliferation by decreasing both IFN-g and tumor necrosis factor-a (TNF-a) production, while increasing IL-10 secretion (23,181). Also, in MLRs, addition of MSCs stimulates indoleamine 2,3-dioxygenase (IDO) activity, resulting in the degradation of tryptophan and inhibition of T-cell proliferation. Concerted actions of chemokines and nitric oxide (NO) were shown to mediate the immunosuppressive effect on T-cell proliferation and cytokine production (145). Species differences in the relative contribution of inducible nitric oxide synthase (iNOS) and IDO in suppressing T-cell function have been described in vitro with mouse and human MSCs (140). Soluble factors are believed to mediate the inhibition of B-cell proliferation and differentiation by MSCs, but these mechanisms have been less characterized (12). MSCs secrete prostaglandin E2 (PGE2), thus inhibiting NK cell proliferation and cytotoxicity (2). Impaired monocyte and DC maturation was associated with decreased secretion of IL-1, CD40, and TNF-a together with production of PGE2 by MSCs (91). Relevant Animal Models for MSC Immunomodulation The immunomodulatory effects of MSCs in vivo were first observed in a baboon model, where expanded donor or third-party bone marrow-derived MSCs delayed rejection of incompatible skin grafts (18). Afterwards, MSC immunomodulation was tested in several animal models of immune disorders. MSCs were shown to alleviate GVHD, a much-feared complication following allogeneic bone marrow transplantation (30,63,81,88,142). Animal models might help predict efficacy of different cell sources, since different therapeutic outcomes were observed in a GVHD rat model when using bone marrow-derived MSCs than conventional short-term cultures or cloned MSCs, the latter being ineffective (88). In experimental allergic encephalomyelitis (EAE), MSCs induce a strong immunosuppression on effector T-cells at the level of secondary lymphoid organs, leading to IL-2reversible T-cell anergy (183), and decrease central nervous system injury while reducing infiltration of T-cells (14). Contrasting results were obtained in experimental collagen-induced arthritis (CIA), where injected MSCs were either beneficial, with decreased serum TNF-a and induced hyporesponsiveness of T lymphocytes (13) or detrimental, with accentuation of the Th1 response (48). Interestingly, in the latter study, an immortalized MSC C3 line was used, further emphasizing the importance of cell source in determining the outcome of treatment. Contrasting data were also reported in murine models of systemic lupus erythematosus (SLE) (25,52,65,151,177, 182,186). When Murphy Roths Large/Fas (TNF receptor superfamily 6) lymphoproliferation mutation (MRL/lpr) mice were treated with human MSCs from healthy donors, T-cell proliferation was affected in a dose-dependent manner with control of autoimmune progression (186), reduction

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of circulating anti-double-stranded DNA (dsDNA) antibodies, and of proteinuria. Transplantation of MSCs from human exfoliated deciduous teeth was also effective in treating SLE-like MRL/lpr mice, by increasing the ratio of Tregs mediated by inhibition of Th17 cells (177). Effective treatment of lupus nephritis was also obtained by transplantation of human umbilical cord blood-derived MSCs in New Zealand Black/White F1 (NZB/WF1) mice (25). In contrast, Youd et al. observed that allogeneic MSCs exacerbated lupus disease in NZBxNZW F1 mice (182). Such discrepancies might be due to differences in cell source and in animal models and highlight the complexity of exogenous MSC–host tissue interactions possibly leading to unpredictability of treatment outcome (see later). The successful use of MSCs in animal models of insulindependent diabetes mellitus (type 1 diabetes) has received much interest (31). MSCs were found to induce the regeneration of recipient-derived pancreatic insulin-secreting cells, inhibit T-cell-mediated immune responses against newly formed b-cells, and ameliorate hyperglycemia and microalbuminuria (16,32,56,58,69,78,96,99,111,166). Moreover, coencapsulation with MSCs improved engraftment and survival of transplanted islets (86,134,170). However, the mechanisms underlying the observed effects of MSCs in vivo are largely unknown. Available data suggest limited engraftment and even poorer persistence of transplanted MSCs at the injury site. When male MSCs were injected directly at the border of the infarcted site in syngeneic female rats, the percentage of donor cells in the heart decreased rapidly from 34–80% of injected cells immediately