Pediatr Nephrol (2013) 28:2249–2254 DOI 10.1007/s00467-013-2413-z

REVIEW

Role of mesenchymal stem cell-derived microvesicles in tissue repair

Stefania Bruno & Giovanni Camussi

Received: 12 October 2012 / Revised: 4 December 2012 / Accepted: 12 December 2012 / Published online: 6 February 2013 # IPNA 2013

Abstract Results from recent studies suggest that the ben- eficial effect of stem cell-based therapy is mainly dependent on a paracrine effect. The paracrine hypothesis implicates the ability of stem cells to limit injury or coordinate repair through the release of soluble factors. Among these factors microvesicles (MVs) have emerged as a mechanism through which stem cells may reprogram injured cells. In fact, MVs released from stem cells may deliver proteins, bio-active lipids and nucleic acids to injured cells. In particular, the transfer of transcripts derived from stem cells may induce phenotypic and functional changes in the recipient cells that promote the activation of regenerative programs.

Keywords Exosomes . Microvesicles . Extracellular vesicles . Mesenchymal stem cells

Introduction

Mesenchymal stem cells, also known as mesenchymal stro- mal cells (MSCs), are a heterogeneous multipotent cell population residing in the bone marrow that can be rapidly expanded in vitro while maintaining their multipotency [1]. A number of studies have focused on alternative sources of

S. Bruno
Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy

G. Camussi
Department of Medical Sciences, University of Torino, Torino, Italy

G. Camussi (*)
Dipartimento di Scienze Mediche, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti 14,
10126 Torino, Italy
e-mail: giovanni.camussi@unito.it

stem cells with multiple differentiating capabilities and easy accessibility [2]. MSCs obtained from adipose tissue or from umbilical cord vein, as well as from dental pulp have been investigated as a potential source of stem cells [3–5]. In the search for alternative sources of stem cells, the isola- tion of stem cells from human amniotic fluids (hAFSCs), and their subsequent differentiation into all three types of germ layer cells, has also been reported [6]. Since the recovery of hAFSC is considered ethically acceptable and does not involve the destruction of human embryos, hAFSCs present an attractive source of stem cells for tissue regeneration [7].

Although MSCs exhibit the capacity to migrate to injured sites and can therefore contribute to tissue repair, the extent of improvement of injured tissues has not been shown to correlate with cellular engraftment and differentiation of MSCs to tissue cells, suggesting that they may play an indirect role in tissue regeneration.

Growing evidence indicates that the therapeutic effect of MSCs depends primarily on their capacity to secrete soluble factors; these include a variety of growth factors, cytokines and chemokines that orchestrate interactions within the mi- croenvironment and influence tissue regeneration. These factors can inhibit apoptosis, stimulate proliferation, pro- mote vascularization and modulate the immune response [8]. In addition to soluble factors, MSCs also secrete extra- cellular vesicles which are small, spherical membrane frag- ments that are involved in cell-to-cell communication and are capable of altering the cell fate and phenotype of recip- ient cells [9].

Extracellular vesicles: origin and modes of action

Many different sub-populations of extracellular vesicles have been described and characterized in detail, including exosomes, ectosomes and apoptotic bodies. Exosomes,

2250

Pediatr Nephrol (2013) 28:2249–2254

derived from endocytic vesicles, have a diameter of 30– 100 nm and are generated following the fusion of multi- vesicular bodies with plasma membranes [10]. Shedding vesicles, also named ectosomes or membrane particles, tend to be 100 nm–1 μm in diameter and bud directly from the plasma membrane. These vesicles are shed into the extra- cellular space in a calcium flux- and calpain-dependent manner [11]. Most extracellular vesicles that are described in the literature, either released in vitro or present in biolog- ical fluids, are heterogeneous and consist of exosomes and shedding vesicles, collectively defined here as microvesicles (MVs).

