Mesenchymal stem cells in health and disease
Antonio Uccelli*‡§, Lorenzo Moretta||‡¶ and Vito Pistoia||
Abstract | Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from many adult tissues. They can differentiate into cells of the mesodermal lineage, such as adipocytes, osteocytes and chondrocytes, as well as cells of other embryonic lineages. MSCs can interact with cells of both the innate and adaptive immune systems, leading to the modulation of several effector functions. After in vivo administration, MSCs induce peripheral tolerance and migrate to injured tissues, where they can inhibit the release of pro-inflammatory cytokines and promote the survival of damaged cells. This Review discusses the targets and mechanisms of MSC-mediated immunomodulation and the possible translation of MSCs to new therapeutic approaches.
Cells of non-lymphoid origin that form the framework of each organ. By expressing various molecules, these cells can support the adhesion, proliferation and survival of distinct cell subsets.
*Department of Neurosciences, Ophthalmology and Genetics, University of Genoa, Italy. ‡ Centre of Excellence for Biomedical Research, University of Genoa, Italy. § Advanced Biotechnology Center (ABC), Genoa, Italy. || IRCSS Giannina Gaslini, Genoa, Italy. ¶ Department of Experimental Medicine (DIMES), University of Genoa, Italy. Correspondence to A.U. e‑mail: email@example.com doi:10.1038/nri2395 Published online 18 August 2008
Friedenstein was the first person to identify multipotential stromal precursor cells; he described the isolation, from the bone marrow, of spindle-shaped, clonogenic cells in monolayer cultures, which he defined as colony-forming unit fibroblasts (CFU-Fs). CFU-F-derived stromal cells can serve as feeder layers for the culture of haematopoietic stem cells (HSCs) and they can differentiate into adipocytes, chondrocytes and osteocytes both in vitro and after transfer in vivo1. Friedenstein’s original observation was the basis of further studies showing that bone-marrow-derived stromal cells are the common predecessors of mesenchymal tissues. Recently, several studies have reported that multipotential stromal precursor cells can also differentiate into cells from unrelated germline lineages (in a process known as transdifferentiation)2,3 (BOX 1; FIG. 1). As a result of their supposed capacity for self-renewal and differentiation, bone-marrow-derived stromal cells were first considered as stem cells by Caplan and named mesenchymal stem cells (MSCs)4. However, in the bone marrow, stromal cells are a rare and heterogeneous population of cells that contain a mixture of progenitors at different stages of commitment to the mesodermal lineage and only a very small number of multipotential self-renewing stem cells, which have recently been identified as sub-endothelial cells that express CD146 (also known as MCAM)5. It is now accepted that most bone-marrow-derived progenitor stromal cells can be considered, after in vitro proliferation, to be MSCs6. In addition to their potential for tissue repair, MSCs have been shown recently to have potent anti-proliferative
and anti-inflammatory effects. In this Review, we discuss the functional features of bone-marrow-derived MSCs, describe their mechanisms of action and elucidate how these findings could be translated to the clinical setting.
Definition of mesenchymal stem cells MSCs, which can alternatively be defined as multipotent mesenchymal stromal cells, are a heterogeneous population of cells that proliferate in vitro as plastic-adherent cells, have fibroblast-like morphology, form colonies in vitro and can differentiate into bone, cartilage and fat cells6 (FIG. 1). Although stromal cells that apparently fulfill these criteria for an MSC have been isolated from almost every type of connective tissue7, MSCs have mainly been characterized after isolation from the bone marrow. Therefore, in this Review, we focus on bone-marrow-derived MSCs. MSCs that are cultured in vitro lack specific and unique markers. There is a general consensus that human MSCs do not express the haematopoietic markers CD45, CD34 and CD14 or the co-stimulatory molecules CD80, CD86 and CD40, whereas they do express variable levels of CD105 (also known as endoglin), CD73 (ecto-5′nucleotidase), CD44, CD90 (THY1), CD71 (transferrin receptor), the ganglioside GD2 and CD271 (low-affinity nerve growth factor receptor), and they are recognized by the monoclonal antibody STRO-1. The variable expression level of these markers that has been observed probably arises from species differences, tissue source and culture conditions.
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These are embryonic tissues of mesodermal origin, consisting of loosely packed, unspecialized cells set in a gelatinous ground substance, from which connective tissue, bone, cartilage and the circulatory and lymphatic systems develop.
Box 1 | The embryonic origin of MSCs might explain their differentiation pathways
Mesenchymal stem cells (MSCs) can differentiate into cells of the mesodermal lineage, such as bone, fat and cartilage cells, but they also have endodermic107 and neuroectodermic differentiation potential2,3 (FIG. 1). This capacity could be explained by the developmental origin of mesenchymal tissues, which includes the mesoderm and, to a lesser extent, the cranial neural crest. Although adult MSCs are commonly considered to be of mesodermal origin108, embryonic MSCs have recently been shown to derive mainly from the neuroepithelium and neural crest109. Indeed, bone-marrow-derived stromal cells are not a homogeneous population, as reflected by their complex transcriptome, which encodes a wide range of proteins involved in different developmental pathways and in a large number of diverse biological processes110,111. Despite evidence that MSCs can transdifferentiate into multiple cell types in vitro and in vivo, the real contribution of MSCs to tissue repair, through significant engraftment and differentiation into biologically and functionally relevant tissue-specific cell types, is still unclear112. The lack of consistent transdifferentiation in vivo might be a result of the limited number of mesenchymal precursor cells derived from non-mesodermal embryonic lineages, as recently indicated by the rapid decrease in the number of MSCs of neuroepithelial origin in the adult bone marrow109. We can assume that in post-natal life, the relative importance of MSCs derived from other developmental lineages decreases due to the increasing importance of mesodermal MSCs. So, although it has been proposed that MSCs could be used for the regeneration of almost any tissue, current scientific evidence supports their use for tissue reconstruction through exclusive differentiation mechanisms only for bone repair113.
The ability of a non-stem cell to transform into a different type of cell lineage, or when an already partially differentiated stem cell transforms into a different cell lineage or type.