after transplantation to less than 5% at 3 weeks (118). In a similar rat model using allogeneic transplantation, neither iron oxide-labeled nor unlabeled cells survived in the infarcted myocardium after 4 weeks (9). In a more recent work on a mouse model with hepatic ischemia– reperfusion injury (52), labeled MSCs were found only in the lungs 1 h after intravenous (IV) infusion. The induction of hepatic injury did not trigger the migration of viable MSCs to the liver, and viable labeled cells completely disappeared from the body within 24 h. The authors conclude that long-term immunomodulatory and regenerative effects of infused MSCs must therefore be mediated by indirect mechanisms or via other cell types. Little information is available in humans. After intracoronary transplantation of radiolabeled unfractionated autologous bone marrow-derived mononuclear cells in a patient with myocardial infarction, the estimated radioactivity uptake by the heart was 5% of the injected radioactivity at 2 h and only 1% at 18 h after transplantation (127). In conclusion, the disproportion between the observed functional effects of MSC administration and the low rates of cell engraftment and persistence supports an indirect primary mechanism

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other than structural integration of transplanted cells at injured tissue. Clinical Applications Studies on biological properties and therapeutic applications of MSCs have been extensively propelled within the past 5 years, including efforts to define this cell population by the use of appropriate markers and extensive use of animal models. Nowadays, there is the urgency to optimize protocols for clinical applications through the collaborative effort of clinicians and laboratory scientists. As mentioned above, GVHD is the area where MSCbased therapy provided the most significant results. This serious complication occurs following hematopoietic stem cell transplantation when donor T-cells react against the immunocompromised recipient (53,57). The first experience was reported by Le Blanc and colleagues in a patient with severe steroid-refractory acute GVHD, who showed complete response after two infusions of ex vivo expanded haplo-identical human MSCs (95). A phase II trial was subsequently published by the same group (94) in 55 patients with steroid-resistant acute GVHD treated using MSCs from haplo-identical donors (16 cases) or from third-party HLA-mismatched donors (69 cases). No adverse reactions were reported either during the treatment or after MSC infusion. Results were encouraging, since a complete response was observed in 30 patients while 9 patients showed a partial response. Patients with complete response also had longer survival. Similar outcomes were reported in another phase II trial (85). A larger phase III double-blind, placebo-controlled clinical trial using commercial allogeneic MSCs (Prochymal®) consisted of two different protocols (104,112). One protocol evaluated the safety and efficacy of Prochymal in conjunction with steroid therapy in 192 patients with newly diagnosed acute GVHD. The primary endpoint of this study was not met, since there was no statistical difference in the proportion of patients surviving at least 90 days that achieved a complete response when Prochymal was added to steroid therapy compared to those receiving steroids alone. The other protocol evaluated the safety and efficacy of Prochymal in conjunction with standard of care for the treatment of patients who had failed to respond to corticosteroid treatment for acute GVHD. The primary endpoint was a durable complete response defined as complete resolution of GVHD for a duration of at least 28 days. This endpoint was not met in the whole group of patients, but Prochymal significantly improved response rates in the subgroups of patients with steroid-refractory hepatic and gastrointestinal GVHD. Importantly, Prochymal showed a stronger trend of improvement in response rates in pediatric patients (86% vs. 57%, p = 0.094, n = 28). No adverse events were registered. Despite the mixed results

