Immunomodulatory Activity of Mesenchymal Stem Cells

Current Stem Cell Research & Therapy, 2011, 6, 00-00

1

Immunomodulatory Activity of Mesenchymal Stem Cells Johanne M. Kaplan*,#, Michele E. Youd# and Tracey A. Lodie# Genzyme Corporation, Framingham, Massachusetts Abstract: Mesenchymal stem cells (MSCs) were discovered as a rare population of non-hematopoietic stem cells that reside in the bone marrow and interact closely with hematopoietic stem cells to support their growth and differentiation. MSCs are multipotent cells that have the ability to differentiate into cells of the mesenchymal lineage including adipocytes, osteocytes and chondrocytes and they have been reported to home to areas of tissue injury and participate in tissue repair. More recently, MSCs have also been described to possess anti-inflammatory and immunomodulatory properties that can affect multiple arms of the immune system. MSCs have been shown to inhibit T and B cell proliferation, downregulate the lytic activity of cytotoxic T lymphocytes and NK cells, inhibit the maturation and antigen-presenting function of dendritic cells and modulate macrophage function through both contact-dependent and contact-independent mechanisms. The administration of MSCs in models of autoimmune disease such as collagen-induced arthritis, EAE and autoimmune diabetes has provided additional evidence for an immunoregulatory role of MSCs supporting their use in controlling autoimmunity. The administration of allogeneic MSCs as immunosuppressive agents represents a viable approach as they appear to be largely non-immunogenic and clinical trials with allogeneic MSCs are currently underway in graftversus-host disease, Crohn’s disease and type I diabetes indications. The immunomodulatory properties, mechanism of action and potential clinical utility of MSCs are reviewed herein.

Keywords: Mesenchymal stem cell, immunomodulation, autoimmunity, graft-versus-host disease, diabetes, lupus. 1. INTRODUCTION Mesenchymal stem cells (MSCs) were first described in 1976 by Friedenstein et al. as adherent fibroblast-like cells found in the bone marrow [1]. MSCs represent a rare population of non-hematopoietic stem cells that interact closely with hematopoietic stem cells in the bone marrow and support their growth and differentiation [2]. In addition to bone marrow, MSCs are found in many adult tissues including skin, adipose tissue, heart and spleen [3-7] and in fetal tissues such as placenta, cord blood, fetal liver and lung [8-10]. MSCs can be isolated from tissues through their ability to adhere to plastic and can be expanded to large numbers ex vivo. MSCs are characterized by their lack of expression of typical hematopoietic surface markers (CD34, CD14, CD45) accompanied by expression of several adhesion molecules (CD44, CD29, CD105, CD73, CD166) [3, 11, 12] and by their ability to differentiate into bone, fat, and cartilage lineages in vitro [3, 13]. Recently, a subpopulation of adult mouse MSCs from the bone marrow has been identified in vivo and shown to express PDGFR
and Sca-1 but lack CD45 and Ter119 [14]. These primary MSCs appear to be located in the perivascular space near the inner surface of the bone and, due to their location, it was postulated that this subpopulation may function to support hematopoeisis. MSCs express various chemokine receptors and have the ability to migrate to sites of inflammation and injury [15-17] *Address correspondence to this author at the Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701, USA; Tel: 508-270-2442; Fax: 508-620-1203; Email: [email protected] # All authors contributed equally to this work 1574-888X/11 $58.00+.00

where it has been hypothesized that they could contribute to tissue repair through transdifferentiation into tissue-specific cell types or through production of factors that promote healing of injured cells and differentiation of endogenous progenitor and stem cells [18-22]. More recently, MSCs have been described to possess anti-inflammatory and immunomodulatory properties which may represent an additional contributing factor to tissue repair through reduction of immune-mediated damage and without requiring long term engraftment. The relative ease in generating large numbers of MSCs from a single bone marrow sample [23] combined with their described lack of immunogenicity and ability to exert immunosuppression across MHC types makes them an attractive mode of therapy for autoimmune diseases and has stimulated numerous pre-clinical studies and clinical testing. The immunomodulatory activity of MSCs on B and T lymphocytes, macrophages, dendritic cells and NK cells has been extensively studied in vitro and is reviewed herein. Although suppressive activity by MSCs is well accepted, conflicting findings on the mechanism of action or immunostimulation by MSCs have been reported depending on the in vitro conditions used, stressing the complexity of the interactions between MSCs and immune cells. In order to more closely approximate the conditions that prevail in a given disease state, in vivo animal models of autoimmunity have been employed to test the impact of MSC treatment and are described herein. The results suggest that treatment with MSCs may not be applicable to all autoimmune diseases and additional study is needed to elucidate the mechanisms and utility of MSC therapy, in particular in Th2 type-driven disease models [24]. Ongoing clinical trials, including graftversus-host disease (GvHD), Crohn’s disease, and type I © 2011 Bentham Science Publishers

2 Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

diabetes mellitus trials, are providing initial insights into the therapeutic potential of MSCs in humans and are also reviewed. 2. IN VITRO STUDIES OF THE IMMUNOMODULATORY ACTIVITIES OF MSCS 2.1. Modulation of T Cell Responses The most extensively studied aspect of immunomodulation by MSCs is their ability to suppress T cell responses (Fig. 1). Numerous in vitro studies have demonstrated the ability of MSCs to inhibit T cell proliferation triggered by mitogenic stimulation such as exposure to PHA or antiCD3/CD28 as well as by antigenic stimulation with allogeneic cells or nominal antigen [25-29]. In most systems, the inhibition is not due to induction of apoptosis or prevention of activation, since upregulation of activation markers is still observed, but rather to the arrest of T cells in the G0/G1 phase of the cell cycle with associated reduction of cyclin D2 and Ki67 expression and upregulation of p27kip1 [30-35]. CD4+ and CD8+ T cells appear to be equally affected [30, 35, 36]. The inhibition of proliferation is not MHC-restricted and can be mediated by allogeneic MSCs [27, 29, 33, 37]. Both contact-dependent and contact-independent mechanisms have been implicated in the T cell inhibitory activity of MSCs sometimes with apparently contradictory results. Differences between studies are likely due to factors such as the state of activation of the T cells, origin and preparation of the MSCs, ratio of T cells to MSCs, and activation stimulus [29, 38-42]. A general picture has emerged whereby MSCs first need to be “licensed” by inflammatory signals in order to exert an immunosuppressive effect [34, 38]. In particular, IFN- and TNF-
produced by activated T cells have been reported to trigger the immunosuppressive activity of MSCs [36, 41, 43-51]. In addition to cytokines, cell to cell contact

Kaplan et al.

between MSCs and T lymphocytes has also been described to enhance licensing of MSCs [34, 52, 53]. Licensed MSCs can then exert their immunosuppressive activity through a variety of mechanisms. No single mediator or pathway can entirely account for the MSC-mediated suppression of T cell activity and the multiple mechanisms described by various investigators may prevail to different degrees depending on the particular conditions under study. The involvement of contact-dependent mechanisms is shown by studies in which physical separation of MSCs and lymphocytes by a membrane in a transwell system was found to reduce the immunosuppressive impact of MSCs [27, 52, 53]. Contactdependent suppression has been variably explained by physical hindrance of the contact between T cells and antigen-presenting cells by MSCs [27] or expression of the negative co-regulatory molecules B7-H1 [43, 44, 53], B7-H4 [54], or HLA-G [52, 55] on the surface of MSCs. Soluble mediators released by licensed MSCs also play an important role in T cell inhibition. In particular, prostaglandin E2 (PGE2) [30, 47, 50], nitric oxide [30, 38, 51, 56], and indoleamine 2,3-dioxygenase (IDO) [30, 38, 43, 45-47, 49] have been found to be upregulated by inflammatory signals in vitro and have been most commonly implicated in the contact-independent, suppressive activity of MSCs. Their involvement is supported by significant reversal of T cell inhibition upon suppression of PGE2 synthesis by indomethacin [43, 47, 50], inhibition of NO production by INOS inhibitors [56], or neutralization of IDO activity through silencing of the IDO gene, addition of the antagonist 1methyl L-tryptophan, or replenishment of tryptophan in cocultures of MSCs and lymphocytes to reverse depletion of this essential amino acid [45, 47, 49]. Other cytokines that have been reported to be similarly upregulated in licensed MSCs and whose neutralization at least partially reverses the inhibitory activity of MSCs include TGF- [25, 30, 47], he-

Fig. (1). Modulation of T cell responses by MSCs. Licensing by inflammatory cytokines, and possibly TLR ligands, triggers the immunosuppressive activity of MSCs which can suppress T cell responses through contact-dependent inhibition, contactindependent inhibition mediated by various soluble cytokines, or through the induction of targets.

