The Elusive Nature and Function of Mesenchymal Stem Cells

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research12 (BOX 2). Nevertheless, many opinion important aspects regarding MSC biology are still unclear. In this Opinion article, we The elusive nature and function of discuss the wellestablished properties of MSCs cultured in vitro, and focus on how these properties relate to recent studies that mesenchymal stem cells are beginning to uncover MSC localization and function in vivo. César Nombela-Arrieta, Jerome Ritz and Leslie E. Silberstein Studying MSCs in vitro Abstract | Mesenchymal stem cells (MSCs) are a diverse subset of multipotent Defining MSCs. Stem cells are classically precursors present in the stromal fraction of many adult tissues and have drawn defined by their multipotency and self intense interest from translational and basic investigators. MSCs have been renewal (BOX 1). Based on these criteria, the operationally defined by their ability to differentiate into osteoblasts, adipocytes central and most disputed issue is the use and chondrocytes after in vitro expansion. Nevertheless, their identity in vivo, of the term MSC. This term was first used heterogeneity, anatomical localization and functional roles in adult tissue to refer to a hypothetical postnatal, multi potent and selfrenewing precursor derived homeostasis have remained enigmatic and are only just starting to be uncovered. from an original embryonic MSC, the func tion of which was to maintain the turnover Stem cells are clonogenic cells that have two colonyforming unit fibroblasts (CFUFs)4, in of skeletal tissues in homeostasis or tissue remarkable features, the ability to differenti analogy to their haematopoietic counterpart) repair during adulthood. As described for ate into multiple mature cell types (multi that could be selected by adherence to plastic HSCs, MSCs would lie at the top of the potency) and the ability to simultaneously surfaces and expanded in vitro. Further stud mesenchymal cell hierarchy and progress replenish the stem cell pool (selfrenewal), ies substantiated the ability of cultured cells through discrete stages of differentiation that allow them to sustain tissue develop derived from single CFUFs to proliferate in an orderly manner to give rise to func ment and maintenance1 (BOX 1). The surge of while preserving the ability to differentiate tionally and pheno typically mature tissues, stem cell research arose when it became clear to osteoblasts, adipocytes and chondrocytes including bone, smooth muscle, tendons that all blood cell components are derived in vitro5. Multilineage capacity and prolifera and cartilage6. from a rare subset of bone marrow (BM) tion in vitro were interpreted at the time as This theoretical model provided an residing haematopoietic stem cells (HSCs), indicative of in vivo multi potency and self attractive conceptual framework in which which can be isolated and assayed in vitro renewal, the hallmarks of ‘stemness’. Thus, the stromal multipotent precursor cell and in vivo. Currently, HSCs are the para the term mesen chymal stem cell (MSC) was described by Friedenstein was rapidly digmatic and bestcharacterized example coined and gained acceptance to refer to regarded as the prototypical MSC, despite of tissuespecific stem cells. these newly identified precursor cells6,7. Since the fact that such a cell had not been shown Soon after the discovery of HSCs, studies their original description, stromal cells cat to strictly fulfil the attributes conveyed in by Friedenstein and colleagues2 reported that egorized as MSCs based on trilineage (osteo the term MSC. First, the biological proper the BM stroma can generate bone, fat cells, blast, adipo cyte and chondrocyte) potential ties of multipotent progenitors had mostly cartilage and reticular cells following hetero in vitro have been isolated from the adherent been inferred from the analysis of clonal topic transplantation (that is, transplantation fraction of many adult and embryonic tissues and nonclonal in vitroexpanded popu into a different tissue from that of origin) in in multiple species8–11 (FIG. 1). lations owing to the inability to isolate and mice. This suggested the existence of non Multipotentiality in vitro, as well as assay them directly from tissues. Until haematopoietic BM multipotent precursor ease of isolation and expansion, rapidly recently, the multipotency and selfrenewal cells with skeletal and adipose potential2,3. positioned MSCs as promising therapeutic of un cultured progenitors had not been fully It was later shown that these precursors were agents in regenerative medicine and made probed using stringent in vitro and in vivo a subset of fibroblastlike cells (defined as them the subject of intensive clinical assays. Furthermore, the existence of a com mon postnatal ‘mesenchymal’ progenitor has been questioned, as bone and muscle derive Box 1 | Criteria used to define stem cells: HSCs and BM‑resident MSCs from different progenitors during embryonic Multipotency development, and because whether MSCs The first criterion used to define stem cells is their ability to differentiate into multiple types give rise to muscle cells in vivo has not been of functionally mature specialized cells. Haematopoietic stem cells (HSCs) can give rise to all convincingly demonstrated to date. For this blood cell components, including neutrophils, lymphocytes, natural killer cells, dendritic cells, reason, alter native names such as osteogenic macrophages and monocytes. Bone marrow (BM)-resident mesenchymal stem cells (MSCs) can or skeletal stem cells have been suggested. 