MVs hold the signature of the cell of origin, as they contain proteins (e.g. receptors, adhesion molecules) and lipids of the cell membrane and cytoplasmic constituents [mRNA, microRNA (miRNA), DNA, proteins]. MVs can function as a cargo for delivering cellular components to other cells, thus inducing alterations in the phenotype and behavior of recipient cells [12]. The results of a recent study by Aliotta et al. [13] indicate that MVs can induce changes in cellular phenotypes that persist in vitro as well as in vivo for extended periods of time. Therefore, MVs may stably alter the phenotype of target cells [13].

The mechanisms whereby MVs influence the fate of target cells are varied. They may directly stimulate the cells by means of interactions with receptors and molecules that are expressed on their surface and which bind to specific ligands expressed by recipient cells [14]. Following ligand interaction, MVs may deliver their contents to the recipient cell. For example, MVs derived from endothelial cells can activate angiogenesis through the transfer of pro-angiogenic molecules, such as growth factors [15]. MVs may repro- gram mutant tissue by direct transfer of membrane- associated wildtype molecules. In particular, it has been recently demonstrated that MVs from different types of MSCs shuttle a cysteine-selective transport channel (cystinosin) that restores function in mutant target cells [16]. MVs may also mediate the horizontal transfer of ge- netic information, such as subsets of mRNA and microRNA (miRNA) specific to the cell of origin [17]. Ratajczak et al. [17] demonstrated that MVs may facilitate the transfer of mRNA and, as a result, influence the behavior of target cells. Specifically, these authors found that MVs produced by murine embryonic stem cells (ESCs) may reprogram hematopoietic progenitors. In particular, MVs may increase the pluripotency of hematopoietic progenitors by delivering mRNA for transcription factors involved in stem cell self- renewal, such as Oct-4, Rex-1, Nanog and GATA-2. The transferred mRNA then becomes translated into protein by recipient cells [17]. MVs can also contain mRNA for recep- tors of specific growth factors; for example, MVs released by bone marrow MSCs (BM-MSCs) contain mRNA for the insulin growth factor 1 (IGF-1) receptor [18]. In an in vitro

model of renal toxic injury induced by cisplatin, the transfer of IGF-1 receptor mRNA through MVs has been shown to increase the proliferation of damaged proximal tubular cells [18].

Recently, Quesenberry and Aliotta suggested that MVs released in the microenvironment around injured cells may represent a signal for stem cell differentiation [19]. In fact, MVs derived from the damaged murine lung may enter into bone marrow cells where they induce a lung-specific phe- notype in these cells [20, 21]. Signals from injured cells may therefore induce stem cell recruitment and differentiation in specific cells of injured organs.

Our group has focused on MVs released from human BM-MSCs, demonstrating that they are responsible for shuttling a specific subset of cellular mRNA, including tran- scripts that are characteristic of mesenchymal cell lineages, as well as transcripts related to several different cell func- tions (e.g. for the control of transcription, cell proliferation and immune regulation) [22]. In addition to mRNA, MVs may also transfer miRNA, a function confirmed by experi- ments demonstrating that the conditioned medium produced by human MSCs derived from ESCs contains MVs that shuttle specific pre-miRNA [23] and miRNA [24]. Specifically, we have shown that MVs released from human BM-MSCs contain mature functional miRNA that can be transferred to target renal tubular cells [25]. Gene ontology analysis has shown that the highly expressed miRNAs in MVs from BM-MSCs are involved in multi-organ develop- ment, cell survival and differentiation.

The protein content of MVs from MSCs has also been characterized recently [26]. MVs from MSCs contain pro- teins that are characteristic of MSCs (e.g. CD29, CD73, CD44, CD105), proteins associated with intra-cellular MV biogenesis and trafficking (RAB protein family) and pro- teins associated with MSC self-renewal and differentiation (TGF-β, MAPK, PPAR, etc.). Gene ontology analyses of such proteins indicate that several biological processes are represented, including vesicle-mediated transport, cell cycle and proliferation, cell migration, morphogenesis and devel- opmental processes.

