Potential therapeutic applications of mesenchymal stromal cells

Pathology (October 2011) 43(6), pp. 592–604

CELLULAR THERAPIES

Potential therapeutic applications of mesenchymal stromal cells STEPHEN LARSEN*

AND IAN

D. LEWIS{z

*Institute of Haematology, Royal Prince Alfred Hospital, Camperdown, New South Wales, {Haematology, IMVS/SA Pathology and Royal Adelaide Hospital, Adelaide, and zSchool of Medicine, University of Adelaide, Adelaide, South Australia, Australia

Summary Mesenchymal stromal cells (MSCs) are a non-homogeneous population of plastic-adherent cells which were initially isolated from post-natal bone marrow. They have the capacity to differentiate to multiple mesodermal lineages including bone, cartilage and adipose tissue. In stringent culture conditions, MSCs can also be induced to differentiate into different cell types of endoderm and neuroectoderm lineages. To date, no specific marker identifies MSCs, although a number of cell surface antigens have been described which enrich for MSCs. Mesenchymal stromal cells possess a number of properties which have generated considerable interest in diverse cellular therapeutic applications. The capacity of MSCs to differentiate into multiple different cell lineages has seen them actively explored for tissue repair, particularly in cardiac, orthopaedic and neurological applications. A large body of data indicates that MSCs possess immunomodulatory properties. Mesenchymal stromal cells are immunosuppressive, interacting with T lymphocytes, antigen presenting cells, B lymphocytes, and natural killer cells. In addition, they are immunoprivileged, allowing transplantation across allogeneic barriers. These immunomodulatory properties have seen infusion of MSCs for the treatment of steroid refractory graft versus host disease, a life threatening complication of haemopoietic cell transplantation, with promising results. Furthermore, these immune functions may lead to roles in the facilitation of engraftment, induction of tolerance and as therapy in autoimmune disease. Abbreviations: aGVHD, acute graft versus host disease; CFU-F, colonyforming units-fibroblast; cGVHD, chronic graft versus host disease; DCs, dendritic cells; EAE, experimental autoimmune encephalomyelitis; FCS, fetal calf serum; GVHD, graft versus host disease; HLA, human leukocyte antigen; HCT, haemopoietic cell transplantation; HSC, haemopoietic stem cells; LTC-ICs, long-term culture-initiating cells; MSCs, mesenchymal stromal cells; SR-GVHD, steroid refractory graft versus host disease; Treg, regulatory T lymphocytes. Key words: Mesenchymal stromal cells, immunomodulatory properties, graft versus host disease, tissue repair.

INTRODUCTION Mesenchymal stromal cells (MSCs) are a rare population of cells initially isolated from post-natal bone marrow. The first description of these cells, more than 40 years ago, described an adherent cell population with fibroblast-like morphology.1 These rare cells are clonogenic, giving rise to colonies termed Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0b013e32834ab72d