1 give rise to osteoblasts, chondrocytes, adipocytes and reticular stroma . Regardless of its inaccuracy13,14, the term Self-renewal MSC has remained prevalent to date to Stem cells also possess the capacity of self-renewal; that is, the ability to undergo numerous cell designate stromal precursors with triline divisions while retaining their stem cell identity. HSCs have been shown to self-renew: HSCs age potential isolated from the BM and, by transplanted into irradiated mice can reconstitute the haematopoietic system, including giving extension, from any other mammalian tis rise to a population of HSCs that can be serially re-transplanted1. BM-resident MSCs have also sue. Of note, the common use of the name been shown to possess this ability, as their transplantation gives rise to ‘ossicles’ composed of bone, cartilage and reticular stroma, as well as to a population of serially re-transplantable MSCs1. MSC to indistinctively refer to both in vivo precursors and their in vitroexpanded

progeny has frequently led to misconcep tions in the field. The International Society for Cellular Therapy, Vancouver, Canada, has recommended the use of the name multipotent mesenchymal stromal cell (also $QPGOCTTQY -KFPG[ 2CPETGCU #FKRQUGVKUUWG 5MGNGVCNOWUENG $TCKP .KXGT abbreviated to MSCs, although not in this Opinion article) for the in vitro cultured cells, restricting the term stem cell to 2GTKXCUEWNCTOWNVKRQVGPV RTGEWTUQTt/5%! des ig nate the proposed in vivo precursors %& or stem cells15,16. 0) 2&)(4β Characterization of mesenchymal stromal cells. Beyond their ability to generate osteo blasts, adipocytes and chondrocytes in vitro5, mesenchymal stromal cells give rise to bone %(7( and cartilage after ectopic implantation in vivo17,18 and have been documented to contribute to bone regeneration in animal 19 Multipotent mesenchymal models of genetic bone disorders . Many stromal cell studies have further reported mesenchymal stromal cell differentiation into multiple other cell types of mesodermal and non mesodermal origin, including endothelial &GZCOGVJCUQPG +UQDWV[NOGVJ[NZCPVJKPG 6TCPUHQTOKPIITQYVJ cells20, cardiomyocytes21, hepato cytes22 and .CUEQTDKECEKFCPF FGZCOGVJCUQPG HCEVQTβCPFFGZCOGVJCUQPG 23,24 KPQTICPKERJQURJCVG KPFQOGVJCEKPGCPFKPUWNKP neural cells . Nevertheless, such multi ! potential capabilities of mesenchymal stro mal cells are not universally accepted. There are concerns because of the lack of globally 1UVGQDNCUVU #FKRQE[VGU %JQPFTQE[VGU 1VJGTEGNNV[RGU standardized methods for their isolation, Figure 1 | MSCs and multipotent mesenchymal stromal cells. The plastic-adherent cellular frac- expansion and identification, as well as the tion of many organs contains stromal progenitor cells that 0CVWTcan giveG4G riseXKGYU to ^colonies/QNGEWNCT%GNN$KQNQI[ of fibroblastic range of assays used to define terminally morphology. This cellular subset, known as colony-forming unit fibroblasts (CFU-Fs), totally or partially differentiated, functionally mature popu corresponds to a proposed multipotent progenitor cell population, most probably heterogeneous in lations. Claims for in vivo differentiation nature and origin, that resides in the proximity of blood vessels in all tissues studied so far and has been into other cell types are equally controver shown to express pericyte-specific markers (CD146, NG2 (also known as CSPG4) and platelet-derived growth factor receptor (PDGFR )). When cultured under the appropriate cell densities, colonies sial, as BMderived mesenchymal stromal β β derived from single CFU-Fs can be isolated and expanded after multiple passages in vitro (indicated cell cultures have been shown to contribute by the curved arrow) without losing their multipotent mesenchymal capacity. These cultured cells, to many tissues following transplantation classically referred to as mesenchymal stem cells (MSCs), are now termed multipotent mesenchymal through fusion with endogenous cells and stromal cells. The hallmark that defines mesenchymal stromal cells is their ability to differentiate into not through differentiation into mature cell osteoblasts, adipocytes and chondrocytes when placed under inductive stimuli. Differentiation types25. How multipotent mesenchymal into multiple non-mesenchymal mature cell types (such as muscle cells, endothelial cells and neural stromal cells really are remains unclear. cells) has been reported