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of the trial, on May 2012, the sponsor company received market authorization from Canada Health Authorities for the treatment of steroid-resistant GVHD in pediatric patients, making Prochymal the world’s first approved drug having stem cells as its active ingredient. This trial highlights the importance of well-defined patient populations in determining the outcome of MSCbased therapy. Moreover, several issues were raised regarding protocols used in current clinical trials for GVHD, including the need to optimize the timing, dose, and frequency of MSC administration. Usually the number of cells varies from 1 × 106 to 2 × 108 per kg of body weight, administered in one to six infusions. An additional concern regards the purity of MSC preparations (128,181), where cells are isolated using the conventional density-gradient centrifugation approach. It is widely recognized that MSCs expanded in culture consist of a mixture of different cells with various degrees of stemness, only a minority of them showing multilineage potential and self-renewal (98,119, 138). A clonal allogeneic MSC preparation was recently developed by Lim et al. (100) and successfully used for the treatment of two patients with steroid-resistant acute GVHD. If safety and efficacy of clonal MSCs are confirmed, they might represent a more reproducible therapeutic tool with respect to nonclonal preparations. Another major area of interest is the potential application of MSCs in treating autoimmune diseases, due to their immunomodulating and anti-inflammatory properties (165). Crohn’s disease is a disorder of unknown etiology characterized by transmural inflammation of the gastrointestinal tract, resulting in fibrosis causing intestinal obstruction or giving rise to perforations and fistulae. In a pilot study, nine patients with refractory Crohn’s disease were treated with autologous bone marrow-derived MSCs (51). Two IV infusions of 1–2 × 106 cells per kilogram of patient body weight were administered 7 days apart. Three patients showed clinical response, while three patients required surgery due to disease worsening. A large company-sponsored phase III study using allogeneic MSC infusion in patients with refractory Crohn’s disease was halted in March 2009 because of negative preliminary results (15). As an alternative therapeutic approach, autologous adipose tissue-derived MSCs in fibrin glue were inoculated into the fistulas in patients with Crohn’s disease, in an attempt to achieve local stimulation of ulcer healing. A preliminary phase I study in five patients showed a fistula healing rate of 75% and no adverse events with a follow-up of 12–30 months (60). These encouraging results were confirmed by a phase II trial in 24 patients (61). Nevertheless, only a low proportion of the stem cell-treated patients with closure after the procedure remained free of recurrence after more than 3 years of follow-up (66). Moreover, a multicenter phase III clinical trial led by the same group

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and including 200 patients showed comparable healing rates (about 50% at 1 year) in patients treated with MSCs, MSCs in fibrin glue, or fibrin glue alone (72). Thus, despite the above-reported positive results in patients with steroidrefractory gastrointestinal GVHD, data on the treatment of chronic inflammatory bowel disease with MSCs have been inconclusive so far, highlighting the unpredictability of response to cell therapy in complex disorders, where unknown individual patient characteristics may play a critical role. In recent years, encouraging results were reported from clinical trials evaluating the treatment of SLE (52) with MSCs in patients refractory to conventional therapy (57). Sun et al. (151) used MSCs for treating four patients with active SLE refractory to prolonged therapy with IV cyclophosphamide and oral prednisone. MSCs improved the disease activity index without side effects in a prolonged follow-up to 12 months and induced expansion of Tregs in the peripheral blood of patients at 3-month follow-up. No complications were developed within 12–18 months of follow-up. Based on these encouraging results, a larger phase I trial in 15 patients with refractory disease was performed by the same group (152). Allogeneic MSCs were obtained from non-HLA-matched family members. Clinical amelioration of disease was achieved during a follow-up period of 3–36 months without side effects. Parameters such as anti-dsDNA antibodies and proteinuria decreased significantly, while the percentage of Tregs was significantly restored from the low levels observed before MSC administration. The same authors (152) also explored the use of umbilical cord blood-derived MSCs in 16 severe SLE patients with improvement of SLEDAI (Systemic Lupus Erythematosus Disease Activity Index) score. With this successful background, the European League Against Rheumatism (EULAR) Stromal Cell group is at present conducting a multicenter trial on lupus nephritis treated with allogeneic MSCs (165). MSC therapy was also