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colony-forming units-fibroblast (CFU-F).2 Subsequently it was demonstrated that these cells were of mesodermal lineage and in appropriate in vitro conditions were able to differentiate into osteoblasts, chondrocytes and adipocytes.3 Initially these cells were designated as mesenchymal stem cells, but as they have not been shown to possess the stem cell characteristics of selfrenewal or multi-potent differentiation capacity in vivo, the term ‘multipotent mesenchymal stromal cells’ is preferred.4 Characterisation of MSCs involves a combination of culture properties, phenotypic marker expression, multi-lineage differentiation capacity and identification of tissue of origin. The International Society of Cellular Therapy produced a position paper in 2006 to harmonise the definition of MSCs.5 The properties of MSCs are: 1. Adherence to plastic in standard culture conditions. 2. Phenotype: positive (95%þ) CD105, CD73, CD90; negative (2%þ) CD45, CD34, CD14 or CD11b, CD79a or CD19, HLA-DR. 3. In vitro differentiation: osteoblasts, adipocytes, chondroblasts. To date, no marker has been identified that specifically identifies MSCs, although a number have been proposed including STRO-1, SSEA-4, CD271, frizzled-9 and CD106.6–10 It has become evident that cells fulfilling the criteria of MSCs can be isolated from a number of tissues including cord blood, amniotic fluid, liver, lung, and placenta.11–15 The true nature of MSCs, including their origin, tissue location and function has yet to be defined. Recent studies have contributed to defining a population of cells thought to be more representative of immature MSCs based on anatomical location and phenotypic marker. Pericytes, isolated from perivascular tissue from different organs expressing CD146, NG2 and PDGF-b demonstrate multi-lineage differentiation ability and are clonal at the single cell level.16 Recently, nestin positive cells isolated from bone marrow have demonstrated CFU-F activity and have been shown to self-renew and play an important role in the environment of the bone marrow niche.17 The in vitro properties of MSCs have generated great interest in their potential for clinical applications. Broadly, MSCs are being explored in two therapeutic areas: tissue repair and immunomodulation. The multi-lineage differentiation capacity has been harnessed for a diverse range of tissue repair applications with particular interest in cardiac, orthopaedic and neurological indications. The unique immune properties of MSCs have enabled use of third party allogeneic cells. Furthermore, these immune properties have seen MSCs used in transplantation medicine and autoimmune diseases.

2011 Royal College of Pathologists of Australasia

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POTENTIAL THERAPEUTIC APPLICATIONS OF MESENCHYMAL STROMAL CELLS

For use in cellular therapy, MSCs need to be isolated and expanded in vitro to obtain sufficient cell numbers for clinical application. Different tissue sources of MSCs are utilised, but bone marrow derived MSCs are the most widely studied. The majority of culture systems rely on plastic adherence of cells, rather than prospective isolation, and most of the properties of MSCs have been derived from cells cultured in vitro. Fetal calf serum (FCS) is the most common component of expansion systems, but is heterogeneous, and careful batch selection is necessary to optimise cell expansion.18 Potential risks of using FCS include immune rejection19 and transmission of zoonoses and prions. To reduce these risks, different serum-free conditions have been tested, with basic fibroblast growth factor and transforming growth factor-b being important components.20,21 Platelet lysate, which contains high concentrations of growth factors, is emerging as a promising alternative to FCS with studies showing high proliferative capacity.22–24 In summary, MSCs can be isolated from different tissue sources and expanded in different culture conditions to produce cellular populations fulfilling the criteria for MSCs. The optimal cell source, isolation method and culture conditions have not been defined, and are a major challenge in the field.

MSCs IN HAEMOPOIESIS Physiological role in the bone marrow microenvironment The coexistence of normal haemopoietic stem cells (HSCs) and a microenvironment, mostly located in the medullary part of bones, is required for normal bone marrow function; this represents the so-called stem cell niche model.25 The bone marrow microenvironment, often called the stroma, consists of a heterogeneous population of non-haemopoietic and haemopoietic cells as well as extracellular matrix, which collectively provide the structural scaffold, the spatial framework and the appropriate physiological cues to control HSC maintenance and function. Ever since the stem cell model of haemopoiesis was established, the putative stromal counterpart of the HSC has been sought and was expected to give rise to the different non-haemopoietic components of the bone marrow. It was Friedenstein et al. who first demonstrated that bone marrow does indeed contain non-haemopoietic cells capable of differentiating into osteoblasts and fibrous tissue in a series of influential experimental works in the late 1960s and 70s.1,26 The development of discrete colonies of plastic-adherent elongated cells after seeding of bone marrow cells, called CFU-F, is analogous to the term established for haemopoietic progenitor colonies.2 The subcutaneous transplantation of the clonal progeny of a single CFU-F could give rise to fibrous tissue, bone and bone containing marrow, suggesting that at least some of the transplanted colonies were able to reconstitute the haemopoietic microenvironment. These in vivo data were further corroborated with the establishment of a system of murine long-term cultures demonstrating that bone marrow stromal cells can maintain haemopoiesis for periods longer than 6 months.27 Simmons and Torok-Storb demonstrated that a subset of human bone marrow stromal cells expressing the STRO-1 antigen possesses haemopoietic supporting ability.28 STRO-l-selected adherent layers were able to support the generation of clonogenic cells and mature haemopoietic cells from a population of CD34þ cells highly enriched in long-term culture-initiating cells (LTC-ICs) over an 8 week period.