but remains a matter of debate. Discrepancies in the reported properties of MSCs might be partially explained by their presence in tissues of diverse precursor conventional mesenchymal stromal cell of their biological properties and in vivo types, heterogeneous in nature and origin, cultures arise from, and contain, a hetero identities is lacking, to date. that seem similar on the basis of their in vitro geneous pool of mesenchymal progenitors An additional important consideration at characteristics. However, heterogeneity or stem cells that can be organized in a hier this point is that mesenchymal stromal cells is obvious at the level of mesenchymal archical manner, analogous to that of other derived from various postnatal or embryonic stromal cell cultures (reviewed in ReF. 26), welldescribed stem cell systems. Beyond tissues using identical culture conditions with the presence of clones of different MSCs, more primitive multipotent cell sub display significant differences in colony morphologies8,27,28, proliferative capacities29, sets with the potential to give rise to cells of morphology, differentiation potential in vitro multidifferentiation capacities and all three germ layers have been proposed to and gene expression8,35–37. This raises the in vivo abilities to generate bone in ectopic be present within the tissueresident pool question of whether MSCs from different implants27,30,31. Singlecellderived clones of cells and copurify with mesenchymal anatomical locations, selected by classic of mesenchymal stromal cells from human stromal progenitors. Nevertheless, it should adherence and in vitro culture methods, are umbilical cord progenitors that display be emphasized that the existence of stem cell biologically equivalent. Collectively, these varying degrees of multipotency and exten populations of such nature, which include results suggest that mesenchymal stromal sive selfrenewal in vitro have been shown to MAPCS (multipotent adult progenitor cell cultures may originate from an array of generate daughter clones that gradually lose cells)33 and MUSe cells (multilineage differ tissuespecific multipotent precursor cells their multilineage differentiation capacity32. entiating stressenduring cells)34, is highly that are present in native tissues and have Together, these observations suggest that controversial, as a detailed characterization diverse degrees of plasticity and selfrenewal.

Box 2 | Therapeutic exploitation of mesenchymal stromal cells reported to label BMresident MSCs in a selective manner. This study showed for the Although clinical interest in cultured mesenchymal stem cells (known as mesenchymal stromal first time that MSCs are the progenitors of cells) initially focused on the potential of their stem cell-like properties for tissue regeneration and mature osteochondral cell types in the BM repair, the discovery of their paracrine properties markedly increased the range of therapeutic under physiological conditions. Nestin+ applications for which they are currently studied. Systemic infusion of mesenchymal stromal cells has proved beneficial in different preclinical models of acute lung injury, myocardial infarction, BMderived MSCs could be cultured under diabetes and multiple sclerosis, as well as renal and hepatic failure74,78. Although the mechanisms nonadherent conditions and could be seri underlying the therapeutic effects of mesenchymal stromal cells in these disease models are not ally transplanted, therefore demonstrating a well characterized, they are thought to arise partially from the release of a combination of multiple robust selfrenewal capacity47. bioactive molecules with anti-inflammatory, antiproliferative, anti-apoptotic and angiogenic Together, these studies have convincingly properties (reviewed in ReF. 12). The current hypothesis is that paracrine factors secreted by shown the selfrenewing and differentiation mesenchymal stromal cells provide protective microenvironmental cues and promote repair by potential of a specific population of MSCs local tissue-resident progenitor populations, thereby explaining the detection of favourable effects in the BM. It remains to be determined 12,74,75 even in the absence of prolonged mesenchymal stromal cell engraftment at sites of injury . whether, and to what extent, the specificity These findings have prompted clinical studies on the therapeutic potential of mesenchymal of these markers and the functional charac stromal cells. For instance, the osteogenic properties of mesenchymal stromal cells have been used to treat children with osteogenesis imperfecta and have shown promising outcomes79,80. teristics of these BMresident MSCs can be On the basis of their immunoregulatory and tissue-protective properties, mesenchymal stromal used to describe MSC populations from cells are also being tested for the treatment and prevention of graft-versus-host disease, Crohn’s different adult tissues. disease and certain haematological malignancies78,81,82. Nevertheless, in most