MSCs and MSC-derived MVs favor renal regeneration

It has been reported that MSC administration effectively ameliorates experimental acute kidney injury (AKI) [27–31] and induces functional improvements in chronic kidney disease (CKD) [32]. MSCs can migrate to injured tissues when transplanted systemically. Homing of MSCs into injured tissues depends on their ability to migrate and interact with the local microenvironment. The triggering of the chemokine receptor CXCR4 by its ligand, stromal de- rived factor, plays an important role in the homing of MSCs

Pediatr Nephrol (2013) 28:2249–2254

2251

to the sites of injury [33, 34]. Moreover, it has been shown that CD44 and glycosaminoglycan hyaluronan (HA) inter- action is involved in MSC migratory capacity [35]. The expression of CD44 is involved in the localization of exog- enous MSCs to injured renal tissue, based on the observa- tions that loss of CD44 expression by mutant MSCs results in reduced MSC localization to injured tissue and subse- quent prevention of renal repair [30]. After systemic admin- istration, MSCs accumulate in the injured kidney, but only few of these permanently engraft within the tubules [27–30]. The beneficial effect of MSC administration is due to tran- sient localization to the damaged tissue. In rats with ische- mia and reperfusion injury, MSCs were found to localize to the peritubular capillaries and further migrate to the tubular interstitium 24 h after systemic administration. However, after 3 days, none of the administered MSC had differenti- ated into tubular or endothelial cells [36]. In the model of glycerol-induced AKI, the majority of injected MSCs dis- appeared from the kidney after just 5 days [37]. This exper- imental evidence indicates that MSCs do not replace renal tubular cells, but mitigate injury by providing paracrine support for repair. This hypothesis is sustained by experi- ments showing that conditioned medium (CM) from murine MSCs mimics the beneficial effects of the cells of origin in the experimental model of cisplatin-induced AKI [38]. In this study, the intraperitoneal administration of MSC-CM limited renal injury by diminishing tubular cell apoptosis and increasing tubular cell survival. It can therefore be concluded that bio-active factors secreted by MSCs are responsible for their capacity to ameliorate kidney function and morphology. In particular, IGF-1 and vascular endothe- lial growth factor (VEGF) were suggested to be involved in the renal-protective effect of MSC administration. Injection of IGF-1 gene-silenced MSCs in the murine model of cisplatin-induced AKI reduced the beneficial effect of MSCs [39]; VEGF knockdown by small interfering RNA

(siRNA) also reduced the effects of MSC infusion in ische- mia and reperfusion AKI [40].

As MVs can influence the behavior of recipient cells by shuttling bio-active molecules, our group and others have recently exploited the possible effects of MSC-derived MVs in tissue regeneration, in different experimental animal mod- els of renal injury (Table 1).

MSC-derived MVs express several adhesion molecules of MSCs, such as CD44, CD29 (β1-integrin), α4- and α5 integ- rins and CD73 [22]. In this study [22], CD44 and CD29 were shown to be instrumental in MV internalization into tubular epithelial cells, as treatment with specific blocking antibodies was found to prevent MV incorporation. Moreover, pre- treatment of MSC-derived MVs with annexin V abrogated their uptake and effect on target cells [16].

In studies carried out by our group, we found that human BM-MSC-derived MVs stimulated the proliferation and apoptosis resistance of tubular epithelial cells in vitro [22, 41]. In vivo, MV treatment accelerated the morphological and functional recovery of glycerol-induced-AKI in SCID mice [22]. In this experimental model, the effect of MV injection was comparable to that obtained with MSCs, indi- cating that MVs mimic the beneficial effects of the cells of origin. This in turn suggests that MVs may mediate, at least in part, the MSC-regenerative potential. In the lethal model of AKI induced by cisplatin, a single administration of MVs improved the survival of SCID mice, without preventing chronic tubular injury. Multiple injections of MVs, at dif- ferent time points after injury, not only further improved the survival of SCID mice but also completely prevented chron- ic tubular injury [41]. We also found that a single adminis- tration of MVs immediately after ischemia and reperfusion injury protected rats from AKI and prevented CKD [42]. After injection, MVs transiently localize within glomeruli and proximal tubules in animals with ischemia and reperfu- sion injury (Fig. 1). In contrast, in normal rats, MVs are