applied to systemic sclerosis (57), a disease characterized by thickening of the skin caused by accumulation of collagen and by injuries to the smallest arteries, based on the principle that MSCs are able to differentiate into endothelial cells, thus contributing to angiogenesis in adult tissues (103). Following the successful treatment of the first two patients (34,65), Akiyama et al. (5) treated five patients with allogeneic MSCs. The cell administration induced T-cell apoptosis, lymphopenia, and Treg expansion. Clinical improvement of the skin score and of the autoantibody titer was reported. The use of allogeneic versus autologous cells represents an unresolved issue in MSC clinical application. Studies in vitro and in vivo showed similar efficacy of the two cell sources in suppressing immune response (18,41,47). As mentioned above, no serious adverse events were reported

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in clinical trials with allogeneic MSCs. The latter cell source would be available “off the shelf,” also allowing better standardization and reducing costs of production. This would increase the number of patients taking advantage of such novel therapies. Moreover, autologous cells are not always suitable for efficient ex vivo expansion. There is however a need of reliable specific potency assays in vitro in order to predict the clinical efficacy of different cell preparations. Two major considerations can be put forward when collectively evaluating the results of the above studies. First, the enormous progress of molecular and cell biology that allowed the isolation, characterization, and production of stem cells as therapeutic tools was not paralleled by a similar improvement in our understanding of tissue biology (157). Thus, interactions between administered cells and host tissue remain largely unknown. Second, on the clinical side, appropriate patient selection is an often overlooked factor, which can critically influence the success of the study. Indeed, especially in designing companysponsored clinical trials, the need to reach a sufficient number of patients in a limited time frame leads to increased variability, both including several participating centers from different countries, and by enrolling patients with different characteristics. Such an attitude can increase the risk of missing primary endpoints and can also limit the chances to understand the underlying mechanisms by including patients with a wide spectrum of pathophysiology. Despite such limitations, significant progress has been made over the past decade, also testified by the raising interest of Big Pharma in cell therapy (146). MICROVESICLES: A PATHWAY OF CELL–CELL COMMUNICATION As described above, MSCs—like several other cell types—exert many of their effects via paracrine signaling. The traditional view of cell–cell communication consists in the release of bioactive molecules, resulting in a complex mixture whose local precise composition is largely undetermined. However, it has been known for many years that cells also secrete membrane vesicles, composed of a lipid bilayer including transmembrane proteins and enclosing cytoplasmic components. Such vesicles can transmit signals by interacting at the cell surface, by internalization into endocytic compartments, or by fusion with plasma membranes (7,105) (Fig. 1). Cell-derived membrane vesicles are generally classified according to their size and intracellular origin. Exosomes are 40–100 nm in size, derived from multivesicular bodies (MVBs), a late endosomal compartment, and are secreted via fusion of multivesicular bodies with the plasma membrane. Exosome production is generally regarded as a constitutive membrane vesicle pathway, although it can increase upon stimulation.

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Shedding vesicles, also known as microvesicles, are a heterogeneous population of membrane vesicles up to 1 mm in size directly derived from the cell membrane of activated cells through the disruption of cortical cytoskeleton. The process is initiated by increased intracellular Ca2+ levels, inhibiting scramblase and floppase to return phospholipids into the inner membrane surface and resulting in altered phospholipid balance of the cell membrane. Interestingly, the targeting of proteins into shedding vesicles seems to be selective (38,117,169). Apoptotic signals also promote the release of membrane particles via the initiation of actin–myosin sliding, resulting in budding and detachment of the membrane from the cytoskeleton. It is a challenge to experimentally distinguish