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Studies from the early 1970s have suggested that osteoblasts have an important role in the regulation of primitive haemopoietic cells. Gong demonstrated that HSCs are preferentially localised near or at the endosteum.29 More recent in vivo experiments have shown that when progenitor cells are injected into irradiated mice they tend to home closer to the endosteum.30 These experiments also interestingly suggest that more immature subsets are found nearer to the endosteum, in comparison to the more mature HSCs, which reside further away.31,32 Taichman showed that murine osteoblast cell lines and primary human osteoblasts produce various haemopoietic cytokines.33 The same investigators have shown that primary human osteoblasts can stimulate growth and maintain clonogenic potential of CD34þ cells in vitro and to support LTC-ICs.34 Visnjic et al. demonstrated that when osteoblast deficiency is induced in mice, haemopoiesis is severely limited with a significant reduction in both myeloid and lymphoid lineages.35 Consistent with this finding, Zhu et al. demonstrated that primary murine osteoblasts support all stages of B lymphopoiesis in vitro,36 a process that requires contact with osteoblasts, and Taichman and Emerson showed that these cells support the survival and limited proliferation of myeloid progenitors.37 Interestingly, it may be that only more primitive subpopulations of mesenchymal osteolineage cells are critical for HSC regulation. Recently it was demonstrated that targeted deletion of Dicer1 (an endonuclease necessary for microRNA biogenesis and RNA process) in murine osteoprogenitors, but not in mature osteoblasts, disrupted haemopoiesis.38 Mice developed a phenotype which recapitulated many features of human myelodysplastic syndrome, including evolution to acute myeloid leukaemia. Collectively, these findings suggest that osteoblasts are a key cell type within the HSC niche and that molecules expressed by these cells may have previously unrecognised roles in regulating haemopoiesis. One molecule that shows high levels of expression in osteoblast cells lining bone is osteopontin.39 Osteopontin is a multidomain, phosphorylated glycoprotein synthesised by many cell types and involved in many physiological and pathological processes, including cell adhesion, angiogenesis, apoptosis, inflammatory responses, and tumour metastasis. Nilsson et al. demonstrated that osteopontin participates in HSC location (it contributes to HSC transmarrow migration toward the endosteal region) and as a physiological-negative regulator of HSC proliferation.40 It should be noted that the endosteal region is highly vascularised and osteoblasts are in close proximity to capillaries and sinusoids. New technologies such as high resolution confocal microscopy and two-photon video imaging demonstrate that bone marrow HSCs selectively localise around sinusoids under steady state condition.31,41 It has been demonstrated that a rare subset of MSCs that surrounds bone marrow blood vessels and harbour the neuro-ectoderm stem cell marker nestin, are spatially associated with HSCs and express high levels of HSC maintenance genes (CXCL12, angiopoietin-1, c-kit ligand, vascular cell adhesion molecule-1, interleukin- 7 and osteopontin).17 Purified HSCs homed to nestinþ MSCs in the bone marrow of lethally irradiated mice and selective in vivo nestinþ cell depletion rapidly reduced HSC/progenitors. Nestinþ MSCs were found to be innervated by the sympathetic nervous system and b3-adrenergic stimulation reduced the expression of HSC maintenance genes. Thus, it has thus been proposed that nestinþ MSCs could serve as mediators for the nervous system regulation of haemopoiesis.