Table 1 Evidence for microve- sicle involvement in tissue regeneration

Microvesicle cell sources
Bone marrow mesenchymal stem cells

Embryonic stem cell-derived mesenchymal stem cells

Human liver stem cells Endothelial progenitors cells

Animal models Reference

Mouse glycerol-induced AKI [22] Rat renal ischemia and reperfusion injury [42] Mouse cisplatin-induced AKI [41] Mouse remnant kidney model [43] Rat gentamicin-induced AKI [44] Rat middle cerebral artery occlusion [47] Mouse myocardial ischemia and reperfusion injury [45]

Rat hepatectomy [49] Rat renal ischemia and reperfusion injury [51] Mouse hind limb ischemia [52]

 

AKI, Acute kidney injury

2252

Pediatr Nephrol (2013) 28:2249–2254

 

Fig. 1 Representative confocal microscopy showing the localization of PKH26-labeled microvesicles (MVs) within the glomerulus and proximal tubules in a rat with ischemia and reperfusion injury, 2 h after MV injection. MVs are seen within the glomerulus and, in particular, in the tubular epithelial cells. Magnification: ×600

captured by the liver and do not accumulate within the kidney [42]. The mechanism of protection has mainly been ascribed to the stimulation of cell proliferation and inhibi- tion of apoptosis. In particular, we found that inhibition of in vitro apoptosis of cisplatin-treated tubular cells was associ- ated with the down-regulation of genes involved in the execution phase of cell apoptosis (caspase 1 and 8) and with the up-regulation of anti-apoptotic genes (Bcl-xL and Bcl2) [41]. The in vitro and in vivo effects of MVs have been partially attributed to RNA delivery, as the inactivation of RNA has been found to abate their properties [22, 40, 41]. Moreover, the transfer of specific miRNA and mRNA and its subsequent translation into proteins in renal tubular cells has been shown both in vitro and in vivo [22, 41].

He et al. recently reported that MSC-derived MVs also protect against renal injury in the mouse remnant kidney model [43]. In this model of 5/6 subtotal nephrectomy, a single MV administration preserves the function of the remnant kidney, ameliorated renal injury and prevented renal fibrosis. Reis et al. [44] have recently reported that BM-MSCs, as well as CM or purified MVs, minimize gentamicin-induced AKI. When CM or MVs were incubated with RNase, the in vivo effects were blunted, confirming that regenerative properties are mediated by RNAs carried by MVs [44].

The therapeutic effect of MVs produced by MSCs has also been reported in experimental models of myocardial ischemia and reperfusion injury (Table 1). In two studies, CM from human ESC-derived MSCs significantly reduced infarct size

in pig and mouse models of myocardial infarction [45, 46]. The results of electron microscopy, ultracentrifugation, mass spectrometry and biochemical studies/assays demonstrated that the active components of CM are exosomes [45]. Therefore, in these cases as well, the authors attributed the therapeutic activity of MSC-CM to exosomes.

MSCs also have a potential therapeutic benefit for the treatment of neurological disease and injury. In a recent study, Xin et al. [47] reported that MSC treatment of rats subjected to middle cerebral artery occlusion leads to a significant increase in miR-133b levels in the ipsilateral hemisphere by exosome-mediated delivery. This study is the first to demonstrate that MSCs communicate with brain parenchymal cells and regulate neurite outgrowth by trans- fer of miRNA to neural cells via exosomes [47].

MVs derived from other cell sources may contribute to tissue regeneration

As shown in Table 1, MVs produced by other stem/proge- nitor cell sources have been studied as potential therapeutic agents in different animal models of tissue injury.

Human liver stem cells (HLSCs) represent a population of MSCs resident in the adult human liver, with a partial hepatic commitment [48]. HLSC-derived MVs induce in vitro proliferation and apoptosis resistance in human and rat hepatocytes [49]. Moreover, in vivo MVs accelerate morphological and functional recovery of the liver in a model of 70 % hepatectomy in rats [49]. In this case as well, the activities of MVs have been found to be at least partly RNA-dependent: RNase pre-treatment reduces their protective effects [49]. In addition, in the same study, spe- cific human proteins translated from MV-transported mRNA were detected in vitro and in vivo in rat hepatocytes.