Immunomodulatory Activity of Mesenchymal Stem Cells

patocyte growth factor (HGF) [25, 30, 47], COX-2 [43], soluble HLA-G [52, 55], IL-10 [57, 58], heme oxygenase-1 (HO-1) in combination with NO [51], galectin-1 and semaphorin-3A [59], leukemia inhibitory factor (LIF) [60], and insulin-like growth factor-binding proteins [30]. Toll-like receptors (TLRs) which recognize pathogenassociated molecular patterns have been found on the surface of MSCs and could represent another trigger in the licensing of MSCs. TLR2,3,4,7 and, to a lesser extent TLR9, were detected by flow cytometry on the surface of human MSCs [61-63]. However, while ligation by TLR ligands was reported to enhance the immunosuppressive activity of MSCs by some investigators [61, 62], a decrease in MSC-mediated suppression was observed by others [63]. The exact role of TLR stimulation in the immunomodulatory activity of MSCs therefore remains unclear. In addition to contact-dependent inhibition and release of soluble mediators, evidence that MSCs suppress T cell responses indirectly through the induction of regulatory T cells (Tregs) or interference with the activity of antigen-presenting cells (APCs; see below) has been reported. Indeed, the percentage of T cells with a Treg phenotype was increased by the addition of MSCs to a mixed lymphocyte reaction (MLR) in the rat and the human system [37, 52, 57], perhaps through the production of LIF [60] or HLA-G [52] by the MSCs. In addition, co-culture of human MSCs and purified T cells in vitro has indicated that MSCs can not only increase the percentage of Tregs but also help maintain their regulatory activity compared to Tregs cultured alone [64]. Interestingly, a recent publication has also shown that human MSCs can inhibit the differentiation of naïve human T cells into Th17 cells and are able to induce a Treg phenotype in fully differentiated Th17 cells by inhibiting the production of pro-inflammatory cytokines including IL-17, IL-22, IFN- and TNF-
, as well as CCL20 from these cells while increasing the production of IL-4, IL-5, IL-13, and IL-10 [65]. In spite of extensive evidence for suppression of T lymphocyte activity by MSCs, it is important to note that MSCs can actually enhance T cell proliferation under certain conditions [29, 39, 40, 42]. For example, it has been reported that low ratios of MSCs added to T lymphocytes undergoing stimulation with mitogens [40], cytokines [39], or allogeneic cells [29, 40, 42] tend to promote T cell proliferation while higher ratios of MSCs have a suppressive effect. Depending on the levels of IFN-, MSCs can also be induced to act as antigen-presenting cells and stimulate MHC Class I- and Class II- restricted T cell responses [41, 66]. Taken together, these findings suggest that the ultimate impact of MSCs on T cell responses is influenced by the particular immunological milieu. 2.2. Modulation of Antigen-Presenting Cells While the ability of MSCs to affect T cell responses directly is well-documented, MSCs can also influence T cell responses indirectly through their effect on antigenpresenting cells (Fig. 2). Results from several different groups have indicated that MSCs can inhibit differentiation of CD34+ precursors and CD14+ monocytes into dendritic cells (DCs) as well as interfere with the activity of mature

Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

3

DCs in both the mouse and the human system [50, 67-74]. In several studies, MSCs cultured with peripheral blood monocytes in the presence of GM-CSF and IL-4 prevented differentiation of the monocytes into dendritic cells [68, 72-74] as shown by reduced expression of DC markers including MHC Class II, CD80 and CD86 co-stimulatory molecules, and the CD83 maturation marker compared to control cultures without MSCs. The resulting cells were also refractory to activation by CD40L, LPS or TNF-
[73], and produced less IL-12 [68, 73]. As expected from this phenotype, the cells were severely impaired in their ability to stimulate proliferation of allogeneic T cells [68, 73]. This impairment of monocyte differentiation into DCs was variably attributed to a block in the G0 to G1 phase of the cell cycle [72] or to the production of inhibitory soluble factors by the MSCs including PGE2 [68], IL-6, and M-CSF [73]. MSCs were also found to affect the activity of mature DCs. Exposure of mature DCs to MSCs or their soluble factors typically resulted in a shift towards a less mature phenotype characterized by a decrease in expression of MHC Class II, CD80, CD86, CD40, and CD83 [67, 69-71, 75], an increase in endocytic activity [67, 70], and reduced production of IL-12 [67, 75]. As observed above, the MSC-induced changes in phenotype reduced the ability of the DCs to stimulate proliferation of allogeneic T cells [67, 70, 71, 75]. The reduction in T cell stimulation could conceivably be attributed to decreased antigen presentation and costimulation by the MSC-exposed DCs, but evidence that these cells may be capable of actively inhibiting T cell function has also been described. For example, Zhang et al. [67] reported that MSC-exposed DCs differentiate into a type of regulatory DCs that can inhibit Con A or allogeneic cellinduced T cell proliferation in a contact-dependent manner through expression of Jagged-2 on the cell surface. In separate studies, Aggarwal and Pittinger [50] showed that MSCs caused mature DC type 1 cells (DC1) to produce less TNF-
and mature DC2 to produce more IL-10 thereby favoring an immunosuppressive profile. Taken together, these results indicate that modulation of APC activity can also contribute to the immunosuppressive activity of MSCs. 2.3. Modulation of Macrophages Little has been published on the effect of MSCs on macrophages but the available evidence suggests that MSCs generally act to dampen the proinflammatory activity of macrophages. Kim et al. [76] reported that human macrophages cultured with MSCs develop a suppressive immunophenotype characterized by decreased production of IL-12 and TNF-
and increased production of IL-10 and IL-6. Similarly, in the mouse, MSCs were found to reduce the production of TNF-
by macrophages activated with lipopolysaccharide (LPS) [77, 78] or silica [78] in vitro. The inhibition was attributed to the release of soluble factors by the MSCs including IL-1 receptor antagonist [78] and PGE2 [79], the latter resulting in the release of immunosuppressive IL-10 by the macrophages themselves. This in vitro antiinflammatory activity of MSCs translated into a benefit in models of bleomycin- or LPS-induced pulmonary inflammation [77, 78] and against sepsis induced by cecal ligation and puncture [79].

4 Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

Kaplan et al.

Fig. (2). Modulation of antigen-presenting cells by MSCs. MSCs can interfere with the activity of antigen-presenting cells though several mechanisms including inhibition of the maturation of CD14+ monocytes into DCs; interference with the function of mature DCs leading to reduced IL- 1 2 production and reduced stimulation of allogeneic T cells; induction of regulatory DCs capable of Jagged 2 contact-dependent inhibition; and modulation of DC cytokine production towards a DC2 immunosuppressive phenotype (increased IL-10, reduced TNF-
production).

Fig. (3). Modulation of NK cell activity by MSCs. The release of inhibitory cytokines by MSCs in vitro reduces the ability of NK cells to proliferate and produce cytokines in response to stimulation with IL-2, IL- 15 and allogeneic cells. The impact of MSCs on NK target cell lysis is variable depending on culture conditions and nature of the target cells.