between exosomes and shedding vesicles because of their overlapping biophysical characteristics and the lack of discriminating markers (38,169). Therefore, in this review, the term “microvesicles” (MVs) is used to refer to a mixed population of vesicles. MVs are commonly isolated by differential centrifugation. Such a procedure is, however, cumbersome and unfeasible to produce the large quantities needed for extensive in vivo investigations or for clinical applications. Methods based on immunoaffinity and immunomagnetic isolation by antibodies directed against surface proteins have been developed (28,35). While this approach may prove feasible for MV quantification (although no specific marker has been identified), it cannot be used for functional studies, since particles cannot be retrieved following binding. Ultrafiltration is another widely used isolation method, and it was shown to result in higher MV recovery when compared to ultracentrifugation (93,108). MVs can be approximately quantified by total protein determination (158), although one should consider possible contamination by proteins present, for example, in culture media. Flow cytometry has been assayed by several investigators, although the size of most particles is below the detection limit of commonly used instruments (168). Recently, Nolte-’t Hoen et al. developed a flow cytometric method to analyze single vesicles (124) based on forward scatter wide-angle detection of fluorescentlabeled MVs, reportedly allowing for both quantitative and qualitative analysis of nanosized vesicles such as exosomes. Composition of MVs More than 4,000 different proteins and 2,400 different RNAs are described in the exosome database ExoCarta (106). Interestingly, phosphatidylserine (PS) has been described as a canonical MV lipid present on the surface, as PS is an important docking site for proteins involved in signaling and fusogenesis, and it may contribute to the selective MV enrichment in proteins involved in signaling and fusion with target cells (150). PS could also be recognized

Figure 1. Immunomodulatory effects of MSC-derived MVs on lymphocytes. MVs can transfer signals to target cells by endocytosis, fusion with cell membrane, and delivery of content into the cell or by ligand–receptor interaction. The effects of MSC-derived MVs on lymphocytes are described in the text. CD4 +CD25+FoxP3+ Treg cells: cluster of differentiation 4-positive cluster of differentiation 25-positive forkhead box P3-positive regulatory T-cells.

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as a signal inducing capture by target cells, as is the case for apoptotic cells (8). MVs of all cellular origins consistently bear adhesion molecules, which could favor their capture by recipient cells, such as intercellular adhesion molecule 1 (ICAM1), lysosomal-associated membrane protein 2 (LAMP2), tetraspanins [such as CD9, CD151, and tetraspanin 8 (Tspan8)], and integrins [integrin a 3 (CD49C), a 3 subunit of very late activation protein 3 (VLA-3) receptor and integrin a 4 (CD49D), a 4 subunit of VLA-4 receptor] (17,122,123,185). Lactadherin is bound to PS exposed at the surface of exosomes (171). When bound to apoptotic cells, milk fat globule-epidermal growth factor (EGF) factor VIII protein (MFGE8)/lactadherin promotes their phagocytosis by macrophages expressing avb3 and avb5 integrins (67); it could thus play a similar role for capture of MVs. Heat shock proteins, cytoskeletal proteins, MV proteins involved in membrane trafficking [“Ras-related in brain” proteins (rabs) and annexins], scaffolding (tetraspanins), transmembrane transport (solute carriers), and translation (ribosomal proteins) are also commonly found in MVs (107). An excellent recent overview provides exhaustive proteomics data from ExoCarta (106). ROLE OF MVs IN IMMUNE RESPONSES The role of MVs in immune responses has been reviewed extensively by the group of Théry (27,160) and by others (79,143,155). Stimulation of Immune Response Under several circumstances, secreted vesicles can have immuno-activatory properties. Macrophages infected by various pathogens (Mycobacterium and Toxoplasma) release MVs containing pathogen-derived proinflammatory molecular determinants inducing the secretion of proinflammatory cytokines by recipient macrophages (20). Cultured cells infected by Mycoplasma also release proinflammatory exosomes that induce polyclonal activation of B- and T-cells (132). MVs isolated from body fluids could exacerbate autoimmune diseases. In rheumatoid arthritis (RA) patients, fibroblasts obtained from synovial fluid secrete exosomes bearing