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Therapeutic use of MSCs to improve haemopoietic stem cell engraftment Given the strong evidence of the role of MSCs and their progeny in haemopoiesis, there is a lot of interest in the possibility that MSCs may have promoting effects on HSC engraftment and repopulation. Several studies have explored the co-transplantation of HSCs and MSCs in animal models and humans, with some evidence that there is increased chimaerism and/or haemopoietic recovery.15,42,43 One of the hurdles that is faced when transplanting MSCs concomitantly with HSCs is that there is good evidence that very few cells make their way to the bone marrow when infused intravenously, but rather are trapped in the lung, presumably due to their large size.44,45 Conversely, Muguruma et al. showed that following intramedullary transplantation of human ex vivo-expanded labelled MSCs into the bone marrow of non-obese diabetic/severe combined immunodeficiency mice, donor-MSCs did engraft into the bone marrow stroma.46 In addition, the presence of human MSCs in murine bone marrow was shown to increase engraftment of primitive human haemopoietic cells and to improve the function of the latter. Koc et al. reported on the infusion of 28 patients with advanced breast cancer with peripheral blood stem cells and culture expanded bone marrow MSCs. The infusion was deemed to be feasible and safe and there was rapid recovery of haemopoiesis, however the study was not randomised so no firm conclusions could be made.42 In the largest reported number of cases, Lazarus et al. conducted a multicentre clinical trial evaluating the safety of allogeneic MSC infusion in 46 patients with haematological malignancies undergoing myeloablative haemopoietic cell transplantation (HCT).47 No acute or long-term MSC-associated toxicities were observed and prompt haemopoietic recovery was observed in most patients. However, when compared to controls, there was no difference. Le Blanc et al. administered MSCs together with allogeneic peripheral blood stem cells in seven patients; three patients for graft failure and four patients were included in a pilot study.48 Co-infusion resulted in fast engraftment of absolute neutrophil count (ANC) and platelets within 30 days and 100% donor chimaerism, even in the three patients regrafted for graft failure/rejection. The possibility that the infusion of MSCs may aid haemopoietic recovery in patients who have graft failure has also been explored. Meuleman et al. reported on six patients with poor haemopoietic recovery following allogeneic HCT.49 MSCs were infused 50–294 days post-transplant with the aim of functionally improving the bone marrow microenvironment. Two patients, both transplanted in first complete remission, showed rapid haemopoietic recovery. MSCs to facilitate ex vivo expansion of cord blood progenitors The groups at the M. D. Anderson Cancer Center and The Brown Foundation Institute of Molecular Medicine have explored using MSCs ex vivo to help facilitate the expansion of haemopoietic progenitors in cord blood units.50 Umbilical cord blood is becoming an important source of haemopoietic progenitors for transplantation in patients lacking human leukocyte antigen (HLA)-matched donors. One disadvantage associated with the use of cord blood for transplantation is the relatively low cell dose available. This contributes, at least in part, to the slower engraftment and an elevated risk of

Pathology (2011), 43(6), October

engraftment failure that is associated with cord blood transplantation.51 The time required for a patient receiving a cord blood transplantation to achieve an absolute neutrophil count of 0.5  109/L can range from 23 to 41 days. Engraftment failure rates in adults as high as 20% have been reported.52 Even with the use of double cords to increase cell dose, the median time to neutrophil engraftment is still significantly prolonged compared to peripheral blood stem cells at 23 days (range 15– 41 days).53 Attempts to increase haemopoietic progenitors using ex vivo expansion have been explored in a peripheral blood stem cell setting with varied success.54–56 Whilst these studies have set the stage for use of this approach with cord blood cells, one concern that exists is that short-term reconstituting haemopoietic progenitors are expanded at the expense of longer-term reconstituting progenitors, thereby significantly impacting the haemopoietic reserve of the graft.57 Robinson et al. have attempted to avoid this problem by co-culturing cord blood cells with MSCs on the basis that these cells are part of the microenvironment,50 as it has been previously shown that contact between primitive haemopoietic progenitors and stromal components of the haemopoietic microenvironment preserve stem cell activity.58 Both family-derived MSCs and an off-the-shelf MSC product (Revascor; Angioblast Systems, USA) have been tested and in both cases time to both neutrophil (15 days) and platelet engraftment (30 and 38 days, respectively) were improved compared to peripheral blood stem cell controls. Cells from expanded units were co-infused with cells from an unexpanded unit; on transplant day þ21, the chimaerism assays revealed that the MSC-expanded unit contributed to engraftment with a mean of 19% of the mononuclear cell, 16% of the T cell, and 14% of the myeloid fractions due to the expanded unit. Interestingly, haemopoiesis was increasingly derived from the unexpanded unit with long-term engraftment provided exclusively by the unexpanded unit by 6 months post-transplant in the vast majority of patients.