A subset of cells that has a self-renewing capacity and under appropriate conditions can give rise to several mature cell lineages.
In animals with three tissue layers, the mesoderm is the middle layer of tissue, between the ectoderm and the endoderm. In vertebrates, the mesoderm forms the skeleton, muscles, heart, spleen and many other internal organs.
The role of MSCs in physiology Formation of HSC niches in the bone marrow. After transplantation into the bone marrow of non-obese diabetic–severe combined immunodeficiency (NOD– SCID) mice, MSCs have been shown to differentiate into pericytes, myofibroblasts, bone-marrow stromal cells, osteocytes, osteoblasts and endothelial cells, all of which constitute the functional components of the HSC niche that support haematopoiesis8. The developing haematopoietic cells are retained in a quiescent state in the bone marrow until, after the appropriate stimulation, they differentiate and are then released in the sinusoidal vascular system. In the bone marrow, the niche stromal cells surround
the HSCs and their progeny9 (FIG. 2). Two types of niche have been described in rodents. The ‘endosteal niche’ is formed by osteoblasts that line the endosteal surface of the trabecular bone, and the ‘vascular niche’ is composed of endothelial cells and CD146+ sub-endothelial stromal cells that lie at the abluminal side of bone-marrow sinusoids5. Stromal cells in both types of niche provide a sheltering microenvironment that supports the maintenance and self-renewal of HSCs by shielding them from differentiation and apoptotic stimuli that would otherwise challenge stem-cell reserves. Moreover, the niche also controls the proliferation and differentiation of HSCs and the release of mature progeny into the vascular system. The regulation of HSC quiescence, through the maintenance of HSCs in the G0 phase of the cell cycle in the endosteal niche, and the control of HSC proliferation, differentiation and recruitment in the vascular niche can be ascribed to bone-marrow stromal cells10,11 (FIG. 2). Anti-proliferative activity. Stromal-cell progenitors can preserve the HSC pool in the bone marrow by maintaining HSCs in a quiescent state; in addition, terminally differentiated stromal cells such as fibroblasts and chondrocytes seem to share anti-proliferative effects with their predecessors, as shown by their ability to inhibit T-cell proliferation12,13. Fibroblast-mediated modulation of T-cell responses is triggered by interferon-γ (IFNγ)14, which indicates that stromal cells in connective tissues might be involved in the homeostasis of peripheral leukocyte populations15. These results support the hypothesis that stromal cells, at all stages of maturation, have antiproliferative features that are in common with physiological stromal-cell niches, including the HSC niche.
Neuron Connective stromal cell Cartilage cell Mesoderm MSC Fat cell Bone cell
Muscle cell Osteoblast Gut epithelial cell Lung cell Endoderm
Figure 1 | The multipotentiality of MSCs. This figure shows the ability of mesenchymal stem cells (MSCs) in the bone-marrow cavity to self-renew (curved arrow) and to Nature Reviews | Immunology differentiate (straight, solid arrows) towards the mesodermal lineage. The reported ability to transdifferentiate into cells of other lineages (ectoderm and endoderm) is shown by dashed arrows, as transdifferentiation is controversial in vivo.
The effects of MSCs on immune cells The immunomodulatory effect of MSCs has only been described recently, following the observation that bone-marrow-derived MSCs suppressed T-cell proliferation16,17. These studies redirected the attention of scientists away from the multipotentiality of MSCs towards their possible regulatory effects on immune cells, and they paved the way for the characterization of the broad immunoregulatory activities of MSCs.
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HSC in G0
Stromal progenitor cell
HSC CD146+ sub-endothelial stromal cell
Sinusoid Mobilized HSC Sinusoidal endothelial cell Vascular niche
Figure 2 | Stromal cells in the haematopoietic-stem-cell niche. In the bone marrow of trabecular bones, multipotential stromal progenitor cells at different stages of maturation contribute to the formation of the haematopoietic stem cell Nature Reviews | Immunology (HSC) niche. In the endosteal niche, stromal progenitor cells, together with osteoblasts, contribute to the maintenance of HSCs in a quiescent state (G0 phase of the cell cycle). Around sinusoids, angiopoietin-1 (ANG1)+CXC-chemokine ligand 12 (CXCL12)+CD146+ sub-endothelial stromal cells, perivascular stromal cells and sinusoidal endothelial cells also regulate HSC maintenance and control HSC proliferation, differentiation and recruitment to the vascular niche. The endosteal niche also contains self-renewing (dividing) HSCs and mobilized HSCs that are recruited to the vascular niche following proper activation.
The microenvironment inside the trabecular bone cavity, which is composed of a specialized population of cells that has an essential role in regulating the self-renewal and differentiation of haematopoietic stem cells (HSCs).
The commitment and differentiation processes that lead from a haematopoietic stem cell to the production of mature cells of all blood lineages — erythrocytes, myeloid cells (macrophages, mast cells, neutrophils and eosinophils), B and T cells, and natural killer cells.
Blood-filled spaces that lack the anatomy of a capillary. Sinusoids generally contain slow-flowing blood, which facilitates cellular interactions. Such vessels are found in the bone marrow and in the liver.
Innate immunity. Myeloid dendritic cells (DCs) have a fundamental role in antigen presentation to naive T cells following DC maturation, which can be induced by proinflammatory cytokines and/or pathogen-associated molecules. During maturation, immature DCs acquire the expression of co-stimulatory molecules and upregulate expression of MHC class I and class II molecules together with other cell-surface markers (such as CD11c and CD83). MSCs have been shown to inhibit the maturation of monocytes, and cord-blood and CD34+ haematopoietic progenitor cells into DCs in vitro18–21 (FIG. 3) . Furthermore, mature DCs incubated with MSCs have decreased cell-surface expression of MHC class II molecules, CD11c, CD83 and co-stimulatory molecules, as well as decreased interleukin-12 (Il-12) production, thereby impairing the antigen-presenting function of the DCs18,20,22–24. MSCs can also decrease the pro-inflammatory potential of DCs by inhibiting their production of tumour-necrosis factor (TNF)22. Furthermore, plasmacytoid DCs (pDCs), which are specialized cells for the production of high levels of type I IFN in response to microbial stimuli, upregulate production of the anti-inflammatory cytokine Il-10 after incubation with MSCs22. Therefore, it is tempting to speculate that the combined effects of MSCs on DCs and pDCs, as described in vitro, could translate into potent anti-inflammatory and immunoregulatory effects in vivo.