Endothelial progenitor cells (EPCs), obtained from the peripheral blood of healthy donors are also able to produce MVs which, after internalization in normal endothelial cells, activate an angiogenic program by the horizontal transfer of mRNA [50]. The administration of a single dose of EPC- MVs immediately after renal ischemia and reperfusion inju- ry in rats was found to confer functional and morphological protection from AKI by improving proliferation and reduc- ing apoptosis of the renal tubular cells [51]. Moreover, EPC- MVs are able to protect against CKD by inhibiting capillary rarefaction, glomerulosclerosis and tubulo-interstitial fibro- sis. Non-specific depletion of miRNA content in MVs by Dicer knock-down in EPCs or depletion of specific pro- angiogenic miRNA (miR-126 and miR-296) by transfection of EPCs with specific antagomir reduced the renal protec- tive effect of EPC-MVs [51]. Moreover, EPC-MVs also reduced injury and enhanced reperfusion in a murine model of hind limb ischemia [52].

Pediatr Nephrol (2013) 28:2249–2254

2253

7. Siegel N, Rosner M, Hanneder M, Freilinger A, Hengstschlager M (2008) Human amniotic fluid stem cells: a new perspective. Amino Acids 35:291–293

8. Caplan A, Dennis JE (2006) Mesenchymal stem cells as trophic mediators. J Cell Biochem 98:1076–1084

9. Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L (2010) Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney Int 789:838–848

10. Keller S, Sanderson MP, Stoeck A, Altevogt P (2006) Exosomes: from biogenesis and secretion to biological function. Immunol Lett 107:102–108

11. Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ (1999) Activated platelets release two types of membrane vesicles: micro- vesicles by surface shedding and exosomes derived from exocyto- sis of multivesicular bodies and alpha-granules. Blood 94:3791– 3799

12. György B, Szabó TG, Pásztói M, Pál Z, Misják P, Aradi B, László V, Pállinger E, Pap E, Kittel A, Nagy G, Falus A, Buzás EI (2011) Membrane vesicles, current state-of-the-art: emerging role of ex- tracellular vesicles. Cell Mol Life Sci 68:2667–2688

13. Aliotta JM, Pereira M, Li M, Amaral A, Sorokina A, Dooner MS, Sears EH, Brilliant K, Ramratnam B, Hixons DC, Quesemberry PJ (2012) Stable cell fate changes in marrow cells induced by lung-derived microvesicles. JEV 1:18163. Available at: http://www. journalofextracellularvesicles.net/index.php/jev/article/view/18163)

14. Théry C, Ostrowski M, Segura E (2009) Membrane vesicles as conveyors of immune responses. Nat Rev Immunol 9:581–593

15. Taraboletti G, D’Ascenzo S, Giusti I, Marchetti D, Borsotti P, Millimaggi D, Giavazzi R, Pavan A, Dolo V (2006) Bioavailability of VEGF in tumor-shed vesicles depends on vesicle burst induced by acidic pH. Neoplasia 8:96–103

16. Iglesias DM, El-Kares R, Taranta A, Bellomo F, Emma F, Besouw M, Levtchenko E, Toelen J, van den Heuvel L, Chu LL, Zhao J, Young YK, Eliopoulos N, Goodyer P (2012) Stem cell micro- vesicles transfer cystinosin to human cystinotic cells and reduce cystine accumulation in vitro. PLoS One 7:e42840

17. Ratajczak J, Miekus K, Kucia M, Zhang J, Reca R, Dvorak P, Ratajczak MZ (2006) Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia 20:847–856

18. Tomasoni S, Longaretti L, Rota C, Morigi M, Conti S, Gotti E, Capelli C, Introna M, Remuzzi G, Benigni A (2012) Transfer of growth factor receptor mRNA via exosomes unravels the regener- ative effect of mesenchymal stem cells. Stem Cells Dev. doi:10.1089/scd.2012.0266