cases these studies are preliminary, and treatment efficacy has not been conclusively established. Some of the major Perivascular localization in vivo. A key problems that still need to be resolved concern the standardization of protocols for the isolation task for assessing the function of MSCs of mesenchymal stem cells and their expansion into mesenchymal stromal cells in vitro, the in vivo is to define their microanatomical safety of such cell-based therapies and the homing and engraftment of mesenchymal stromal localization in situ in diverse organs. efforts cells to their target tissues. to track the identity of tissueresident MSCs have consistently suggested that these cells lie adjacent to blood vessels48. evidence for Studying MSCs in vivo BM; these include the use of combinations such association came from initial observa After years of investigating MSCs out of of markers such as STRO1 and CD106 tions that pericytes (also known as Rouget their native context, little has been learned (ReFs 39,40), SSeA4 (also known as FUT4)41, cells or mural cells), which are defined by regarding the identity and function of their CD56, CD271, MSC antigen 1 (MSCA1) their perivascular location and morphology, precursors in vivo. It is important to note and D7FIB (a fibroblast or epithelial cell display MSClike features49. Pericyte that the fundamental biological properties marker)42–44. derived cultures are similar to mesenchymal of mesenchymal stromal cells are likely to Recent studies have provided valuable stromal cell cultures in terms of morphol be altered by culture conditions and thus insight into the identity and physiology of ogy and cellsurface antigen expression, and should not be directly ascribed to their pre BMresident MSCs by using new markers to can be induced to differentiate into not only sumed in vivo counterpart, as has often been track and purify MSCenriched populations osteo blasts, chondrocytes and adipocytes the case in the published literature. Progress and assay them in vivo. Of note, unlike the but also smooth muscle cells and myocytes in our understanding of bona fide MSCs haematopoietic system, in which haemato under appropriate conditions50–52. Cells largely relies on having the capacity to rec poietic reconstitution of myeloablated expressing some mesenchymal stromal cell ognize progenitor cells in situ, prospectively recipients is the gold standard to assess HSC markers were found to localize to blood isolate them and finally assay their multi selfrenewal and differentiation at a single vessel walls in human bone marrow and potency and selfrenewal capacity in vivo. cell level, no universally accepted assay has dental pulp53. Conversely, MSClike cultures been established to date to probe for the were generated from cells enriched directly Prospective isolation, multipotency and activity of MSCs in vivo. from tissues based on expression of pericyte self-renewal. The unequivocal identification Using the capacity to form bone and specific markers54. However, evidence that of MSCs in vivo has been hindered by their assemble a functional BM stroma at hetero pericytes and MSCs are biologically equiva extremely low frequency in tissues38 and topic sites as an indication of MSC potential lent has remained indirect for a long time. the lack of a distinct MSCspecific immuno in vivo, one group identified a population A recent study identified a combination phenotype to enable their iso lation. Indeed, of CD146+ perivascular selfrenewing of markers, such as NG2 (also known as cultured human mesenchymal stromal osteoprogenitors that were present in the CSPG4), CD146 and PDGFRβ, that seemed cells express a panel of cell surface markers outermost connective tissue layer covering to specifically label pericytes in a range of (such as CD73, CD90 and CD105) and lack BM microvessels (known as the adventitia)45. human organs, including fetal and adult endothelial or haematopoietic cell markers Furthermore, in mice combined expression skin, pancreas, heart, brain, lung, bone (CD31, CD34 and CD45)16. Nevertheless, of surface cell antigen 1 (SCA1) and platelet marrow and placenta. longterm cultures these are not homogeneously expressed derived growth factor receptorα (PDGFRα) derived from prospective pericytes isolated throughout stromal cultures and they specifies a subset of nonhaematopoietic directly from these organs, based on specific vary with isolation protocols and passage; cells that resides close to arteries and expression of those markers, displayed simi therefore, they are not necessarily repre gives rise to osteoblasts, reticular cells and lar morphological features to cultured mes sentative of MSCs in vivo. Several labelling adipo cytes in vivo upon transplantation enchymal stromal cells, as well as trilineage strategies have been used to successfully into an irradiated recipient46. Finally, the potential in vitro and osteogenic potential enrich for CFUFs in human and mouse neural stem cell marker nestin was recently in vivo55.