Natural killer (NK) cells are important effector cells of innate immunity and they have a key role in antiviral and anti-tumour immune responses owing to their cytolytic activity and production of pro-inflammatory cytokines25. The function of NK cells is tightly regulated by cell-surface receptors that transduce either inhibitory or activating signals. NK-cell-mediated lysis of target cells requires the expression of ligand(s) by the target cell that are recognized by activating NK-cell receptors, together with low-level to absent expression of MHC class I molecules by the target cell, which are recognized by the MHC-class-I-specific inhibitory receptors of NK cells26,27. MSCs can inhibit the cytotoxic activity of resting NK cells by downregulating expression of NKp30 and natural-killer group 2, member D (NKG2D), which are activating receptors that are involved in NK-cell activation and target-cell killing28 (FIG. 3). Freshly isolated, resting NK cells proliferate and acquire strong cytotoxic activity after culture with Il-2 or Il-15. However, when resting NK cells are incubated with these cytokines in the presence of MSCs, NK-cell proliferation and IFNγ production are almost completely abrogated28,29. Similar to resting NK cells, pre-activated NK cells had decreased proliferation, IFNγ production and cytotoxicity after culture with MSCs in vitro22,28–32. However, when the susceptibility of NK cells to MSCmediated inhibition of proliferation was compared, preactivated NK cells were found to be more resistant to the effects of MSCs than were resting NK cells28. Conversely, both autologous and allogeneic MSCs have been shown to be killed by cytokine-activated, but not resting, NK cells in vitro28,30,31 (FIG. 3). The susceptibility of human MSCs to NK-cell-mediated cytotoxicity depends on the low level of cell-surface expression of MHC class I molecules by MSCs and the expression of several ligands that are recognized by activating NK-cell receptors29. Incubation of MSCs with IFNγ partially protected them from NK-cell-mediated cytotoxicity through the upregulation of expression of MHC class I molecules by MSCs28. Together, these findings support the possibility that, following encounter with MSCs in vivo, activated NK cells could undergo limited functional inhibition that does not compromise their ability to kill MSCs. As IFNγ protects MSCs from NK-cell-mediated lysis28, a microenvironment rich in IFNγ might favour the inhibition of NK-cell function by MSCs, whereas in the absence of IFNγ, the balance would be tilted towards the elimination of MSCs by activated NK cells. However, the in vivo relevance of these interactions might be limited only to cases of MSC transplantation. Neutrophils are another important cell type of innate immunity that, in the course of bacterial infections, are rapidly mobilized and activated to kill microorganisms. After binding to bacterial products, neutrophils undergo a process known as the respiratory burst. MSCs have been shown to dampen the respiratory burst and to delay the spontaneous apoptosis of resting and activated neutrophils through an Il-6-dependent mechanism33 (FIG. 3). previous studies have established a link between the downregulation of the respiratory burst and an increase
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Resting NK cell
burst and apoptosis
PGE2 IDO sHLA-G5 MSC PVR or nectin-2 DNAM1 ULBP3 or MICA NKG2D
Loss of DC-stimulated NK-cell activation
IL-6 PGE2, IDO, TGFβ1, HGF, iNOS and HO1
TNF Monocyte pDC CD4+ T cell CD8+ T cell B cell Regulatory T cell
Activated NK cell
Killing Immature DC
Defective antigen presentation
Figure 3 | Possible mechanisms of the interactions between MSCs and cells of the innate and adaptive immune systems. a | Mesenchymal stem cells (MSCs) can inhibit the proliferation and cytotoxicity of resting natural Nature E2 (PGE killer (NK) cells and their cytokine production in vitro. These effects are mediated by prostaglandin Reviews2|),Immunology indoleamine 2,3-dioxygenase (IDO) and soluble HLA-G5 (sHLA-G5) released by MSCs. Killing of MSCs by cytokine-activated NK cells involves the engagement of NKG2D (natural-killer group 2, member D) expressed by NK cells with its ligands ULBP3 (UL16-binding protein 3) or MICA (MHC class I polypeptide-related sequence A) expressed by MSCs, and of NK-cell-associated DNAM1 (DNAX accessory molecule 1) with MSC-associated PVR (poliovirus receptor) or nectin-2. b | MSCs inhibit the differentiation of monocytes to immature myeloid dendritic cells (DCs), skew mature DCs to an immature DC state, inhibit tumour-necrosis factor (TNF) production by DCs and increase interleukin-10 (IL-10) production by plasmacytoid DCs (pDCs). MSC-derived PGE2 is involved in all of these effects. The immature DCs are susceptible to killing by cytokine-activated NK cells. The effect of MSCs on DCs impairs the stimulatory effect of mature DCs on resting NK cells and compromises antigen presentation to T cells, which cannot then undergo proliferation and clonal expansion. Finally, MSCs dampen the respiratory burst and delay the spontaneous apoptosis of neutrophils by constitutively releasing IL-6. c | Direct inhibition of CD4+ T-cell function depends on the release by MSCs of several soluble molecules, including PGE2, IDO, transforming growth factor-β1 (TGFβ1), hepatocyte growth factor (HGF), inducible nitric-oxide synthase (iNOS) and haem oxygenase-1 (HO1). Defective CD4+ T-cell activation impairs helper function for B-cell proliferation and antibody production. The inhibition of CD8+ T-cell cytotoxicity and of the differentiation of regulatory T cells mediated directly by MSCs are related to the release of sHLA-G5 by MSCs. In addition, the upregulation of IL-10 production by pDCs results in the increased generation of regulatory T cells through an indirect mechanism. MSC-driven inhibition of B-cell function seems to depend on soluble factors and cell–cell contact, but little is known about the identity of the molecules involved.