19. Quesenberry PJ, Dooner MS, Aliotta JM (2010) Stem cell plastic- ity revisited: the continuum marrow model and phenotypic changes mediated by microvesicles. Exp Hematol 38:581–592

20. Aliotta JM, Sanchez-Guijo FM, Dooner GJ, Johnson KW, Dooner MS, Greer KA, Greer D, Pimentel J, Kolankiewicz LM, Puente N, Faradyan S, Ferland P, Bearer EL, Passero MA, Adedi M, Colvin GA, Quesenberry PJ (2007) Alteration of marrow cell gene ex- pression, protein production, and engraftment into lung by lung- derived microvesicles: a novel mechanism for phenotype modula- tion. Stem Cells 25:2245–2256

21. Aliotta JM, Pereira M, Johnson KW, de Paz N, Dooner MS, Puente N, Ayala C, Brilliant K, Berz D, Lee D, Ramratnam B, McMillan PN, Hixson DC, Josic D, Quesenberry PJ (2010) Microvesicle entry into marrow cells mediates tissue-specific changes in mRNA by direct delivery of mRNA and induction of transcription. Exp Hematol 38:233–245

22. Bruno S, Grange C, Deregibus MC, Calogero RA, Saviozzi S, Collino F, Morando L, Busca A, Falda M, Bussolati B, Tetta C, Camussi G (2009) Mesenchymal stem cell-derived microvesicles protect against acute tubular injury. J Am Soc Nephrol 20:1053– 1067

Conclusions

Based on the fact that, in regenerative therapies MVs mimic the beneficial effects of the cells from which they originate, MVs could represent an important potential therapeutic tool. As they may influence the behavior of recipient cells by delivering their bioactive cargo, it may be possible to exploit this effect in tissue regeneration and repair. MVs for use in regenerative therapy could be obtained on a large scale from producer stem/progenitor cells and suitably altered to obtain engineered MVs to enhance tissue regeneration. For example, producer cells might be modified to over-express specific antigens capable of delivering MVs to a specific organ or tissue. Moreover, the use of MVs in place of stem/progenitor cells could represent a novel therapeutic strategy, avoiding the possible adverse effects of cell administration, such as pulmo- nary occlusion at the moment of systemic cell delivery, or long-term mal-differentiation of transplanted cells.

However, several problems need to be addressed before MSC-released MVs can be considered as a potential thera- peutic tool. First, it is necessary to develop strategies to obtain sufficient amounts of MVs. A preliminary kinetic study we undertook based on NanoSight detection of MVs in MSC-conditioned medium revealed that the timing of collection is critical. In fact, in our study, after 12 h, a single MSC released about 7,000 MVs, whereas after 24 h only 2,500 MV/cell were detectable, due to a re-uptake of MVs by MSCs. In addition, an enhanced release could be obtained after appropriate stimulation of MSCs. Moreover, further studies are needed to define biosafety, fields of application and effective doses.

References

1. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147
2. Kuci S, Kuci Z, Latifi-Pupovci H, Niethammer D, Handgretinger R, Schumm M, Bruchelt G, Bader P, Klingebiel T (2009) Adult stem cells as an alternative source of multipotential (pluripotential) cells in regenerative medicine. Curr Stem Cell Res Ther 4:107–117
3. Breymann C, Schmidt D, Hoerstrup SP (2006) Umbilical cord cells as a source of cardiovascular tissue engineering. Stem Cell Rev 2:87–92
4. Graziano A, Aquino R, Laino G, Papaccio G (2008) Dental pulp stem cells: a promising tool for bone regeneration. Stem Cell Rev 4:21–26
5. Murohara T, Shintani S, Kondo K (2009) Autologous adipose- derived regenerative cells for therapeutic angiogenesis. Curr Pharm Des 15:2784–2790
6. De Coppi P, Bartsch G Jr, Siddiqui MM, Xu T, Santos CC, Perin L, Mostoslavsky G, Serre AC, Snyder EY, Yoo JJ, Furth ME, Soker S, Atala A (2007) Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol 25:100–106