active membrane-bound TNF-a, which binds to T lymphocytes and renders them resistant to activation-induced cell death (184). Qazi et al. (131) found abundant exosomes in bronchoalveolar fluid from sarcoidosis patients, displaying proinflammatory activity such as increased secretion of IFN-g and IL-13 by PBMCs, and IL-8 by epithelial cells. Exosomes from mature DCs pulsed with male antigen peptide speed up male skin graft rejection by female mice, showing in vivo priming and effector differentiation of activated CD4+ T-cells (147). Exosomes secreted by DCs can also induce humoral responses against the antigens that were fed to DCs before exosome purification (6,35, 130), leading, in the case of the parasite Toxoplasma gondii, to strong protection against acute or congenital

infection (6). In fact, feeding DCs with intact antigen (rather than peptide) allows them to secrete more efficient exosomes, able to stimulate both T- and B-cells in vivo, leading to both memory Th1 and immunoglobulin responses (130). Thus exosomes can induce effector immune responses in vivo. In addition, careful evaluation of immune responses in the first cancer patients treated with exosomes from their own DCs (55) recently suggested a promoting effect of these exosomes on the patient’s NK cell activity (172). Exosomes carry both antigenic material and MHC– peptide complexes required for the initiation of immune responses by APCs. In addition to whole or partially processed antigens, secreted vesicles also expose MHC–peptide complexes, which can be presented to T lymphocytes. Exosomes are not only capable of presenting antigens directly but are also able to transfer both the MHC II molecule and the antigen to neighboring DCs that can in turn present the antigen to T-cells (159). Tumor-derived membrane vesicles stimulate the immune response by transferring tumor antigens to DCs (176) as well as MHC–peptide complexes, leading to antigen-specific T-cell activation in vitro. Exosomes secreted by virtually any cell type bear MHC class I molecules that could potentially induce CD8 T-cell activation and seem to carry antigens, which have to be processed by the recipient APCs. With respect to exosomes secreted by APCs, several groups have reported that DC-derived exosomes could induce activation of CD8+ CTL clones, either by themselves (55,167), or when incubated with DCs expressing allogeneic MHC class I, showing that exosomes bear functional preformed MHC I–peptide complexes (26,76). In addition, more efficient T-cell activation was obtained with exosomes purified from mature rather than immature DCs, suggesting that costimulatory molecules present in exosomes participate in costimulation of T-cells (1). APC-derived exosomes also bear large amounts of MHC II molecules (1). Exosomes bearing specific MHC II–peptide complexes can be obtained by loading APCs with antigen (135) or peptide (115,147,159). Such exosomes can activate cognate clones (135), T-cell lines (147), or activated CD4+ T-cells (159) by themselves, but need to be captured by recipient DCs to activate naive CD4+ T-cells (147,159). Recipient DCs may also use MHC–peptide complexes from exosomes as a source of peptides to load on their own MHC molecules (115). Inhibition of Immune Responses As mentioned earlier, tumor-derived MVs can stimulate DCs to initiate the immune response, but are, on the other hand, able to exert immunosuppressive properties (36,129) resulting in tumor cell escape from immune surveillance. MVs or exosomes obtained from tumor cell lines or tumor-bearing patients were shown in vitro to

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induce T-cell apoptosis via Fas ligand (FasL) (10,77) and galectin-9 (89). In addition, tumor-derived exosomes can inhibit IL-2-induced T-cell proliferation (158), and/or promote differentiation into Tregs (153), reduce CD8+ T-cell proliferation (175), decrease NK cell cytotoxicity by displaying NK cell receptor D (NKG2D) ligands leading to downregulation of the receptor (11,101), impair myeloid precursor differentiation into DCs (178), and induce myeloidsuppressor cells (39). Vesicles secreted by immune cells can also display immunosuppressive properties. Activated T-cells secrete exosomes bearing FasL, which induce apoptosis of bystander T-cells (114). Exosomes in plasma of pregnant women bear FasL and reduce CD3z expression by T-cells (154). Another mechanism of immunosuppression has been observed for exosomes secreted by placental explants: they bear NKG2D ligands, and induce reduced cytotoxicity of NK and CD8+ T-cells in vitro (71). Thus, placental MVs seem