A large increase in oxygen consumption and the generation of reactive oxygen species that accompanies the exposure of neutrophils to microorganisms and/or inflammatory mediators.
in the life span of neutrophils34. MSC-mediated preservation of resting neutrophils might be important in those anatomical sites where large numbers of mature and functional neutrophils are stored, such as the bone marrow and lungs35. Adaptive immunity. After T-cell receptor (TCR) engagement, T cells proliferate and exert several effector functions, including cytokine release and, in the case of CD8+ T cells, cytotoxicity. The proliferation of T cells stimulated with polyclonal mitogens, allogeneic cells or specific antigen is inhibited by MSCs16,17,22,36–45 (FIG. 3). This inhibition is not MHC restricted as it can be mediated by both autologous and allogeneic MSCs. MSCmediated inhibition of T-cell proliferation depends on the arrest of T cells in the G0/G1 phase of the cell cycle41,45. MSCs do not promote T-cell apoptosis, but instead support the survival of T cells that are subjected to overstimulation through the TCR and are committed
Activation-induced cell death
A process by which activated, T-cell-receptor-restimulated T cells undergo cell death after engagement of cell-death receptors, such as CD95 or the tumour-necrosis factor receptor, or after exposure to reactive oxygen species.
to undergo CD95–CD95-ligand-dependent activationinduced cell death45. The MSC-mediated anti-proliferative effect on T cells is associated with the survival of T cells in a state of quiescence that can be partially reversed by Il-2 stimulation38. Inhibition of T-cell proliferation by MSCs has been reported to lead to decreased IFNγ production both in vitro22 and in vivo38 and to increased Il-4 production by T helper 2 (TH2) cells, which indicates a shift in T cells from a pro-inflammatory (IFNγ-producing) state to an anti-inflammatory (Il-4-producing) state22. An important T-cell effector function is the MHCrestricted killing of virus-infected or allogeneic cells, which is mediated mainly by CD8+ cytotoxic T lymphocytes (CTls). MSCs have been shown to downregulate CTl-mediated cytotoxicity46 (FIG. 3). Human MSCs pulsed with viral peptides or transfected with mRNA from tumour cells were protected from lysis by CTls in vitro. pre-treatment with IFNγ increased the cell-surface
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expression of MHC class I molecules by MSCs but was ineffective at restoring CTl-mediated killing47,48, which indicates that although MSCs inhibit CTl activity they are not CTl targets. Regulatory T cells are a specialized subpopulation of T cells that suppress activation of the immune system and thereby help to maintain homeostasis and tolerance to self antigens. MSCs have been reported to induce the production of Il-10 by pDCs, which, in turn, triggered the generation of regulatory T cells22,24. In addition, after co-culture with antigen-specific T cells, MSCs can directly induce the proliferation of regulatory T cells through release of the immunomodulatory HLA-G isoform HlA-G5 (ReF. 32) (FIG. 3). Taken together, these findings indicate that MSCs can modulate the intensity of an immune response by inhibiting antigen-specific T-cell proliferation and cytotoxicity and promoting the generation of regulatory T cells. In principle, from a clinical perspective, excessive inhibition of T-cell responses by MSCs would render the host vulnerable to infectious agents. However, fail-safe mechanisms might exist; for example, MSCs express functional Toll-like receptors (TlRs) that, after interaction with pathogen-associated ligands, induce the proliferation, differentiation and migration of MSCs and their secretion of chemokines and cytokines49–51, and it has been shown that MSCs lose the ability to inhibit T-cell proliferation due to impaired Notch signalling after triggering of TlR3 and TlR4 (ReF. 52). Therefore, it is possible that pathogenassociated molecules might reverse the suppressive effects of MSCs on T cells, thereby restoring efficient T-cell responses to pathogens52, but it is also possible that tissue stromal cells can instruct local immune responses after pathogen infections53. The second main cell type involved in adaptive immune responses is b cells, which are specialized for antibody production. Studies of the interactions between MSCs and b cells have produced different results, possibly as a result of the experimental conditions used41,54–57. Most published works to date indicate that MSCs inhibit b-cell proliferation in vitro41,54,56 (FIG. 3). MSCs can also inhibit b-cell differentiation and the constitutive expression of chemokine receptors56. These effects seem to depend on the release of soluble factors56 and on cell–cell contact, possibly mediated by interactions between programmed cell death 1 (pD1) and its ligands54. However, other in vitro studies have shown that MSCs could support the survival, proliferation and differentiation to antibody-secreting cells of b cells from normal individuals57,58 and from paediatric patients with systemic lupus erythematosus57. Regardless of the controversial in vitro effects, it should be emphasized that b-cell responses are mainly T-cell dependent and therefore the final outcome of the interaction between MSCs and b cells in vivo might be significantly influenced by the MSC-mediated inhibition of T-cell functions. Such an assumption is supported by the results of a study of experimental autoimmune encephalomyelitis (eAe) in mice injected with a proteolipid protein (plp) peptide, which is a model of multiple sclerosis. In this model, the production of antigen-specific antibodies in vivo was inhibited by the infusion of MSCs, in addition to a significant downregulation of plp-specific T-cell responses, which indicates that the two events were closely linked59. paradoxically, despite their broad immunosuppressive activities, it is possible that MSCs could function as non-professional antigen-presenting cells (ApCs). low concentrations of IFNγ upregulate the expression of MHC class II molecules by MSCs, which indicates that they could act as ApCs early in an immune response when the levels of IFNγ are low60,61. However, this upregulation of MHC expression by MSCs, together with the ApC function, was progressively lost as IFNγ concentrations increased. Such a mechanism could allow MSCs to function as conditional ApCs in the early phase of an immune response and later switch their function to immunosuppression60. Most of the immunomodulatory activities of MSCs described here have been documented by in vitro experiments. As MSCs are derived from stromal progenitor cells that reside in the bone marrow, their potential role in the control of physiological immune responses is unknown, despite the fact that the bone marrow might be a site for the induction of T-cell responses to blood-borne antigens62. However, it is possible