2.4. Modulation of B Cell Responses In vitro studies of the impact of MSCs on B lymphocytes have produced mixed results with some investigators reporting inhibition of B cell proliferation and antibody production by MSCs [32, 53, 80-85] and others demonstrating an increase in B cell responses in the presence of MSCs [86, 87]. This indicates further work is needed to elucidate the effect of a variety of factors including differences in culture conditions, ratios of MSCs to target cells, sources of B lymphocytes (fractionated vs unfractionated), and stimuli used to activate B cells. For example, Rasmusson et al. [87] observed that the addition of MSCs to purified human B cells stimulated with viral antigens or lipopolysaccharide (LPS) resulted in the suppression of IgG secretion when a strong degree of stimulation was applied but actually enhanced IgG production when a weaker stimulus was used. In separate studies, Corcione et al. [84] showed that MSCs inhibited the proliferation and production of IgM, IgG, and IgA by puri-

fied human B cells stimulated with a cocktail of CpG2006, recombinant CD40L, anti-Ig, IL-2, and IL-4 but only at MSC:B cell ratios of 1:1 to 1:2 with lower ratios being without effect. Interestingly, Traggiai et al. [86] using similar conditions (human MSC:B cell ratio of 1:1, stimulation with CpG2006, recombinant CD40L, anti-Ig, and IL-2) reported a strong stimulatory effect of MSCs resulting in increased proliferation, differentiation into antibody-producing cells and isotype switching using B lymphocytes from both normal donors and systemic lupus erythematosus patients. Similarly, we have observed that co-culture of mouse MSCs with purified plasma cells from normal or lupus-prone New Zealand black (NZB)
New Zealand white (NZW)F1 (NZB/WF1) mice promoted plasma cell survival and antibody secretion [24]. MSC production of Th2-type cytokines (IL-6, IL-10) [86, 87], which are known to promote B cell proliferation and differentiation as well as plasma cell survival [88], may contribute to the stimulatory effect of MSCs although cell to cell contact has also been reported to be involved [86, 87].

Immunomodulatory Activity of Mesenchymal Stem Cells

Where examined, suppression of B lymphocyte function by MSCs appeared to be largely contact-independent and to involve soluble factors [80, 82, 84]. Clearly, the impact of MSCs on B lymphocytes involves a complex set of interactions and is dependent on particular environmental conditions. 2.5. Modulation of Natural Killer Cell Activity MSCs have also been reported to impact NK cell activity (Fig. 3), an essential component of the innate immune response against virally-infected cells, tumor cells, and allogeneic cells. It has been consistently reported that co-culture with MSCs inhibits the proliferation of NK cells in response to various stimuli including IL-2 [89, 90], IL-15 [91], or allogeneic cells [37]. The inhibition appears to be mediated by soluble factors and could be at least partially reversed by inhibitors of TGF- [91] or PGE2 [91] or a combination of PGE2 and IDO inhibitors [89]. MSCs were also found to inhibit cytokine production (IFN-, IL-10, TNF-
) by NK cells cultured in IL-2 or IL-15 [50, 89-91]. Findings regarding the impact of MSCs on the cytolytic activity of NK cells are inconsistent and the discrepancies are likely due to differences in assay conditions and in the particular choice of target cells used to assess cytolysis. For example, Spaggiari et al. [89] reported that NK cells cocultured with MSCs in the presence of IL-2 displayed reduced cytolytic activity against a range of HLA Class I+ and Class I- targets while others observed reduced cytolysis by MSC-exposed NK cells against HLA Class I+ targets [91] but not HLA Class I- targets [91, 92]. The nature of the HLA Class I- target cells was not the same in each study and Spaggiari et al. [89] have suggested that some HLA Class Itarget cells may also express activating NK receptors thus rendering them particularly susceptible to NK cell lysis and making it more difficult to detect any decreases in NKmediated lysis. A similar situation could be envisaged in vivo with some NK targets being more sensitive than others to the inhibitory influence of MSCs. The mechanism by which MSCs can impair the development of NK cell cytolytic activity remains to be fully elucidated but appears to involve both contact-dependent [91] and contact-independent mediators such as soluble HLA-G5 [52], PGE2, and IDO [89] and may involve down-regulation of activating NK receptors such as NKp30, NKp44, or NKG2D [89]. MSCs can not only affect NK cell function but can also be subject to their activity. It has been generally observed that MSCs are not lysed by freshly isolated NK cells [90-92] even though they typically express low levels of MHC class I as well as ligands for activating NK receptors which contribute to NK susceptibility. However, upon activation, NK cells are in fact capable of lysing MSCs [90, 91]. Exposure to IFN- increases MHC Class I expression on MSCs and concurrently decreases their susceptibility to NK-mediated lysis [90], suggesting that the fate of MSCs in a host would likely depend on the particular immunological environment. 3. PRE-CLINICAL IN VIVO STUDIES The ability of MSCs to suppress immune responses, particularly T cell responses, in vitro has led investigators to ask whether MSCs can be used to attenuate deleterious immune

Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

5

responses in vivo by testing MSC therapy in mouse models of autoimmune disease. Because Th2-type cytokines, such as IL-10 [57, 58], have been shown to contribute to MSC suppression of T cell responses and since MSCs have been shown to shift the cytokine profile from a Th1-type to a Th2type in a MLR in vitro [50], the treatment of Th1-type autoimmune diseases has been explored primarily, as discussed below. Some data suggest that MSCs may not provide a benefit in a Th2-type disease setting and this work is also described. 3.1. MSC Treatment in Murine Models of Graft-VersusHost Disease Evidence that MSCs have the potential to control autoimmune and inflammatory responses comes from work testing MSC therapy in graft-versus-host disease (GvHD), in studies ranging from murine models to clinical trials. GvHD is a complex disease affecting multiple organs and is a major cause of morbidity and mortality after allogeneic bone marrow or hematopoietic stem cell transplantation. GvHD occurs because donor immune cells from the transplant recognize the recipient as foreign and essentially mount an alloreactive immune response against the recipient’s organs. Acute or chronic GvHD can develop and, although donor T cells and NK cells are thought to mediate disease in both [93], the timing, pathology, and immune mechanisms involved are not the same. Consequently acute and chronic GvHD are considered distinct illnesses rather than chronic GvHD developing from acute GvHD. Because of this, treatments that provide benefit in the acute setting may not be efficacious in the chronic condition and vice versa. Many murine models of GvHD exist, each replicating different aspects of GvHD pathology [93, 94]. Because the extensive literature describing MSC treatment of GvHD was reviewed recently [95], the current understanding will be discussed briefly. The first indication that stromal cells from bone marrow might be protective in a GvHD setting came from work by Miyashima and colleagues who in 1996 showed that a bone graft given along with a bone marrow transplantation protected mice from developing GvHD [96]. Since then, numerous studies have been performed and the majority tested MSC treatment in typical acute GvHD models and in general show MSC treatment to be efficacious (Table 1). Collectively, the MSC source (i.e. bone marrow or adipose tissue) did not seem to matter. A range of MSC doses and timing of delivery were tested and the data suggest that earlier intervention and multiple doses of MSCs might be most efficacious at improving survival, target organ pathology, and/or disease scores although this was not absolute. For example, Min and colleagues only delivered one dose of MSCs and saw no benefit unless the MSCs were engineered to overexpress IL-10 [97]. In addition, Badillo et al. [98] and Lu at al. [99] employed the same GvHD model but Lu et al. delivered 2-13 fold more MSCs than Badillo et al. on the same days and saw a significant improvement in overall clinical score although survival was not affected. When multiple low doses of MSCs were given early on, MSCs were not efficacious in the Badillo et al. study but were efficacious in a study by Yañez et al. [100]. Whether the lack of efficacy was due to MSC clearance is unclear. Badillo and others have shown

6 Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

Table 1.

Kaplan et al.

The Prevention and Treatment of GvHD in Murine Models

Author

Mouse Model

Miyashima 1996 [96]

lpr > B6

Chung 2004 [104]

C3H > B/c

Sundres 2006 [101] Yañez 2009 [100]

Min 2007 [97]

Tisato 2007 [102]

Conditioninga TBI 8.5 Gy T-dep BM 2
107

MSC Sourceb

MSC Administrationc Dose

Time

lpr bone graft

Trans-plant

-

0

+

C3H BM

i.v.