Collectively, these results strongly suggest C $QPGOCTTQY D 5KPWUQKF that the precursors of cultured mesenchyma l *5% Dendritic stromal cells preferentially reside close #FKRQE[VG cell to blood vessels in vivo, a trait that is not 1UVGQRTQIGPKVQT 6EGNN unique to MSCs but that is pertinent to other 2TGQUVGQDNCUV /5% /5% multipotent stem or progenitor cells present $EGNN in adult tissues. In this respect, HSCs (dis cussed below), as well as white fat progenitor *5% %#4% 0CVWTCN cells and skeletal muscle stem cells (other MKNNGTEGNN illdefined tissue progenitors), have been reported to reside in perivascular spaces of 1UVGQDNCUV bone marrow, adipose tissue and skeleta l $QPG muscle microvessels, respectively56–59. Figure 2 | Proposed biological functions of BM-resident MSCs in vivo. a | Bone marrow (BM)- Nevertheless, it is important to note that the derived mesenchymal stem cells (MSCs) differentiate into osteoblasts,0CVWTG4G XKGYUadipocytes^/QNGEWNCT%GNN$KQNQI and reticular cells[ terms pericyte and MSC are not equivalent (indicated by bold arrows), which provide the supportive environment for haematopoietic develop- or interchangeable. Although widely used ment and are thought to be responsible for the natural turnover of these mesenchymal cell types in to refer to cells surrounding blood vessels, the bone marrow. Osteoblasts are key components of haematopoietic stem cell (HSC) niches and have the word pericyte strictly refers to cells been proposed to directly interact with, and positively regulate quiescence of, some HSCs in the BM, whereas adipocytes negatively regulate HSC activity. HSCs have also been shown to lie adjacent to adjacent to capillaries and postcapillary 49 CXCL12-abundant reticular cells (CARCs), which are poorly characterized cells with adipogenic and venules ; however, multipotent MSClike osteogenic potential and may correspond to, or originate from, BM-resident MSCs. In addition to giving precursors have been isolated from the walls rise to a haemosupportive environment, BM-resident MSCs expressing the neural stem cell marker of other vascular types, including arteries nestin have been shown to physically associate with HSCs in perivascular BM ‘dual stem cell niches’ and and veins60,61. Furthermore, because peri to regulate HSC homeostasis. b | BM-resident MSCs are found in perivascular areas of BM microenvi- cytes show an extensive tissue distribution ronments, where they may associate with cells of the immune system, including dendritic cells, T cells along diverse microvascular beds and have and B cells. Furthermore, mesenchymal stromal cells, which are thought to directly derive from MSCs many proposed functions (including vessel in vivo, are known to regulate the function of lymphocytes (B cells and T cells), dendritic cells and stabil ization, phagocytosis and regulation of natural killer cells. It is therefore thought that BM-resident MSCs may regulate immune responses vascular integrity and tone49), it is likely that occurring in the BM in vivo. functionally heterogeneous, nonequivalent cell subsets are included under the vague term pericyte. Thus, despite being peri Osteoblasts have been postulated to crucially the adherent fraction of BM stroma promote vascular, not all MSCs can be referred to contribute to HSC niches and to regulate survival and proliferation of HSCs ex vivo67. as pericytes, and not all pericytes exhibit HSC homeostasis through direct celltocell In addition, MSCs are isolated from all fetal MSCspecific properties. interactions62,63. Although the existence of an haematopoietic sites even before the HSC osteoblastic HSC niche is controversial64, it colonization of those tissues10,68. It is there MSCs and haematopoiesis seems clear that, either directly or through fore thought that MSCs have a key role in the The stromal compartment of the BM the secretion of soluble factors, osteoblasts organization of HSC niches, either through was the first biological material from are essential constituents of the BM micro direct interaction or, as proposed in ReF. 13, which MSCs were isolated. Since