that MSC-mediated modulation of immune responses could occur in vivo following the infusion of in vitro-cultured MSCs after transplantation. If this hypothesis is correct, then infused MSCs could interfere with the interactions between DCs and NK cells. Mature DCs stimulate the proliferation and cytotocixity of NK cells and their cytokine production, whereas immature DCs are killed by NK cells25. The dual immunosuppressive effects of MSCs on DCs and resting NK cells could result in the accumulation of immature DCs in vivo that are potentially amenable to NK-cellmediated elimination, but also in the inhibition of NK-cell proliferation, cytotoxicity and cytokine production (FIG. 3). However, as discussed earlier, activated NK cells can kill MSCs. Therefore, the functional outcome in vivo would be determined by the cytokine microenvironment in which tripartite NK-cell–DC–MSC interactions take place. In the absence of IFNγ, activated NK cells could kill both immature DCs and MSCs. by contrast, in an IFNγ-enriched milieu, MSC-mediated inhibition of immune cells would prevail and target both DCs and NK cells. Such interactions between MSCs and immune cells might occur in vivo after MSC transplantation, but it should also be emphasized that the modulation of DC differentiation and function by tissue stromal cells could be viewed as an important mechanism that regulates a local immune response53. As DCs are the main ApCs for T-cell responses, MSC-mediated suppression of DC maturation would preclude efficient antigen presentation to and therefore the clonal expansion of T cells (FIG. 3). Direct interactions of MSCs with T cells in vivo could lead to the arrest of T-cell proliferation, inhibition of CTl-mediated cytotoxicity and generation of CD4+ regulatory T cells. As a consequence, impaired CD4+ T-cell activation would translate into defective T-cell help for b-cell proliferation and differentiation to antibody-secreting cells. These
A non-classical MHC class Ib molecule that is involved in the establishment of immune tolerance at the maternal–fetal interface, the major soluble isoforms of which are HLA-G1 and HLA-G5.
A signalling system comprising highly conserved transmembrane receptors that regulate cell-fate choice in the development of many cell lineages, and so are crucial for the regulation of embryonic differentiation and development.
A term that denotes both proliferating plasmablasts and non-proliferating plasma cells. The term is used when both cell types might be present.
A chronic inflammatory and demyelinating disease of the central nervous system. Multiple sclerosis involves an autoimmune response against components of myelin, which is thought to contribute to disease pathogenesis.
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indirect effects on b cells might be reinforced by the direct inhibitory activities of MSCs on b cells (FIG. 3). Although these speculations are largely based on in vitro studies, the potential in vivo interactions described here should be considered when designing MSC-based cellular therapies. regulatory T cells32,48. Cell contact between MSCs and activated T cells induces Il-10 production, which, in turn, has an essential role in stimulating the release of soluble HlA-G5 by MSCs32. However, inhibition of any one of these molecules does not result in a complete loss of the immunosuppressive activity of MSCs, and their relative contribution to the immunosuppressive effects varies between different studies. Therefore, it is clear that none of these molecules has an exclusive role and that MSC-mediated immunoregulation is a redundant system that is mediated by several molecules.
Mechanisms of immunosuppression by MSCs Although a large number of studies have documented the immunosuppressive activities of MSCs, the underlying mechanisms are only partially known. Contact-dependent mechanisms and soluble factors are thought to collaborate for the induction of MSC-mediated immunosuppression. The first step in the interaction between MSCs and their target cells involves cell–cell contact mediated by adhesion molecules, as indicated by studies showing that the inhibition of T-cell proliferation by MSCs requires engagement of the inhibitory molecule pD1 by its ligands54. Several soluble immunosuppressive factors have been reported to be involved in MSC-mediated immunoregulation, either produced constitutively by MSCs or released following cross-talk with target cells. examples of molecules belonging to the latter group are nitric oxide and indoleamine 2,3-dioxygenase (IDO), which are only released by MSCs after triggering by IFNγ produced by target cells55,63. IDO induces the depletion of tryptophan from the local environment, which is an essential amino acid for lymphocyte proliferation. MSC-derived IDO was reported to be required to inhibit the proliferation of IFNγ-producing TH1 cells55 and, together with prostaglandin e2 (pGe2), to block NK-cell activity29,55. Furthermore, IFNγ, alone or in combination with the pro-inflammatory cytokines TNF, Il-1α or Il-1β, stimulates the production by mouse MSCs of chemokines that attract T cells and of inducible nitric-oxide synthase (iNOS), which inhibits T-cell activation through the production of nitric oxide43. MSCs from mice deficient for the IFNγ receptor IFNγR1 do not have immunosuppressive activity, which highlights the crucial role of IFNγ in this model43. Other soluble factors, such as transforming growth factor-β1 (TGFβ1), hepatocyte growth factor (HGF), Il-10, pGe 2, haem oxygenase-1 (HO1), Il-6 and soluble HlA-G5, are constitutively produced by MSCs16,22,32,37,42,44,48,64. In addition, the production of some of these molecules can be increased by cytokines released by target cells through their interaction with MSCs. For example, TNF and IFNγ have been shown to increase the constitutive production of pGe2 by MSCs22. Of these constitutively produced factors, Il-6 was shown to dampen the respiratory burst and to delay the apoptosis of human neutrophils by inducing phosphorylation of the transcription factor signal transducer and activator of transcription 3 (ReF. 33), and it inhibited the differentiation of bone-marrow progenitor cells into DCs65. Another important molecule involved in MSCmediated regulation of the immune response is HlA-G5. The production of soluble HlA-G5 by MSCs has been shown to suppress T-cell proliferation, as well as NK-cell and T-cell cytotoxicity, and to promote the generation of
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Clinical applications of MSCs Although pioneer in vivo studies with MSCs have focused mainly on their ability to facilitate the engraftment of transplanted HSCs66 and to promote the structural67 and functional repair of damaged tissues owing to their ‘stem-cell-like’ properties68, the data discussed here on the immunomodulatory properties of MSCs support their possible use as a therapy for immune-mediated diseases.