1
105

0

+

5
105

0

+

3
106

0

0

4
106

0

+

TBI 8.75 Gy BM 1
107 SP 5
10

5

TBI 8 Gy B6 >B/c

BM 3
10

6

C57BL/6 BM

i.v.

CD3+ 5
105 B6 > B6D2F1

B6 > B6D2F1

Human > NOD/scid

TBI 11 Gy BM 1
107

0+7+14

+

14+21+28

0

1
106

1

-

2
106

1

+

5
104

B6D2F1 AD

SP 2
107 TBI 11 Gy BM 1
10

C57BL/6 BM

i.v.

IL-10 C57BL/6 BM MSCs

i.v.

2
106

1 0

0

Human UBC

i.v.

3
106

0+7+14+21

+

35+38+41+44

0

7

SP 2
107 TBI 2.5 Gy PBMCs 2
107

1
10 Badillo 2008 [98]

TBI 9 Gy B6 > B6
B/c

BM 1
107

C57BL/6 BM

i.v.

0

0

0

0

5
104

2

0

10

0

21

0

0+7+14

0

SP 3
106

5

0

0

5
105

2

+

20

+

30

+

TBI 10 Gy

0

±

BM

2

+

T cells

20

+

Balb/c BM B/c > B6

IFN
-/- > B6 Lu 2009 [99] Jeon 2010 [103] Joo 2010 [106]

6

1.5
105

1
10

Polchert 2008 [105]

Resultd

Route

i.v.

IFN
tx Balb/c BM MSCs

i.v.

5
105

0

+

Balb/c BM

i.v.

1
105

2

0

C57BL/6 BM

i.v.

2
106

0

+

Human BM

i.v.

1
106

0

0

7

0

5
105

0

±

6

0

+

2
106

0

+

TBI 8.5 Gy B6 > B6
B/c

BM 5
106 SP 2.5
10

7

TBI 9 Gy B6 > B/c

BM 5
106 SP 2
107 TBI 8 Gy

C3H > B/c

BM 5
10

6

SP 1
106

C3H10T1/2 cell line

i.v.

1
10

a, GvHD was induced by delivery of donor bone marrow (BM) and spleen (SP) cells following total body irradiation (TBI). b and c, MSCs derived from mouse BM or adipose tissue (AD) or human umbilical cord blood (UBC) were delivered by intravenous (i.v.) injection at the indicated doses and days post-conditioning. d, Results of the studies are summarized as follows: 0, no effect; +, beneficial effect on survival, overall clinical score, and/or organ pathology; -, worsening of disease; ±, slight improvement in survival, overall clinical score, and/or organ pathology. lpr: MRL/lpr; B6: C57BL/6; B/c: Balb/c; B6D2F1: (C57BL/6
DBA/2)F1; B6
B/c: (C57BL/6
Balb/c)F1; C3H: C3H/HeJ; T-dep: T cell-depleted; CD3+: CD3+ T cells; PBMCs: human peripheral blood mononuclear cells; Gy: gray

Immunomodulatory Activity of Mesenchymal Stem Cells

that MSCs persist in the recipient’s tissues for several weeks post-injection although the amount of MSCs in GvHD target organs is low compared to the lung or bone marrow [98, 101, 102]. Why some treatments worked and others did not is unknown but most likely reflects differences in the model used (e.g. acute vs. chronic; strain of the recipient) and the conditioning applied (e.g. different susceptibility to irradiation among mouse strains; composition of donor cells) [94], and as mentioned above, the timing and dose of MSC delivery. Sudres et al. [101] and Jeon et al. [103] both used the fully allogeneic C57BL/6 into Balb/c acute GvHD model and neither observed enhanced survival but Sudres did report improvement in small intestine pathology. In studies where MSCs were shown to provide a benefit, a reduction in in vivo T cell responses [99, 102], serum IFN- production [104, 105], and an increase in Tregs [106] were shown. Not only were MSCs shown to dampen IFN- levels, but IFN- has also been directly implicated in activating MSCs to become immunosuppressive cells in GvHD as demonstrated by the ability of IFN- pre-treated MSCs to inhibit disease transferred by IFN- deficient T cells [105] and the failure of IFN-R1 deficient MSCs to prevent GvHD [36]. Another variable is whether the MSCs grown in different laboratories are functionally equivalent in vivo. Some laboratories qualified their MSCs by characterizing the phenotype of the cells, by showing the ability of MSCs to differentiate into other mesenchymal lineages, and by demonstrating their ability to suppress T cell responses in vitro [100, 102, 104106]. According to Badillo et al., it is unclear how these in vitro activities accurately reflect in vivo function given that the ability for MSCs to suppress T cell responses in vitro did not perfectly correlate with in vivo efficacy in all disease models [98]. Despite these discrepancies, it is worthwhile to further the understanding of how MSCs impact GvHD in animal models, which may contribute to improvements in clinical application of MSC therapy to treat GvHD. 3.2. MSC Treatment in the Collagen-Induced Arthritis Model Because MSCs were shown to inhibit T cell responses, the first attempt at testing the ability of MSCs to inhibit deleterious T cell responses in a classical autoimmune disease comes from reports evaluating MSC treatment in the collagen-induced arthritis (CIA) model (Table 2). Unlike spontaneous autoimmune disease models, the CIA model is an induction model where arthritis is elicited by stimulating potent antigen specific Th1- and Th17-type T and B cell responses after immunization with collagen II (CII) protein in adjuvant. The collagen-specific immune cells infiltrate the joint and induce a rheumatoid arthritis-like disease. Several groups have tested MSC therapy in the CIA model with mixed results [107-112]. A significant reduction in clinical scores, autoreactive T cell responses, and CIIspecific IgG levels was observed in several studies where bone marrow- or adipose tissue-derived MSCs were delivered either by intravenous or intraperitoneal injection before or after disease onset [107-109]. An accompanying decrease in TNF-
[107-109], IFN- [107], and IL-1 [108] levels in

Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

7

the serum and a reduction of these cytokines, along with a decrease in IL-6, IL-12, IL-17, MIP-2 and RANTES, were also observed in the joint [108]. IL-10 has been reported to contribute to MSC immunosuppression in vitro [57, 58] and although IL-10 levels in the serum from MSC-treated mice were relatively unchanged, IL-10 levels in culture supernatants of T cells re-stimulated with CII in vitro were reduced when cultured with [107] or increased [108] when cultured without MSCs, if the T cells were isolated from CIA mice treated with MSCs. IL-10 was significantly increased in the joint and in supernatants from synovial cells cultured with MSCs [108], suggesting that, in the target tissue, IL-10 might be contributing to the attenuation of the autoimmune response by MSCs. In addition to a modulation of IL-10 production in vitro, the proliferation of CII-specific T cells after re-challenge with CII in vitro and IFN- levels in the supernatants from these cultures were reduced [108]. MSCs have also been shown to induce Tregs in vitro [37, 52, 57] and the number of Tregs and Treg-associated cytokines (i.e. IL-10 and TGF- levels) were increased in MSC-treated CIA mice [107, 108]. In addition, T cells isolated from mice with CIA disease and then cultured with MSCs, but not T cells cultured alone, were able to prevent disease development when delivered 2 days after CIA induction in adoptive transfer studies [108]. CII-specific B cell responses were also impaired as shown by a reduction in anti-CII antibodies in the serum [108]. In contrast to these findings, other reports show that MSCs either have no effect, worsen disease symptoms [110, 112], or provide a clinical benefit if transduced with an IL10 expressing retrovirus [111]. Again, there were important differences in experimental conditions such as the type and dose of MSCs administered and the method of disease induction. For example, Djouad et al. administered the immortalized C3H/HeJ-derived fibroblast cell line C3H/10T1/2 [113], which the authors refer to as an MSC cell line but failed to characterize, while other investigators used culture-expanded primary MSCs. Schurgers et al. [112] delivered syngeneic or allogeneic mouse MSCs but saw no effect. Here the authors induced disease with one intradermal CII injection but saw no benefit when one or two doses of MSCs were delivered whereas one large [107, 109] or several smaller daily doses [108] of MSCs protected mice from developing disease after intravenous CII injection. In these studies, anti-CII antibody responses and serum cytokine levels were shown to be unaffected or enhanced [110] or decreased [111] in mice that were not protected by MSC therapy, suggesting that although MSCs did modulate the immune response, this modulation did not translate into a clinical benefit. Collectively, these data suggest that MSCs can provide a benefit in the CIA model but more work needs to be performed to determine the mechanism(s) by which MSCs are mediating protection in this setting. 3.3. MSC Treatment in Experimental Autoimmune Encephalomyelitis Models Because therapeutic approaches targeting T cells have been used successfully in experimental autoimmune encephalomyelitis (EAE) models [114-116] of multiple sclerosis, the impact of MSC therapy in EAE models has been ex-

8 Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

Kaplan et al.