IMMUNOMODULATORY PROPERTIES OF MSCs The interest generated by the properties and potential uses of mesenchymal stromal cells relates to their differentiation capacity and immunological properties. MSCs are immunoprivileged and immunomodulatory. Many of the functions and properties of MSCs have been derived from specific experimental conditions with, in some cases, contrasting functions observed in different conditions. This has led to the interpretation of function being dependent on specific environmental influences as well as direct cell to cell interaction. Evidence is accumulating that function relies predominantly on the secretion of soluble factors.59 Immunoprivilege Although MSCs express MHC class I molecules and can be induced by interferon (IFN)-g to express class II molecules, they have minimal immunogenicity, and escape recognition by alloreactive cells.60–62 This may be directly related to their immunosuppressive properties by inhibition of effector cells that mediate rejection. This immunoprivileged state has been exploited in therapeutics with the use of allogeneic MSCs being transplanted across HLA barriers. This phenomenon also exists across species with data showing xenogeneic MSCs are not rejected by the host.63 Initial studies showed MSCs were not lysed by cytotoxic T lymphocytes or natural killer cells (NKCs).64,65 However,

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POTENTIAL THERAPEUTIC APPLICATIONS OF MESENCHYMAL STROMAL CELLS

recent data have challenged this with evidence showing autologous and allogeneic MSCs are susceptible to interleukin (IL)-2 activated NKC lysis.66–68 MSCs express class I HLA molecules, which are protective against NKC mediated lysis. The expression level is not great enough to be protective, as upregulation following exposure to IFN-g protects against lysis.68 Immunosuppression A large body of data shows that MSCs modulate the immune function of different cellular components that play roles in adaptive and innate immunity. Based on in vitro studies, the predominant property of MSCs appears to be immunosuppressive (Fig. 1). T lymphocytes play a critical role in adaptive immunity and regulate multiple facets of the immune system. MSCs affect several T cell functions. Human MSCs suppress the proliferation, cytokine production, and cytolytic activity of T cells that have been stimulated by alloantigens and non-specific mitogens.62,69–71 This has also been demonstrated in an in vivo model with MSC infusion prolonging the survival of skin grafts.72 The inhibition of T cell proliferation is dependent on direct cell-cell interaction73 and production of soluble factors.74,75 Data support cross-talk between MSCs and T lymphocytes with production of soluble factors by MSCs induced by T cells.76 Different mediators secreted by MSCs contribute to T lymphocyte inhibition including transforming growth factor-b, hepatocyte growth factor, nitric oxide, indoleamine 2,3-dioxygenase, and Jagged-1.69,77–80 MSCs also have a suppressive effect on cytotoxic T cells.64 Regulatory T lymphocytes (Treg), defined as CD4þ T cells expressing CD25 and Fox P3 induce peripheral tolerance and