to contribute to fetomaternal tolerance (154). Membrane vesicles secreted by immature DCs also induce tolerogenic, rather than effector, immune responses (126). Such exosomes promote graft survival (126) and reduce inflammation in a model of arthritis (87) or of inflammatory bowel disease (179). Exosomes secreted by normal immature DCs also reduce inflammation in a model of septic shock (109), promoting phagocytosis of sepsis-induced apoptotic cells by macrophages and reducing the release of proinflammatory cytokines (110). ROLE OF MSC–MVs IN REGENERATION The regenerative stimulus induced by MSCs has been described in several tissues (75,84). Stimulation of cell proliferation, inhibition of apoptosis, and stimulation of angiogenesis were reported following MSC transplantation into a variety of injured tissues. Initially, these findings were interpreted as the capacity of MSCs to “transdifferentiate” and replace the cells in the host tissue. More recently, however, it has been recognized that the underlying mechanisms can be better characterized as paracrine effects. Cardiac regeneration induced by MSCs has been extensively studied (92,162,163). It was shown that infusion of media conditioned by MSCs reduces ischemia/ reperfusion (I/R) injury and improves cardiac repair by reducing inflammation, oxidative stress, and apoptosis, and by stimulating angiogenesis (162,163). Lai et al. (92) showed that the cardioprotective factor was present in the MV fraction isolated from MSC supernatant. Purified MSC–MVs strongly reduced infarct size in a mouse model of myocardial I/R injury even at low concentrations, suggesting that the regenerative effect of MSCs on injured myocardium was mainly mediated by secreted MVs. In experimental models of acute renal injury, human MSC–MVs are as effective as MSCs in accelerating recovery from glycerol-induced acute kidney injury in severe combined immunodeficient

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(SCID) mice in vivo, stimulate proliferation, and inhibit apoptosis of tubular epithelial cells in vitro, effects that are abolished by RNase treatment of the MVs (21,40). The latter finding suggests that the regenerative response is mediated through the transfer of mRNA. In a murine model of hypoxic pulmonary hypertension, IV delivery of MSC–MVs suppressed the hypoxic pulmonary influx of macrophages and the induction of proinflammatory and proproliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, inhibiting vascular remodeling and pulmonary hypertension, while fibroblast-derived exosomes had no effect. Moreover, MSC–MVs inhibited signal transducer and activator of transcription 3 (STAT3)-mediated hyperproliferative signaling in isolated human pulmonary artery endothelial cells, pointing to a direct effect on hypoxic vascular cells (97). ROLE OF MSC–MVs IN IMMUNOMODULATION Some recent studies suggest that the immunomodulatory activity of MSCs could be at least partially mediated by their ability to release MVs. We observed that the inhibitory effects of MSCs on B-cell proliferation and differentiation in a CpG-stimulated PBMC coculture system could be fully reproduced by MVs isolated from MSC culture supernatants in a dose-dependent fashion (22) (Fig. 1). A dose-dependent inhibitory activity of MSC–MVs was also observed for immunoglobulin M (IgM), IgG, and IgA production. Moreover, in the same coculture system, 7-aminoactinomycin D (7-AAD)-negative and annexinpositive MSC–MVs isolated from MSCs were internalized in a subset of CD86/CD19-positive cells corresponding to activated B lymphocytes. The effect of MVs on T-cells was investigated by Mokarizadeh et al. (113). These authors showed that MVs isolated from murine BMSCs inhibited the proliferation of both syngenic and allogeneic T lymphocytes. Additionally, they demonstrated that these microparticles were able to induce apoptosis in activated T-cells. Interestingly, this inhibition was associated with an increased proportion of regulatory T CD4+-CD25+-forkhead box P3-positive (FoxP3+) cells (Fig. 1). Moreover, an increased secretion of IL-10 and transforming growth factor-b1 (TGF-b1) by cultured splenic cells added with MSC–MVs was observed. These results suggest that MSC–MVs can induce tolerogenic signaling. We recently observed similar results in human PBMC cultures treated with human T-cell activator CD3/CD28 beads (unpublished). In vitro results are also supported by in vivo observations in an animal model of inflammatory bowel disease (46) induced by dextran sulfate sodium (DSS). Mice injected daily with MSC–MVs showed less weight loss, improved disease activity index, and a less severe reduction in colon length when compared to DSS/vehicle-treated controls. Real-time RT-PCR analysis performed on RNA extracted from colon tissue revealed a strong inhibition of the