Characterization of human MSCs. The use of MSCs for clinical purposes takes advantage of their poor immunogenicity in vitro36, in preclinical studies17 and in human studies69, which supported the possible use of MSCs obtained from allogeneic donors in the clinic. In most acute clinical conditions for which the use of MSCs might be considered, allogeneic MSCs would be the only option, as the limited time frame for clonal expansion would not allow for the time-costly in vitro proliferation of autologous MSCs. However, some subacute conditions, such as autoimmune diseases, might allow enough time for autologous MSCs to be harvested and cultured in vitro. In addition, some reports have recently challenged the tenet that allogeneic MSCs are poorly immunogenic70,71, indicating that in some cases an autologous MSC source could be preferable. Despite some early reports that questioned the functional integrity of stromal cells from patients with osteo articular diseases72, recent studies have shown that MSCs from patients with autoimmune disease have a normal ability to support haematopoiesis73 and immunomodulatory activity12, and have a normal cell-surface and molecular phenotype74. The functional impairment of inhibition of T-cell proliferation by MSCs derived from patients with aplastic anaemia75 or multiple myeloma76 is probably the result of an intrinsic abnormality of the bone-marrow microenvironment. These results are consistent with the possibility of using autologous MSCs for clinical purposes. Migratory features of MSCs. The use of stem cells for tissue repair requires that they can easily access the target organ to exert their therapeutic effect. In some cases, in situ administration can directly achieve this goal. In other cases, this possibility is hampered by the anatomical location of the damaged tissue, such as the central nervous system (CNS) or, more importantly, by the systemic nature of the illness (for example, multiple sclerosis). For many conditions, the ideal
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A state of T-cell unresponsiveness to stimulation with antigen. It can be induced by stimulation with a large amount of specific antigen in the absence of the engagement of co-stimulatory molecules.
WNT proteins are glycoproteins related to the Drosophila melanogaster protein Wingless, a ligand that regulates the temporal and spatial development of the embryo. WNT-mediated signalling has been shown to regulate cell-fate determination, proliferation, adhesion, migration and polarity during development. In addition to their crucial role in embryogenesis, WNT proteins and their downstream signalling molecules have been implicated in tumorigenesis and have causative roles in human colon cancers.
treatment should be able to provide both systemic and local therapeutic effects. Transplantation experiments in non-human primates have shown that MSCs can spread to many tissues after intravenous administration77. Systemically administered MSCs seem to preferentially home to the site of injury, where they support functional recovery78. Similar to immune cells, MSCs can extravasate from the blood vessels as a result of the expression of adhesion molecules on their surface. MSCs show coordinated rolling and adhesion behaviour on endothelial cells in a p-selectin- and vascular cell-adhesion molecule 1 (vCAM1)-dependent manner79. Accordingly, MSCs migrate in response to several chemokines that bind to cognate receptors expressed on their cell surface80 and lead to the activation of matrix metalloproteinases that degrade the basement membrane and allow subsequent extravasation81. These results show that the systemic delivery of MSCs could be a useful means to administer these cells in the clinic. Preclinical studies. The ability of MSCs to modulate immune responses in vivo was first investigated by bartholomew and colleagues, who showed that injection of allogeneic MSCs prolonged skin-graft survival in primates17. In the eAe model of multiple sclerosis, systemic injection of MSCs at disease onset ameliorated myelin oligodendrocyte glycoprotein (MOG)-induced eAe and decreased the infiltration of the CNS by T cells, b cells and macrophages. Importantly, T cells from the lymph nodes of MSC-treated mice did not proliferate
Astrocyte Neuron Microglial cell MSC + + + _
Oligodendrocyte Neural precursor cell
Figure 4 | The bystander effects of MSCs on neural cells. A schematic model depicting the potential interactions between mesenchymal stem cells (MSCs) and Nature Reviews | Immunology neural cells in the central nervous system, mainly through bystander mechanisms. MSCs can rescue neurons and oligodendrocytes from apoptosis through the release of trophic and anti-apoptotic molecules, and they can have anti-inflammatory and anti-proliferative effects on microglial cells and astrocytes, resulting in the induction of a neuroprotective microenvironment. In addition, MSCs can promote the proliferation and maturation of local neural precursor cells, leading to their differentiation into mature neurons and oligodendrocytes.
after in vitro re-challenge with MOG peptide, which indicates the induction of T-cell anergy38. Moreover, systemically injected MSCs were found to inhibit the in vivo production of pathogenic plp-specific antibodies and to suppress the encephalitogenic potential of plp-specific T cells in passive-transfer experiments. In this model, the MSCs not only migrated to the lymphoid organs but also to the inflamed CNS, where they had a protective effect on the neuronal axons in situ. Importantly, this effect was unrelated to the transdifferentiation of MSCs into neural cells59,82 (FIG. 4). A protective effect of MSCs on injured neurons is also supported by the results of studies of other neurological diseases. In these studies, regardless of the type of CNS injury, the therapeutic effect of MSCs depended on the release of anti-apoptotic, anti-inflammatory and trophic molecules, as occurred in the case of stroke in rats78, and, possibly, on the recruitment of local progenitors and their subsequent induction to differentiate into neural cells83 (FIG. 4). MSCs have also been shown to favour oligodendrogenic fate decisions by neural precursor cells84. It is of note that these results from CNS disorders are in line with other in vivo studies for a wide range of experimental diseases (TABLe 1). For example, in an experimental mouse model of diabetes induced by streptozotocin, it was observed that MSCs promoted the endogenous repair of pancreatic islets and renal glomeruli85. Similarly, co-infusion of MSCs and bonemarrow cells inhibited the proliferation of β-cell-specific T cells isolated from the pancreas of diabetic mice and restored insulin and glucose levels through the induction of recipient-derived pancreatic β-cell regeneration in the absence of transdifferentiation of MSCs86. These studies show that the in vivo administration of MSCs is clinically efficacious through the modulation of pathogenic b- and T-cell responses and through potent bystander effects on the target tissue. Other studies have provided additional insights into the bystander effects of MSCs mediated by cytokines. For example, in a model of acute renal failure, administration of MSCs increased the recovery of renal function through the inhibition of production of proinflammatory cytokines, such as Il-1β, TNF and IFNγ, and through an anti-apoptotic effect on target cells87. Similarly, the anti-inflammatory activity of MSCs was shown in a model of lung fibrosis, where they inhibited the effects of Il-1α-producing T cells and TNFproducing macrophages through the release of Il-1 receptor antagonist (Il-1RA)88. Another important therapeutic effect of MSCs is mediated by the release of trophic factors such as the WNT-associated molecule secreted frizzled-related protein 2 (SFRp2), which leads to the rescue of ischemic cardiomyocytes and the restoration of ventricular functions89. It is of note that in all of these studies, the beneficial effect of MSCs on the injured tissue occurred despite limited levels of engraftment and transdifferentiation, which indicates that the multipotentiality (that is, stemcell-like property) of these cells is not required for their clinical effect.