Table 2. The Treatment of Collagen-Induced Arthritis with MSCs

Author

Djouad 2005 [110]

Mouse Model

DBA/1

Disease Inductiona

100
g bCII in CFA s.c. d 0; 100 μg bCII in IFA s.c. d 21

C3H10T1/2 cell line

vIL-10

Choi 2008 [111]

DBA/1

100
g mCII in CFA s.c. d 0; 50 μg mCII in CFA s.c. d 21

DBA/1

100
g bCII in CFA s.c. d 0; 50 μg bCII in IFA s.c. d 14

DBA/1

2010 [109]

DBA/1

DBA/1

0

0

i.v.

4106

21

0

0

0

21

-

0

0

5106

DBA/1 BM

i.v. i.v.

110

i.p. i.a.

100
g cCII in CFA s.c. d 0; 100 μg cCII in CFA s.c. d 21

i.p.

0

+ +

1106

21+28+35

0

6

21+28+35

+

1106

24-28

+

1106

30-34

+

22

+

22

+

110

6

24-28

+

6

24-28

+

After onset

+

-1

0

DBA/1 AD

i.p.

110

rat BM

i.p.

2106

100
g cCII in CFA s.c. d 0

16

-

16+23

0

i.v.

1106

16+23+30

0

i.v.

110

6

-1

0

i.p.

1106

16

0

16+23

0

16+23+30

0

16+23+30

0

16+23+30

0

IFNR-/- DBA/1 BM

C57BL/6 BM

Time

21

200
g cCII in CFA s.c. d 0; 100 μg cCII in CFA s.c.

DBA/1 BM

Schurgers 2010 [112]

1106

i.p.

C57BL/6 AD

Mao

i.v.

C57BL/10 BM

Human AD González 2009 [108]

Dose

1106

vIL-10 DBA/1 BM MSC

Resultd

Route

i.v.

C3H10T1/2 Augello 2007 [107]

MSC Administrationc

MSC Sourceb

i.p.

110

6

a, Disease was induced by subcutaneous (s.c.) injection of bovine (b), mouse (m), or chicken (c) collagen II (CII) in complete or incomplete Freund’s adjuvant (CFA or IFA) on the indicated day (d). b and c, MSCs derived from mouse bone marrow (BM) or mouse and human adipose tissue (AD) were delivered by intravenous (i.v.), intraperitoneal (i.p.), or intraarticular (i.a.) injection at the indicated doses and days post disease induction. d, Results of the studies are summarized as follows: 0, no effect; +, beneficial effect on survival, overall clinical score, and/or organ pathology; -, worsening of disease.

plored (Table 3). Like the CIA model, the EAE model is an induction model where the disease is induced by active immunization with myelin protein or peptides in adjuvant thereby eliciting antigen specific T cell responses in the central nervous system. Many EAE models exist [117] and the ones used to test the potential of MSC therapy include the chronic-progressive and relapsing/remitting models where disease is induced in C57BL/6 or SLJ mice by injection of myelin oligodendrocyte glycoprotein (MOG) or proteolipid protein (PLP) antigens, respectively. Systemic delivery of syngeneic or allogeneic mouse MSCs [118-122] or human MSCs [123, 124] has been shown to attenuate disease symptoms. Demyelination and axonal loss [120-122, 124], macro-

phage and T cell infiltration in the central nervous system [120-122, 124], serum INF- and TNF-
levels [118, 119], and myelin-specific autoantibodies [118, 121] were reduced in mice treated with MSCs. Autoreactive T cell responses were dampened by MSC treatment as indicated by a reduction in proliferation and Th1-type cytokine production and a shift to Th2-type cytokines when T cells from MSC-treated mice were re-stimulated in vitro with MOG or PLP antigens [120, 121, 123]. Along with Th1 cells, Th17 T cells, major producers of IL-17, are thought to play a major role in mediating disease in EAE [125, 126] because IL-17 has been shown to play a critical role in the induction of the demyelination process

Immunomodulatory Activity of Mesenchymal Stem Cells

Table 3.

Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

9

The Treatment of Experimental Autoimmune Encephalomyelitis with MSCs Mouse Model

Disease Inductiona

Zappia 2009 [122]

C57BL/6

200
g MOG in IFA s.c. d 0; 400 ng PT i.v. d 0+2

Gerdoni 2007 [121]

SJL

Gordon 2008 [124]

C57BL/6

Kassis 2008 [120]

Author

200
g PLP in IFA s.c. d 0; 300 ng PT i.v.

MSC Administrationc

MSC Sourceb Route

Resultd

Dose

Time 3+8

C57BL/6 BM

i.v.

110

6

+

24

0

10+15+24

+

C57BL/6 BM

i.v.

1106

12

+

MOG in adjuvant s.c. d 0; 200 ng PT i.p. d 0

Human BM

i.p.

1106

6

+

C57BL/6

300
g MOG in CFA s.c. d 0+12; 300 ng PT i.p. d 0+2

C57BL/6 BM

i.v.1

1106

10

+

i.v.

110

6

10

+

C57BL/6

200
g MOG in CFA s.c. d 0; 500 ng PT i.p. d 1+2

Human BM

i.v.

3106

Human BM

i.v.

C57BL/6 BM

d 0+2

SJL

200
g PLP in CFA s.c. d 0; 500 ng PT i.p.

+ 16+26

+

3106

16

+

i.p.

2106

15+27+45

+

CCL2-/-C57BL/6 BM

i.p.

2106

15+27+45

0

C57BL/6 BM

i.p.

2106

16+28

+

Balb/c BM

i.p.

16+28

+

INF
tx Balb/c BM MSCs

i.p.

16+28

0

Bai 2009 [123]

16

d 1+2 Rafei 2009 [118]

Rafei 2009 [119]

C57BL/6

1mg/ml MOG in CFA s.c. d 0+12; PT i.p. d 0+2

C57BL/6

1mg/ml MOG in CFA s.c. d 0+12; PT i.p. d 0+2

2106 2106

a, Disease was induced by subcutaneous (s.c.) injection with myelin oligodendrocyte glycoprotein (MOG peptide 35-55) or proteolipid protein (PLP peptide 139-151) in complete or incomplete Freund’s adjuvant (CFA or IFA) followed by pertussis toxin (PT) on the indicated day (d). b and c, MSCs derived from mouse or human bone marrow (BM) were delivered by intravenous (i.v.), intraperitoneal (i.p.), or intraventricular (1) injection at the indicated doses and days post disease induction. d, Results of the studies are summarized as follows: 0, no effect; +, beneficial effect on overall clinical score, organ pathology, and/or lymphocyte infiltration. Tx: treated.