2254

Pediatr Nephrol (2013) 28:2249–2254

23. Chen TS, Lai RC, Lee MM, Choo AB, Lee CN, Lim SK (2010) Mesenchymal stem cell secretes microparticles enriched in pre- microRNAs. Nucleic Acids Res 38:215–224
24. Koh W, Sheng CT, Tan B, Lee QY, Kuznetsov V, Kiang LS, Tanavde V (2010) Analysis of deep sequencing microRNA ex- pression profile from human embryonic stem cells derived mesen- chymal stem cells reveals possible role of let-7 microRNA family in downstream targeting of hepatic nuclear factor 4 alpha. BMC Genomics 11[Suppl 1]:S6
25. Collino F, Deregibus MC, Bruno S, Sterpone L, Aghemo G, Viltono L, Tetta C, Camussi G (2010) Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs. PLoS One 5:e11803
26. Kim HS, Choi DY, Yun SJ, Choi SM, Kang JW, Jung JW, Hwang D, Kim KP, Kim DW (2012) Proteomic analysis of microvesicles derived from human mesenchymal stem cells. J Proteome Res 11:839–849

38. Bi B, Schmitt R, Israilova M, Nishio H, Cantley LG (2007) Stromal cells protect against acute tubular injury via an endocrine effect. J Am Soc Nephrol 18:2486–2496

39. Imberti B, Morigi M, Tomasoni S, Rota C, Corna D, Longaretti L, Rottoli D, Valsecchi F, Benigni A, Wang J, Abbate M, Zoja C, Remuzzi G (2007) Insulin-like growth factor-1 sustains stem cell mediated renal repair. J Am Soc Nephrol 18:2921–2928

40. Tögel F, Zhang P, Hu Z, Westenfelder C (2009) VEGF is a mediator of the renoprotective effects of multipotent marrow stro- mal cells in acute kidney injury. J Cell Mol Med 13:2109–2114

41. Bruno S, Grange C, Collino F, Deregibus MC, Cantaluppi V, Biancone L, Tetta C, Camussi G (2012) Microvesicles derived from mesenchymal stem cells enhance survival in a lethal model of acute kidney injury. PLoS One 7:e33115

42. Gatti S, Bruno S, Deregibus MC, Sordi A, Cantaluppi V, Tetta C, Camussi G (2011) Microvesicles derived from human adult mes- enchymal stem cells protect against ischaemia-reperfusion-induced acute and chronic kidney injury. Nephrol Dial Transplant 26:1474–

27. Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M,
Rottoli D, Angioletti S, Benigni A, Perico N, Alison M, Remuzzi
G (2004) Mesenchymal stem cells are renotropic, helping to repair 1483

the kidney and improve function in acute renal failure. J Am Soc

Nephrol 15:1794–1804

28. Morigi M, Introna M, Imberti B, Corna D, Abbate M, Rota C,Rottoli D, Benigni A, Perico N, Zoja C, Rambaldi A, Remuzzi A, Remuzzi G (2008) Human bone marrow mesenchymal stem cells accelerate recovery of acute renal injury and prolong survival in mice. Stem Cells 26:2075–2082
29. Herrera MB, Bussolati B, Bruno S, Morando L, Mauriello- Romanazzi G, Sanavio F, Stamenkovic I, Biancone L, Camussi G (2004) Mesenchymal stem cells contribute to renal repair on acute tubular epithelial injury. Int J Mol Med 14:1035–1041
30. Herrera MB, Bussolati B, Bruno S, Fonsato V, Romanazzi GM, Camussi G (2007) Exogenous mesenchymal stem cells localize to the kidney by means of CD44 following acute tubular injury. Kidney Int 72:430–441
31. Duffield JS, Park KM, Hsiao LL, Kelley VR, Scadden DT, Ichimura T, Bonventre JV (2005) Restoration of tubular epithelial cells during repair of the postischemic kidney occurs independently of bone marrow-derived stem cells. J Clin Invest 115:1743–1755
32. Choi S, Park M, Kim J, Hwang S, Park S, Lee Y (2009) The role of mesenchymal stem cells in the functional improvement of chronic renal failure. Stem Cells Dev 18:521–529
33. Ji JF, He BP, Dheen ST, Tay SS (2004) Interactions of chemokines and chemokine receptors mediate the migration of mesenchymal stem cells to the impaired site in the brain after hypoglossal nerve injury. Stem Cells 22:415–427
34. Wynn RF, Hart CA, Corradi-Perini C, O’Neill L, Evans CA, Wraith JE, Fairbairn LJ, Bellantuono I (2004) A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 104:2643–2645
35. Zhu H, Mitsuhashi N, Klein A, Barsky LW, Weinberg K, Barr ML, Demetriou A, Wu GD (2006) The role of the hyaluronan receptor CD44 in MSC migration in the extracellular matrix. Stem Cells 24:928–935
36. Tögel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C (2005) Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mecha- nisms. Am J Physiol Renal Physiol 289:F31–F42
37. Hauser PV, De Fazio R, Bruno S, Sdei S, Grange C, Bussolati B, Benedetto C, Camussi G (2010) Stem cells derived from human amniotic fluid contribute to acute kidney injury recovery. Am J Pathol 177:2011–2021