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Table 1 | Biological effects of MSCs in preclinical models of disease
Co-transplantation with human HSCs Myocardial infarction Skin-graft rejection Stroke Melanoma Acute renal failure EAE Diabetes EAE Rheumatoid arthritis
Sheep Mouse Monkey Rat Mouse Rat Mouse Mouse Mouse Mouse
Mechanism of MSC effects
Route of MSC administration
Systemic Local Systemic Systemic Local Systemic Systemic Systemic Systemic Systemic
66 68 17 78 91 87 38 85 59 105
Haematopoietic Support engraftment and increased haematopoiesis organs Heart Skin CNS Skin Kidney CNS Pancreas and renal glomeruli CNS Joint Generation of new myocytes and vascular structures Inhibition of T cells Release of trophic factors and induction of neurogenesis Inhibition of tumour-specific T cells by CD8 T cells Inhibition of pro-inflammatory cytokine production and induction of anti-apoptotic and trophic factors Inhibition of myelin-specific T cells and induction of peripheral tolerance Induction of local progenitor cells and inhibition of macrophage infiltration Inhibition of production of myelin-specific antibodies and encephalitogenic T cells; decreased axonal loss Inhibition of T cells and of production of pro-inflammatory cytokines; induction of regulatory T cells Decreased retinal degeneration through anti-apoptotic and trophic molecules Inhibition of production of pro-inflammatory cytokines Inhibition of production of pro-inflammatory cytokines and increased production of IL-10 Tubular-cell regeneration through IGF1 secretion Anti-apoptotic and mitogenic effect by the WNT-related molecule SFRP2 Inhibition of leukocyte invasion through the release of cytokines and chemokines Induction of local progenitors and inhibition of β-cell-specific T cells
Retinal degeneration Acute lung injury Acute lung injury Acute renal failure Myocardial infarction Hepatic failure Diabetes
Rat Mouse Mouse Mouse Rat Rat Mouse
Eye Lung Lung Kidney Heart Liver Pancreas
Local Systemic Local Systemic Local MSC-conditioned medium Systemic
117 88 118 119 89 120 86
CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; HSC, haematopoietic stem cell; IGF1, insulin-like growth factor 1; IL, interleukin; MSC, mesenchymal stem cell; SFRP2, secreted frizzled-related protein 2.
(GVHD). A disease that results from the immunological attack by donor allogeneic T cells that are transferred with the allograft (such as bone marrow, liver or gut) of target recipient organs or tissues (such as the skin or gut). GVHD occurs in graft recipients who cannot eliminate the host-reactive donor T cells, owing to immunosuppression, immunological immaturity or tolerance of the recipient.
The discovery of the immunoregulatory effects of MSCs has led to studies investigating the impact of MSCs on tumour growth, possibly through their effects on anti-tumour immune responses. Inhibition or, more frequently, stimulation of tumour-cell proliferation in vitro and/or tumour growth in vivo by MSCs has been reported90–94. These inconsistent results might be related to the heterogeneous nature of the MSC populations and the different experimental tumour models used, in which the microenvironment probably influences the behaviour of the MSCs95. Two main mechanisms are probably involved in the enhancement of tumour growth by MSCs. First, the cross-talk between MSCs and tumour cells could support tumour progression, for example through integration into tumour-associated stroma94, and second, the suppressive effects of MSCs on the immune system of tumour-bearing hosts might facilitate tumorigenesis, as shown for the inhibition of melanoma rejection, which is possibly mediated by regulatory CD8+ T cells91. Irrespective of the possible interactions between cancer cells, immune cells and MSCs, the potential risk of stimulating the growth of a previously undetected cancer by MSCs must be considered.
Clinical studies. On the basis of their capacity to engraft into the recipient’s bone after systemic administration, MSCs have been used for the first time to treat children with severe osteogenesis imperfecta, resulting in increased growth velocity and total body mineral content, and fewer fractures96. Systemic infusion of allogeneic MSCs has also led to encouraging results in patients with cancer who underwent high-dose chemotherapy, through the acceleration of bone-marrow recovery97. However, the in vivo immunosuppressive effect of infused MSCs has only been successfully shown so far in acute, severe graftversus-host disease (GvHD)69, for which the effect was probably due to the inhibition of donor T-cell reactivity to histocompatibility antigens of the normal tissues of the recipient. However as histocompatibility antigens are also expressed by leukaemia cells, MSCs might impair the therapeutic graft-versus-leukaemia effect, as shown by the recent report of effective prevention of GvHD but higher incidence of relapses in patients with leukaemia who were co-transplanted with MSCs and MHC-identical allogeneic HSCs98. Modulation of host alloreactivity led to accelerated bone-marrow recovery in patients cotransplanted with MSCs and haploidentical HSCs99. The
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Polymorphic peptides derived from normal cellular proteins that can be recognized in the context of MHC molecules. They get their name because they are responsible for the compatibility, or rather the lack of it, of the tissues of genetically different individuals. Immune responses specific for these polymorphic antigens can result in graft-versus-host reactions, graft rejection or beneficial anti-tumour responses.
immunomodulatory potential of MSCs is currently being tested for the treatment of Crohn’s disease, where these cells could also contribute to the regeneration of gastrointestinal epithelial cells100. These results indicate that the clinical use of MSCs has therapeutic potential but they also indicate some opportune cautions about their uncontrolled use101.