[126]. Serum IL-17 was reduced in MSC-treated mice [118, 119] and T cells from EAE mice produced less IL-17 when re-challenged with autoantigen in vitro in the presence of MSC-conditioned medium [118]. The CCL2/CCR2 pathway has been shown to play an important role in EAE development [127-130] and MSCs are known to produce CCL2 [15], suggesting that MSC-derived CCL2 might contribute to immunoregulation by these cells. In accordance with this, MSC-mediated inhibition was shown to be dependent on MSC-derived CCL2 expression since CCL2-/- MSCs were not effective [118]. In addition, autoreactive T cells from MSC-treated mice appeared to be anergic since their antigen specific response to re-challenge in vitro was poor [118, 122]. Data suggest that MSCs are providing neuroprotection through modulation of the immune response but whether they may also provide a benefit by differentiating into neuronal cells is not known. Although MSCs have been shown to differentiate into cells that express neuronal markers [131, 132] and display a neuron-like functional phenotype [133] in

vitro, it is unclear whether this occurs in vivo and in the setting of EAE disease. Collectively, these data show that MSC treatment is beneficial in EAE and provide support for the evaluation of MSC treatment in multiple sclerosis patients. Clinical trials in this indication are currently underway. 3.4. MSC Treatment in Type 1 Diabetes Mellitus Disease Models Only recently has MSC therapy been tested in mouse models of type 1 diabetes (Table 4). Type 1 diabetes mellitus is an autoimmune disease characterized by the infiltration of autoreactive T cells into the pancreas leading to beta cell destruction, a loss in insulin production, and a rise in glucose levels. MSC therapy has been tested in the streptozotocin (STZ) and non-obese diabetic (NOD) mouse models. STZ is a chemical that induces beta cell destruction when delivered in multiple low doses to wild type mice [134]. In STZ-induced diabetic mice treated with one (500,000 cells) [135] or two (2106/dose) [136] doses of MSCs, hypergly-

10 Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

Table 4.

Kaplan et al.

The Treatment of Type 1 Diabetes Models with MSCs

Author Lee 2006 [136]

Disease

Mouse Model

Inductiona

NOD/scid

35 mg/kg STZ i.p. d 1-4

MSC Administrationc

MSC Sourceb

human BM

Dose

Time

i.v.1

2
106

10+17

+

1
105

15

+

4

50 mg/kg STZ i.p. d 1-5 TBI 2.5 Gy d 15 Urbán 2008 [145]

C57BL/6

B6D2F1 BM

TBI 2.5 Gy d 15 Ezquer 2008 [135]

C57BL/6

40 mg/kg STZ i.p. d 1-5

Madec 2009 [148]

NOD

TBI 7.5 Gy 5
106 diabetic TC

Fiorina 2009 [151]

NOD

i.v.

B6D2F1 BM C57BL/6 BM

Balb/c BM

Balb/c BM

Balb/c BM NOR BM NOD BM

5
10

15

+

2.5
104

15

0

i.v.

2
105

15

0

i.v.

5
105

25

+

i.v.

1
105

wk 4

±

i.p.

1
105

wk 4

±

1
10

3

0

±

1
10

4

0

±

1
105

0

+

1
10

6

0

5
10

5

wk 10-13

i.v.

1
10

6

2x/wk a.d.o.

+

i.v.

5
105

wk 10-132

+

i.v.

5
105

wk 10-132

0

i.v.

6

2x/wk a.d.o.

0

B6 BM 1
106 d 15 50 mg/kg STZ i.p. d 1-5

Resultd

Route

i.v.

i.v.

1
10

+ 2

±

a, Disease was induced by intraperitoneal (i.p.) injection of streptozotocin (STZ) for 4-5 days. In certain studies, diabetic mice were subject to bone marrow transplantation by administration of C57BL/6 (B6) bone marrow (BM) cells or pre-diabetic mice were given T cells (TC) from diabetic NOD mice to induce disease after total body irradiation (TBI). b and c, MSCs derived from mouse or human BM were delivered by intravenous (i.v.), intraperitoneal (i.p.), or intraventricular (1) injection at the indicated doses and days post disease induction or once a week (2) at the indicated weeks of age (wk) or twice a week after disease onset (a.d.o.). d, Results of the studies are summarized as follows: 0, no effect; +, beneficial effect on hyperglycemia; ±: some improvement in the incidence of diabetes and in insulitis scores. B6D2F1: (C57BL/6
DBA/2)F1; Gy: gray.

cemia was reversed, the number of insulin positive islets was increased, and the number of infiltrates in the pancreas was reduced. The observation that the glucose tolerance test was only slightly improved [135] suggests that in the presence of high glucose levels, the residual beta cell function was not enough to normalize the blood glucose levels. The authors propose that MSCs are mediating the regeneration of the islets since hyperglycemia was reversed and since they were able to visualize islets. Whether MSCs differentiated into beta cells is not clear although it has been reported that MSCs can be induced to express insulin and form islet-like clusters under certain conditions [137-140]. The observation that total urine albumin was decreased [135] in MSC-treated mice and that the kidneys appeared more normal histologically [136] suggests that the MSCs were also providing a benefit to the kidneys. These data support other reports demonstrating that MSC treatment can attenuate or protect animals from developing other diabetes-related complications [141-144]. Urbán and colleagues [145] also reported diabetic STZ-treated mice to benefit from MSC treatment but only when the MSCs accompanied a bone marrow transplant. The STZ model has also been used in islet transplantation ex-

periments and has shown that MSCs can protect allogeneic islets from rejection when co-transplanted [146, 147]. Although the STZ model has provided evidence that MSC therapy might be useful in treating type 1 diabetes, this model does not account for ongoing autoimmune destruction like the NOD model discussed below. Unlike the STZ model, NOD mice spontaneously develop a type 1 diabetes-like autoimmune disease. One dose of MSCs (100,000 cells) administered to 4 week old prediabetic NOD mice provided some protection from disease and, even though the effect was not robust under the conditions tested, the same MSCs conferred protection against the transfer of diabetes by T cells isolated from diabetic NOD mice when higher doses of MSCs were used [148] . The recipient mice were irradiated before T cell and MSC transfer which might have positively affected MSC suppression since total body irradiation has been shown to increase MSC engraftment in various organs [149, 150]. More Tregs were present in mice receiving the diabetogenic T cells and MSCs compared to those receiving T cells alone, suggesting that Tregs are involved in the MSC-mediated suppression of the

Immunomodulatory Activity of Mesenchymal Stem Cells

disease. Most likely, MSC treatment at 4 weeks of age was too early and too few cells were delivered because, when multiple MSC doses (500,000 cells/dose) were administered to NOD mice starting at 10 weeks of age before disease onset, better prevention was observed. In addition, MSC treatment temporarily reversed hyperglycemia in mice with new onset disease if multiple MSC doses were delivered within a few days of diabetes onset [151]. A reduction in insulitis was observed and MSCs were shown to suppress diabetesspecific T cell proliferation [148, 151] and IFN- production in vitro [151] while increasing IL-10 release. The preferential presence of exogenously delivered MSCs in the pancreatic draining lymph node and the pancreas suggests that MSCs can influence the autoreactive T cell response during T cell activation in the lymph node or once the activated T cells reach the pancreas. Th17 cells have recently been shown to play a pathogenic role in NOD mice [152, 153] but whether MSCs are inhibiting this cell type in the NOD model with a consequent effect on disease remains to be determined. MSCs derived from NOD mice as opposed to normal mice were not protective against the development of diabetes [151]. This finding is not necessarily surprising since several reports suggest that MSC derived from humans and mice with autoimmune disease [154-157] or cancer [158] are defective. The authors also provide evidence to suggest that MSC derived from an autoimmune-prone background might become tumorigenic since NOD MSC-treated mice developed tumors while Balb/c MSC-treated mice did not. It is important to note that only 20% (6/29) of the NOD MSCtreated mice in the prevention study developed tumors, and while 100% of the animals in the reversal study showed tumor growth, the group consisted of only 3 mice, too few to be conclusive. Although several reports in the literature show MSC transformation in vivo [159-161], only late passage MSCs (immortalized MSCs) administered to immunodeficient mice were found to be tumorigenic. Primary early passage MSCs were not tumorigenic and, to minimize the potential for transformation, only early passage MSCs are being used in the clinic. Because defective MSCs, or those derived from individuals predisposed to autoimmune disease or cancer, might contribute to disease development, it might be advantageous to screen bone marrow donors from which MSCs are derived for biomarkers of disease if possible. Collectively, these data suggest that allogeneic MSCs from normal individuals might be useful in treating type 1 diabetes in the clinic. A clinical trial evaluating MSC therapy in recent onset type 1 diabetic patients is underway as described below. 3.5. MSC Treatment in Inflammatory Bowel Disease Models Only in the last 2 years has the impact of MSC treatment been evaluated in inflammatory bowel disease (IBD) models (Table 5). In general, IBD encompasses two syndromes, Crohn’s disease and ulcerative colitis, and it is now appreciated that these diseases result from chronic mucosal immune responses to gut microflora that are normally harmless and are considered “autoimmune” responses because of the persistence of the mucosal antigens. Inflammatory responses of innate immune cells, such as macrophages, drive excessive

Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

11

activation of mucosal T cell (and B cell) responses that cause tissue damage and this has led to therapies that target innate immune mediators (i.e. anti-TNF-
) and T cells (i.e. antiCD4). Although there is no single mouse model that recapitulates Crohn’s disease or ulcerative colitis completely, many murine models of mucosal inflammation have been generated that use chemicals or genetically modified animals (transgenic or knock-out mice) to induce disease and can be classified as Th1- or Th2-type T cell driven models [162] To date, four pre-clinical studies have been reported using chemically-induced IBD to test the impact of MSC therapy in murine [163-165] and rat [166] models. Human or mouse as well as rat syngeneic and allogeneic MSC treatment protected animals from developing acute colitis and attenuated chronic colonic inflammation, restored mucosal tissues, diminished weight loss, and led to increased survival. Pro-inflammatory cytokines and chemokines, such as TNF-
, IFN-, IL-1, IL-6, IL-17, MIP-2 and RANTES, were reduced in the sera [164] and in colonic sites [163-166] whereas IL-10 was upregulated. T cell and macrophage infiltration in the colon [163-165] and Th1 responses to restimulation in vitro [164, 165] were reduced with MSC treatment and an increase in Tregs was observed in mesenteric lymph nodes and in the colon [163-165], suggesting Treg involvement in the MSC effects. Tregs were found to contribute to MSC immunomodulation in vivo because MSC-treated mice were no longer protected when given antiCD25 antibodies [165]. In addition, CD25-depleted CD4+ T cells from MSC-treated mice adoptively transferred disease whereas mice administered non-depleted T cells were partially protected and those given non-depleted T cells from control mice were not [164, 165]. The presence of the exogenous MSCs in the inflamed colon and mesenteric lymph nodes [164-166] correlated with the restoration of the colonic architecture and attenuation of immune responses at these sites. These data show that MSC treatment ameliorates IBD in animal models and support the study of MSC therapy for IBD. This concept is currently being investigated in the clinic in Crohn’s disease patients as discussed later. 3.6. MSC Treatment in Lupus Disease Models As described above, MSC treatment of autoimmune diseases has primarily focused on diseases predominantly driven by Th1- and Th17-T cell responses. Whether MSCs can be used to treat autoimmune diseases in general is unclear. To begin to address this, the therapeutic potential of MSCs has been tested recently in mouse models of systemic lupus erythematosus (SLE) (Table 6), a disease characterized by the development of pathogenic Th2-type T cell and B cell responses leading to the formation of pathogenic autoantibodies against various cellular components, most notably double-stranded DNA (dsDNA), a hallmark of the disease [167]. Some reports have indicated that MSCs can inhibit B cell proliferation and antibody production in vitro [32, 53, 80-84] suggesting that treatment with MSCs might provide a benefit in a lupus setting. The therapeutic potential of MSCs has been tested in two well-characterized models of SLE, the MLR/lpr [168, 169] and the NZB/WF1 [24] models, with contradictory findings. In some studies, a reduction in the levels of anti-dsDNA and

12 Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

Kaplan et al.

Table 5. MSC Treatment of Inflammatory Bowel Disease Models

Author

Mouse Model

Tanaka

Disease Inductiona 4% DSS oral d 0-7

2008 [166]

3 mg TNBS i.r. d 0 González 2009 [164]

Balb/c

5 mg TNBS i.r. d 0 1.5 mg TNBS i.r. d 0+9

MSC Administrationc

MSC Sourceb

Rat BM

Human AD

Dose

Time

i.v.

5
106

0+2+4

+

3
105

0

+

i.p.

Human AD

i.p.

Human AD

i.p.

Human AD

i.p.

5% DSS oral d 0-7 C57BL/6

Balb/c BM

3% DSS oral d 0-7+17-24

2009 [163]

1
10

6

0

+

6-14

+

3
10

5

0

±

1
10

6

0

+

1
106

0

+

1
105

Gonzalez-Rey 2009 [165]

Zhang

Resultd

Route

C57BL/6

3% DSS oral d 0-7

i.p.

5
10

0

5

1
106

2

± +

5
10

6

1
10

6

2

+

6

2

+

7

+

+

Balb/c BM

i.p.

1
10

Human AD

i.p.

1
106

7+24

+

Human BM

i.p.

2
10

6

1

+

Human GV

i.p.

2
106

1

+

a, Disease was induced by oral administration of dextran sulfate sodium (DSS) in the drinking water (oral) or by intrarectal (i.r.) delivery of trinitrobenzene sulfonic acid (TNBS). b and c, MSCs derived from rat, mouse, or human bone marrow (BM), adipose tissue (AD), or gingival tissue (GV) were delivered by intravenous (i.v.) or intraperitoneal (i.p.) injection at the indicated doses and days post disease induction. d, Results of the studies are summarized as follows: 0, no effect; +, beneficial effect on survival, body weight, overall clinical score, and/or organ pathology; ±, slight improvement in survival, body weight, clinical score, and/or organ pathology.

anti-nuclear antigen antibody, deposition of complement and immune complexes, and infiltration of T cells in the kidney of MRL/lpr mice treated with one or two doses of allogeneic C3H/HeJ-derived MSCs or human MSCs was observed [168, 169]. These data suggest that MSCs mediated protection by affecting T cell and antibody responses. A decrease in IL-4 and an increase in IFN-
levels in the serum suggested a shift from a Th2-type to a Th1-type response. IL-17 and Th17 cells contribute to lupus pathology [170] and IL17-producing T cells and serum IL-17 levels were decreased in MSC-treated mice. In addition, an increase in Tregs was observed. Together, the changes led to a drop in the percentage of CD138+ plasma cells in the bone marrow and ultimately in serum anti-dsDNA autoantibody levels. In contrast to the studies above, we have found that MSC treatment of NZB/WF1 mice with allogeneic Balb/c MSCs did not protect the mice from disease but in fact exacerbated disease [24]. MSCs were delivered every other week starting either prior to the onset of overt disease or during active disease. MSC administration worsened disease and enhanced the production of autoantibodies against dsDNA. The increase in autoantibody titers was accompanied by an increase in plasma cells in the bone marrow, increased immune complex deposition in the glomeruli, more severe kidney pathol-

ogy, and greater proteinuria. Co-culturing MSCs with plasma cells isolated from NZB/WF1 mice led to an increase in IgG antibody production, suggesting that MSCs might be augmenting plasma cell survival and function in MSCtreated animals. MSC therapy may not be beneficial in diseases primarily driven by Th2-type T cell and B cell responses such as lupus, and further study is needed. Several reasons may explain why the findings in MRL/lpr and NZB/WF1 studies appear contradictory and are difficult to compare. These include the number of MSC doses given, the source of MSCs, and the models being tested. For example, in the MLR/lpr studies, only one or two doses of C3H/HeJ-derived MSCs were delivered whereas multiple doses of Balb/c-derived MSCs were administered over the course of the NZB/WF1 study. C3H/HeJ mice are inherently defective in TLR4 and whether these cells are comparable to Balb/c MSCs in immunomodulation capability is unknown. MSCs from various mouse strains have been shown to differ in their growth rate, differentiation capacity, and phenotype [171] which might translate to differences in in vivo efficacy. In addition, MRL/lpr mice harbor a mutation in the lpr gene which codes for the Fas antigen, a protein involved in cell death. The lpr mutation results in a defect in apoptosis and on certain genetic backgrounds [172],

Immunomodulatory Activity of Mesenchymal Stem Cells

Table 6.

Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

13

The Treatment of Lupus Models with MSCs

Author Zhou 2008 [169] Sun 2010 [168] Youd 2010 [24]

Mouse Modela

MSC Administrationc

MSC Sourceb

Resultd

Route

Dose

Time

1
106

1x a.d.o.

+

wk 9+16

+

MRL/lpr

human BM

i.v.

MRL/lpr

C3H/HeJ BM

i.v.

NZB/W F1

Balb/c BM

i.p.

0.1
106/10 g body weight

wk 21+23+25 1
106

+27+31+33+35

-

wk 32+34+36 (a.d.o.)

-

a-c, MRL/lpr or (New Zealand black (NZB)
New Zealand white (NZW))F1 (NZB/W F1) mice were treated with mouse or human bone marrow (BM) derived MSCs by intravenous (i.v.) or intraperitoneal (i.p.) injection at the indicated doses either after disease onset (a.d.o.) or at the indicated weeks (wk) of age. d, Results of the studies are summarized as follows: +, reduction in autoantibody levels, immune complex deposition, proteinuria, and/or renal pathology; -, increase in autoantibody levels, immune complex deposition, and proteinuria, and more severe renal pathology

such as the MRL background, allows for polyclonal lymphocyte activation and expansion, autoantibody production, immune complex deposition, and nephritis. NZB/WF1 mice carry susceptibility genes but no mutation has been genetically introduced that causes disease to develop. 4. CLINICAL STUDIES Human MSCs have been used in systemic transplantation in the clinic for many indications including treatment of damaged tissues in children with severe osteogenic imperfecta [173] and in acute myocardial infarction patients [174], as well as to replace enzymes in Hurler’s syndrome [175]. Furthermore, the properties of MSCs give rise to their potential use in a broad range of inflammatory and immunemediated conditions such as GvHD and Crohn’s disease. To date, there are over 100 MSC clinical trials either registered or active on clinicaltrials.gov. For the majority of these trials, results are not yet available. This review will focus on data available from clinical trials in which MSCs were used for bone marrow recovery following ablation and hematopoietic stem cell transplantation (HSCT), immune-mediated diseases such as GvHD and Crohn’s disease, and in autoimmune disorders such as type 1 diabetes mellitus and multiple sclerosis (MS). 4.1. MSC Co-Transplantation for Hematopoietic Recovery Following HSCT MSCs have been suggested to be the precursor cells in the bone marrow stroma that promote hematopoiesis. Animal data have shown that adult human MSCs promote hematopoietic engraftment in immunodeficient mice [176]. Therefore, it has been postulated that MSCs may enhance engraftment after high dose chemotherapy and autologous or allogeneic hematopoietic stem cell transplantation. One of the first examples of the use of ex vivo-expanded autologous MSCs for bone marrow recovery came from Lazarus and colleagues who demonstrated that infusion was welltolerated by 15 patients with hematological malignancies in complete remission [177]. In further studies, they used single infusions of autologous human MSCs in patients with advanced breast cancer to promote hematopoietic recovery

after autologous stem cell transplant [178]. These investigators went on to report the results of an open-label, multicenter trial of co-transplantation of culture-expanded MSCs and hematopoietic stem cells from HLA-identical sibling donors after myeloablative therapy in an attempt to facilitate engraftment and lessen graft-versus-host disease. The infusions were well-tolerated, however, the engraftment of neutrophils and platelets was similar to historical reports [179]. More recently, Le Blanc and colleagues were the first to report the successful use of HLA-haploidentical MSCs to enhance engraftment when co-transplanted with HSCT [180]. Macmillan et al. confirmed the safety of haploidentical MSC cotransplantation with HSCT with an overall observed reduction in mortality from 45% to 22% [181]. 4.2. Graft-Versus-Host Disease Clinical Trials A promising area of clinical application is the use of systemic transplantation of MSCs in treatment of GvHD, a life threatening immunological reaction that occurs in certain patients following bone marrow transplantation. The rationale behind the use of MSCs in this context is the increasing evidence that MSCs exert immune-regulatory effects. In 2004, Le Blanc et al. reported that third party haploidentical MSCs successfully treated refractory, severe acute GvHD in a 9 year old boy who had received an HLA identical peripheral blood stem cell transplant from an unrelated donor [182]. This was the first case report to suggest efficacy of MSCs in reducing inflammation in GvHD. This was followed by a pilot study using MSCs to treat steroid refractory GvHD after allogeneic HSCT. Here, 8 patients with steroid refractory GvHD were treated with a combination of identical sibling, haploidentical, and HLA-mismatched donor MSCs. Overall survival was significantly improved in the MSC-treated group, resulting in resolution of GvHD in 6/8 patients [183]. In 2008, Le Blanc et al. reported the results of a Phase II multicenter study of 55 steroid refractory GvHD patients treated with expanded MSCs [184]. Thirty out of 55 patients had a complete response. Among those patients who had a complete response, there was a significant improvement in the 2-year overall survival rate (53% vs. 16% for patients with a partial response and/or no response) and a

14 Current Stem Cell Research & Therapy, 2011, Vol. 6, No. 4

significant decrease in the 1-year transplant-related mortality. These results were not reproducible in a 13 patient study by von Bonin and colleagues using MSCs grown in platelet lysate-containing medium [185], suggesting the need for larger, controlled trials with MSCs grown under uniform GMP conditions. Based on these early results of MSC therapy for steroid refractory GvHD, Osiris Therapeutics tested whether the addition of unrelated, culture-expanded human MSCs (Prochymal®) used in combination with standard corticosteroid therapy for the treatment of de novo acute GvHD would be beneficial. The study was an open-label, randomized, multicenter, phase II trial evaluating two different dose levels of Prochymal® in combination with standard of care. The 32 patient study measured induction of response, overall response of acute GvHD by day 28, and long-term safety. Ninety-four percent of evaluable patients had an initial response to Prochymal® (29/31), where 24 patients (77%) had a complete response and 5 (16%) had a partial response. Of the patients who achieved a complete response, there was no statistical difference between the high and low dose of Prochymal®, and 22 of the 24 (92%) complete response patients survived to day 90. A comparison of response between the two donors for the majority of the MSCs administered showed no difference [186]. At 6 months, 61% of patients who achieved a complete response still had a durable response requiring no additional clinical intervention [33]. Previously published historical data showed that less than 35% of patients achieve this endpoint when treated with steroids alone [187, 188]. This study represents the first prospective trial of third-party, unmatched MSCs for the treatment of de novo acute GvHD. These results are similar to the aforementioned reports of MSCs used in the treatment of steroid refractory GvHD [182, 183]. The results of this study provide evidence that MSCs can effectively induce a response in a high percentage of GvHD cases when used in combination with existing therapy, and may improve overall outcome. Most recently, Osiris Therapeutics conducted a Phase III, randomized placebo controlled study of 244 patients with steroid refractory GvHD. Prochymal® was evaluated in combination with standard of care in a trial of patients with steroid refractory GvHD. Patients received 8 infusions of 2 x 106 MSCs/kg over 4 weeks, with an additional 4 infusions administered weekly after day 28 in patients who had a partial response, defined as improvement in at least one organ without progression in others. The results of this study illustrate the heterogeneity of steroid refractory GvHD with the more severe patients showing the most benefit. The results for a subset of these adult patients treated with Prochymal® who had multi-organ GvHD (skin, liver, and gut) showed an overall complete or partial response rate of 63% vs 0% for the placebo treated group (n=22) at day 28. In addition, patients treated with Prochymal® had less progression of liver GvHD at weeks 2 and 4 compared to the placebo-treated group. The incidence of infections was not different between arms. These results suggest that the addition of Prochymal® produced significant improvement without additive toxicity in patients with steroid refractory GvHD involving liver or gastrointestinal organs [189].

Kaplan et al.

This study was additionally analyzed to stratify the pediatric patient population (