43. HeJ,WangY,SunS,YuM,WangC,PeiX,ZhuB,WuJ,ZhaoW (2012) Bone marrow stem cells-derived microvesicles protect against renal injury in the mouse remnant kidney model. Nephrol- ogy 17:493–500

44. Reis LA, Borges FT, Simoes MJ, Borges AA, Sinigaglia-Coimbra R, Schor N (2012) Bone marrow-derived mesenchymal stem cells repaired but did not prevent gentamicin-induced acute kidney injury through paracrine effects in rats. PLoS One 7:e44092

45. Lai RC, Arslan F, Lee MM, Sze NS, Choo A, Chen TS, Salto- Tellez M, Timmers L, Lee CN, El Oakley RM, Pasterkamp G, de Kleijn DP, Lim SK (2010) Exosome secreted by MSC reduces myocardial ischemia/reperfusion injury. Stem Cell Res 4:214–222

46. Lai RC, Chen TS, Lim SK (2011) Mesenchymal stem cell exo- some: a novel stem cell-based therapy for cardiovascular disease. Regen Med 6:481–492

47. Xin H, Li Y, Buller B, Katakowski M, Zhang Y, Wang X, Shang X, Zhang ZG, Chopp M (2012) Exosome-mediated transfer of miR- 133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth. Stem Cells 30:1556–1564

48. Herrera MB, Bruno S, Buttiglieri S, Tetta C, Gatti S, Deregibus MC, Bussolati B, Camussi G (2006) Isolation and characterization of a stem cell population from adult human liver. Stem Cells 24:2840–2850

49. Herrera MB, Fonsato V, Gatti S, Deregibus MC, Sordi A, Cantarella D, Calogero R, Bussolati B, Tetta C, Camussi G (2010) Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats. J Cell Mol Med 14:1605–1618

50. Deregibus MC, Cantaluppi V, Calogero R, Lo Iacono M, Tetta C, Biancone L, Bruno S, Bussolati B, Camussi G (2007) Endothelial progenitor cell derived microvesicles activate an angiogenic pro- gram in endothelial cells by a horizontal transfer of mRNA. Blood 110:2440–2448

51. Cantaluppi V, Gatti S, Medica D, Figliolini F, Bruno S, Deregibus MC, Sordi A, Biancone L, Tetta C, Camussi G (2012) Micro- vesicles derived from endothelial progenitor cells protect the kid- ney from ischemia-reperfusion injury by microRNA-dependent reprogramming of resident renal cells. Kidney Int 82:412–427

52. Ranghino A, Cantaluppi V, Grange C, Vitillo L, Fop F, Biancone L, Deregibus MC, Tetta C, Segoloni GP, Camussi G (2012) Endo- thelial progenitor cell-derived microvesicles improve neovascula- rization in a murine model of hindlimb ischemia. Int J Immunopathol Pharmacol 25:75–85