An alloimmune attack against recipient haematopoietic neoplasms, which is mounted by donor immune cells in an allogeneic haematopoietic stem-cell transplant. With the exception of T-cell-depleted haploidentical allogeneic stem-cell transplants, wherein graft-versus-leukaemia effects can be mediated by alloreactive natural killer cells, the graft-versus-leukaemia response is mediated by αβ T cells in the donor allograft.
Conclusions and future perspectives Overall, the current data indicate that although bonemarrow-derived MSCs were first proposed for therapeutic purposes in regenerative medicine on the basis of their stem-cell-like qualities, their therapeutic effect can result from other characteristics, such as their antiproliferative and anti-inflammatory properties. MSCs seem to nonspecifically target cells of the immune system. Ultimately, the immuno suppressive activity of MSCs provides a tool for inducing peripheral tolerance following systemic injection and seems to depend on the capacity to ‘freeze’ immunocompetent cells through the inhibition of cell division, thereby preventing their responsiveness to antigenic triggers while maintaining them in a quiescent state. In addition, the evidence of clinical efficacy of MSCs in different experimental models almost only during the acute phase of disease and the limited evidence of transdifferentiation indicate that the therapeutic effectiveness of MSCs relies heavily on their ability to modify the microenvironment of injured tissues. These events occur through the release of anti-inflammatory cytokines, and anti-apoptotic and trophic molecules that promote the repair and protection of damaged tissues. So, the therapeutic plasticity of MSCs might be seen as a recapitulation of the physiological activity of stromal cells in the HSC niche. Here, stromal cells contribute to the generation of the niche by regulating the size of the pool of HSCs and by providing signals necessary to maintain HSCs in a non-proliferating state and refractory to differentiation stimuli, but also to support HSC survival through trophic and anti-apoptotic molecules10,11,102,103.
Box 2 | Safety issues
Mesenchymal stem cells (MSCs) have been used clinically for a decade, mainly in the field of haemato-oncology and musculoskeletal disorders to treat life-threatening conditions including graft-versus-host disease. Few adverse effects have been attributed to MSC administration. However, the limited number of patients who have been treated with MSCs does not allow firm conclusions on the safety of MSCs to be reached. Furthermore, any adverse effects might be masked in patients undergoing allogeneic bone-marrow transplantation and receiving high-dose chemotherapy, as well as other possibly toxic concomitant treatments. A potential risk of treatment with MSCs might paradoxically arise from the ability of MSCs to suppress immune responses and promote tumour growth and metastasis91,94. A further risk associated with MSC proliferation in vitro is the acquisition of cytogenetic abnormalities, which occurs only after long-term passage in culture114, and subsequent differentiation into tumour cells after in vivo administration, as has been shown to occur in rodents115. Although concerns that MSCs might transform into tumorigenic cells still exist, there is a general agreement that bone-marrow-derived MSCs can be safely cultured in vitro with no risk of malignant transformation116 and, so far, there have been no reports in humans of the formation of tumours by in vitro-cultured cells, thus making MSCs amenable for use in transplantation.
The final outcome of the immunomodulatory activity of MSCs is likely to be significantly influenced by the microenvironmental cues encountered following in vivo administration. Indeed, the microenvironment dictates the final effect of MSCs on target cells, as exemplified by the opposite outcomes that can arise from the interaction of MSCs with DCs and NK cells in the presence of high or low concentrations of IFNγ. To elucidate these microenvironmental cues further, studies should address the impact of MSCs on physiological immune response in vivo — for example, on TH1- and TH2-cell responses or responses against viral and bacterial pathogens — and examine the molecular pathways that are activated in MSCs by environmental triggers. Also, it will be relevant to understand the mechanisms that regulate the behaviour of MSCs under conditions of stress, such as during inflammation and tissue injury. Another fundamental question involves whether, after in vivo administration, MSCs can engraft into tissues where they might exert their bystander effects inside ectopic niches, as has been shown for neural stem cells104. limited data are currently available that convincingly show the persistence of MSCs in vivo in tissues. A key related issue that needs to be addressed for clinical purposes concerns the immunogenicity of MSCs, which might restrict engraftment in allogeneic environments. Although other studies will need to address in more detail the cues that might regulate the immunogenicity of MSCs, it should be emphasized that in many reports, allogeneic59,105 or xenogeneic78,85 MSCs have been effectively used to evade host immune surveillance. This behaviour could also be explained by the possibility that MSCs exert their therapeutic effects through a ‘touch and go’ mechanism — that is, through their rapid migration to the damaged organ and subsequent clearance following the release of stress-induced therapeutic molecules. Despite the limitations in our existing knowledge of this matter, the capacity of MSCs to exert their therapeutic plasticity through bystander mechanisms also might indicate that persistent engraftment at the site of damage is not a mandatory prerequisite for having an effect on injured cells and possibly local progenitors during acute stress conditions. In this case, it will be important to understand whether MSCmediated trophic activity occurs through the activation of Notch and wNT signalling on target cells. Indeed, Notch and wNT signalling can promote the self-renewal and maintenance of HSCs106. A final issue of crucial importance concerns the safety profile of injected MSCs. Although their use in most haemato-oncological conditions has been considered safe so far, their long-term effects on immune function and tumorigenic risk are still unknown (BOX 2). An understanding of these issues will allow for the translation of our basic knowledge of MSC biology into the design of clinical therapies. Future clinical trials with MSCs for the therapy of autoimmune diseases and in organ transplantation might be the ideal clinical setting in which to obtain robust information about the therapeutic effectiveness of MSCs.
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