then, environment and have regulatory roles at through their reported ability to organize BMderived MSCs have been the most many stages of haematopoietic development vascular networks, which are key structural widely studied and are the best character (reviewed in ReF. 65). The BM stroma is and functional components of haemato ized, and they are now thought to be key also composed of MSCderived adipocytes, poietic sites45,69. Indeed, the existence of a regulators of BM physiology (FIG. 2). which function as negative regulators of ‘dual stem cell niche’, in which MSCs and early haematopoietic progenitors through HSCs directly interact in perivascular spaces Precursors of the haematopoietic microenvi- unknown molecular mechanisms66. of the BM, is conceptually attractive and has ronment. During adulthood, the sustained Hence, MSCs are the source of two been proposed in two recent publications. production of blood cells occurs primarily in coexisting mature cell types with apparently HSCs have been shown to colocalize with a the BM. MSCs have long been proposed to antagonistic properties on HSCs. Many open subset of poorly characterized fibroblastic be the in vivo precursors of some of the non questions remain concerning the precise reticular cells that are defined by high expres haematopoietic components of the BM that developmental stages that MSCs undergo sion of CXC chemokine ligand 12 (CXCl12; regulate haematopoiesis, such as osteoblasts, during differentiation in situ, the pathways also known as SDF1) and are known as adipocytes and fibroblastic reticular cells2. governing lineage commitment decisions CXCl12abundant reticular cells (CARCs)70. Consequently, MSCs are likely to contribute in vivo and the global impact of the balance These cells have osteogenic and adipogenic to the homeostasis of the haematopoietic of osteoblast and adipocyte production in potential and might therefore correspond compartment in vivo through the regulatory haematopoietic environments (FIG. 2). to, or immediately derive from, MSCs71. properties of their mature progeny (FIG. 2). Moreover, an association between HSCs Inside the BM microenvironment, HSCs HSC niche components. Multipotent, and BMresident MSCs has been visualized are thought to reside in confined niches, im mature BMresident MSCs have long using mice expressing nestin tagged with which are created by surrounding cells, solu been proposed to provide modulatory sig green fluorescent protein (nestin–GFP)47. ble factors and extracellular matrix proteins nals to haematopoietic progenitors, based Selective depletion of CARCs or nestin– that ultimately promote HSC maintenance. on the fact that mixed cultures derived from GFP+ MSCs, both of which express high

levels of HSCregulatory factors, had a direct therapeutic potential (BOX 2) rapidly pro 5. Pittenger, M. F. et al. Multilineage potential of adult human mesenchymal stem cells. Science 284, impact on HSC numbers and homeostasis, liferated, whereas fundamental biological 143–147 (1999). further indicating a role for BMresident questions regarding their in vivo counterpart 6. Caplan, A. I. Mesenchymal stem cells. J. Orthop. Res. 9, 641–650 (1991). 47,71 MSCs in HSC biology . Notably, nestin– populations remained largely unanswered. 7. Prockop, D. J. Marrow stromal cells as stem cells for GFP+ BMresident MSCs are directly In our view, the term MSC is misleading, nonhematopoietic tissues. Science 276, 71–74 (1997). innervated and respond to signals from the in that it has been widely used to refer to 8. da Silva Meirelles, L., Chagastelles, P. C. & Nardi, N. B. sympathetic nervous system (SNS)47, thus a heterogeneous pool of tissuespecific Mesenchymal stem cells reside in virtually all postnatal organs and tissues. J. Cell Sci. 119, 2204–2213 providing a link by which HSC homeostasis multipotent perivascular progenitors, which (2006). is regulated by the SNS, as had been pre are likely to possess diverse in vivo functions 9. Kuznetsov, S. A. et al. Circulating skeletal stem cells. J. Cell Biol. 153, 1133–1140 (2001). 