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induction of inflammatory cytokines with respect to untreated animals. Collectively, these data suggest that MVs isolated from MSCs could reproduce the immunomodulatory effect of MSCs. IMPLICATIONS FOR THERAPY Results and considerations reported above suggest that MSC–MVs could offer several advantages as therapeutic tools with respect to MSCs. MSCs can exhibit either an anti-inflammatory or proinflammatory phenotype depending on the balance between the cytokines/chemokines released into the surrounding microenvironment (84). For instance, as reported above, MSCs were shown to have both inhibitory (41,43) and stimulatory (136,164) effects on B-cell proliferation, differentiation, and antibody production. The effect of MSCs on B-cells appears to be indirect, that is, mediated by other cell types, thus again strongly dependent on the inflammatory environment (41). As reported above, we confirmed MSC-mediated inhibition of B-cell function in PBMCs (22), but we also observed MSC-mediated stimulation of both proliferation and differentiation when using purified B-cells, in agreement with the work of Traggiai et al. (164). Thus, we confirmed that MSCs can exert opposite effects on B-cell function depending on the environment. However, we observed that MSC–MVs suppress B-cell function both in PBMCs and in purified B-cell coculture (Muraca and Fierabracci, unpublished). This preliminary observation is in agreement with the hypothesis that MSC–MVs could provide simpler interactions with the target cells by a onehit stimulus (156), that is, not mediated by other cell types. This is also consistent with our observations that MSC–MVs are taken up specifically by activated B-cells (22). Another possible advantage of using MVs rather than MSCs could be to avoid possible long-term maldifferentiation of engrafted cells or tumor generation (161). Cell products could also be easier to characterize and standardize with respect to the cells themselves. Phase I trials involving MVs derived from DCs for immunotherapy of advanced cancer (55,116) showed that large-scale, clinical-grade production, isolation, and infusion of MVs is feasible and safe. A critical issue will be defining the effective therapeutic dosage of MSC–MVs. In our experience, administration of MVs isolated from the supernatant of 2 × 106 MSC cultures results in significant clinical and biochemical improvements in a mouse model of inflammatory bowel disease (IBD) (46). In a 70-kg human, this therapeutic dosage would theoretically correspond to the isolate from the supernatant of about 6–8 × 109 MSCs. A similar cell mass can be easily cultured in bioreactors. Immortalization of MSCs by Myc (29) has been proposed in order to ensure production of MSC–MVs in sufficient quantity to support clinical applications. It was also shown that MSC lines expressing high levels of CD81, a protein belonging to the

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tetraspanin family, release more MVs in culture medium, an observation that could help select optimal cell phenotype for MSC–MV production (180). Clinical-grade isolation and purification of MVs from large volumes (1–4 L) of supernatant has been described (93). Collectively, these considerations support the feasibility of future therapeutic applications of MSC–MVs. However, additional studies are needed to explore the role and mechanisms of MSC–MVs as modulators of immune response before they can be proposed for clinical use in place of the parent cells. CONCLUSIVE REMARKS Increasing experimental evidence points to the possibility of using MSC-derived MVs both as a stimulus for the regeneration of injured tissues and for immunomodulation, two closely related processes. Clearly, these encouraging results deserve additional investigations before any claim for the use of MV–MSCs as a novel therapeutic approach can be addressed. However, this line of investigation should be pursued, since MVs could represent a safer and more reproducible therapeutic tool than their parent cells. ACKNOWLEDGMENTS: We thank Massimo Dominici (University of Modena and Reggio Emilia, Modena, Italy) for helpful discussion. The financial contribution of CryoSave (Zutphen, Netherlands) is gratefully acknowledged. The authors declare no conflicts of interest.

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