72,73 viously demonstrated . Further studies are and differentiation potential but have similar 10. Mendes, S. C., Robin, C. & Dzierzak, E. Mesenchymal needed to fully understand where and how features after in vitro culture. Among these progenitor cells localize within hematopoietic sites throughout ontogeny. Development 132, 1127–1136 HSCs, MSCs and the SNS interact. progenitors, the only wellcharacterized cells, (2005). in terms of biological properties and in vivo 11. Javazon, E. H., Beggs, K. J. & Flake, A. W. Mesenchymal stem cells: paradoxes of passaging. Immunomodulatory agents. One of the stem cell features, are BMresident MSCs, Exp. Hematol. 32, 414–425 (2004). most remarkable and unforeseen aspects of which sustain the homeostatic turnover of 12. Meirelles Lda, S., Fontes, A. M., Covas, D. T. & Caplan, A. I. Mechanisms involved in the therapeutic mesenchymal stromal cells pertains to their skeletal cell types in the BM in vivo. properties of mesenchymal stem cells. Cytokine immunomodulatory activity (reviewed in Major challenges at hand are to define Growth Factor Rev. 20, 419–427 (2009). 13. Bianco, P., Robey, P. G., Saggio, I. & Riminucci, M. ReFs 74,75). In vitro, mesenchymal stromal the biological equivalence and hierarchical “Mesenchymal” stem cells in human bone marrow cells inhibit T cell activation, dendritic cell relationships between progenitors in diverse (skeletal stem cells): a critical discussion of their nature, identity, and significance in incurable skeletal differentiation and B cell proliferation, and anatomical locations, understand their disease. Hum. Gene Ther. 21, 1057–1066 (2010). impair the cytolytic potential of natural killer developmental origin, characterize their 14. Bianco, P., Robey, P. G. & Simmons, P. J. Mesenchymal stem cells: revisiting history, concepts, and assays. cells. Immunosuppression after MSC infu multipotential capabilities and elucidate Cell Stem Cell 2, 313–319 (2008). sion in vivo has also been documented in their in vivo roles during homeostasis and 15. Horwitz, E. M. et al. Clarification of the nomenclature 12,74 for MSC: The International Society for Cellular Therapy diverse animal models of disease . These tissue repair. Resolving these problems position statement. Cytotherapy 7, 393–395 effects are partially explained by the ability will require comprehensive experimental (2005). 16. Dominici, M. et al. Minimal criteria for defining of mesenchymal stromal cells to secrete approaches, including the use of stringent multipotent mesenchymal stromal cells. The a vast array of soluble mediators, some of in vivo assays to define the multipotency of International Society for Cellular Therapy position statement. Cytotherapy 8, 315–317 (2006). which have immunomodulatory properties: MSC populations, advanced in vivo micro 17. Haynesworth, S. E., Goshima, J., Goldberg, V. M. & for example, interleukin10 (Il10), prosta scopy techniques to track their distribution Caplan, A. I. Characterization of cells with osteogenic potential from human marrow. 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Stem Cells 27, soidal locations with dendritic cells and 577–605 (2009). 23. Arthur, A., Rychkov, G., Shi, S., Koblar, S. A. & circulating B cells76,77, it seems plausible that Jerome Ritz and Leslie E. Silberstein are also at the Center for Human Cell Therapy, Division of Molecular Gronthos, S. Adult human dental pulp stem cells MSC–immune cell interactions may be of differentiate toward functionally active neurons under and Cellular Medicine, Children’s Hospital Boston, appropriate environmental cues. Stem Cells 26, physiological relevance, a possibility that Karp Research Building, One Blackfan Circle, 1787–1795 (2008). merits further investigation (FIG. 2). Boston, Massachusetts 02115, USA. 24. Phinney, D. G. & Prockop, D. J. Concise review: mesenchymal stem/multipotent stromal cells: the Correspondence to L.E.S. state of transdifferentiation and modes of tissue Concluding remarks e-mail: [email protected] repair — current views. Stem Cells 25, 2896–2902 (2007). 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