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inhibit pro-inflammatory immune responses. In contrast to their inhibitory actions on CD4þ and CD8þ T lymphocytes, MSCs promote the expansion and function of Treg cells.81,82 Dendritic cells (DCs) are key components of the immune system, functioning as antigen presenting cells by presenting peptides and protein to T lymphocytes resulting in T cell induction and production of pro-inflammatory cytokines. MSCs modulate the proliferation and differentiation of monocyte-derived DCs, DCs derived from HSC, and slan DCs.83–86 The function of DCs is also inhibited as MSCs suppress the up-regulation of co-stimulatory molecules on DCs including MHC class II, CD40 and CD86 and suppress secretion of tumour necrosis factor-a, IL-12, IL-6, and macrophage-colony stimulating factor.83,87–89 NKCs have an important role in the maintenance of innate immunity, contributing to the elimination of virus infected and malignant cells. MSCs inhibit NKC proliferation, cytotoxicity and cytokine production.67,68 MSCs co-cultured with freshly isolated NKCs do not inhibit target cell lysis67 but NKCs cultured for 4–5 days are impaired.90 Evidence presented to date shows the anti-proliferative activities of MSCs is mediated by soluble factors such as HLA-G582 but the inhibition of NKC cytotoxicity requires cell to cell interaction.67 B lymphocytes play an important role in adaptive immunity with critical functions of antibody production, cytokine production and stimulation of T lymphocytes.91 MSCs inhibit B lymphocyte proliferation, differentiation, activation, antibody secretion and chemotaxis. T cell dependent stimulation of B cells, either by pokeweed mitogen, IL-4 or anti-CD40 antibody normally induces B cell proliferation but this is inhibited in the presence of MSCs.92,93 Utilising BXSB mice, a murine model of human systemic lupus erythematosus (SLE), allogeneic

Fig. 1 Immunomodulation by mesenchymal stromal cells (MSC). HGF, hepatocyte growth factor; HLA-G5, human leukocyte antigen-G5; IDO, indoleamine 2,3dioxygenase; IL-6, interleukin-6; M-CSF, macrophage-colony stimulating factor; NO, nitrous oxide; PGE2, prostaglandin-E2; TGF-b, transforming growth factor-b.

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MSCs inhibit B cell proliferation, activation and function.94 Although some studies have shown MSCs impair B lymphocyte differentiation and antibody production,91,95 other studies have shown MSCs promote B lymphocyte proliferation and function.65,96

IN VIVO EFFECTS OF MSCs Graft versus host disease Allogeneic HCT is a potentially curative treatment for patients with malignant and non-malignant haematological disorders. Graft versus host disease (GVHD) remains a major cause of morbidity and mortality following transplantation. Prevention of GVHD involves appropriate donor selection and administration of prophylactic immunosuppressive agents. Acute GVHD (aGVHD) usually occurs before day 100 posttransplant and can manifest as dermatitis, enteritis and hepatitis. Donor derived T cells mediate GVHD through alloreactive mechanisms due to differences in histocompatibility antigens between donor and recipient. Host antigen presenting cells present recipient peptide, which activates donor T cells, inducing proliferation and activation. These activated T cells induce apoptosis of target through cytokine and enzyme production and also recruit other effector cells including NKCs, macrophages and granulocytes. Mild to moderate aGVHD is usually controlled by the addition of corticosteroids to the immunosuppressive regimen. However aGVHD which does not respond to the addition of corticosteroids has an adverse outcome. Multiple different approaches to control steroid refractory aGVHD (SR-GVHD) have been tried. All rely on intense immunosuppression targeting donor T cells or effector mechanisms, including administration of cytokines, global T cell immunsuppression (anti-thymocyte globulin) or monoclonal antibodies. However, no therapy has been shown to be consistently effective and mortality from SR-GVHD approaches 80% with death due to either progressive GVHD or infection secondary to the effects of immunosuppression. Murine models of GVHD are well established and have produced insights into the pathophysiology of the disease and also potential therapies. However, results of MSC infusion in the prevention of GVHD have produced contrasting results. In a major histocompatibility complex allogeneic murine HCT model, irradiated MSCs infused at the time of bone marrow transplant reduced the severity of GVHD in recipient mice.97 In contrast, no benefit of MSC infusion in the prevention of GVHD was shown in a similar experimental model, despite the MSC population showing potent in vitro immunosuppressive properties.98 A possible explanation for this finding was the demonstration that MSCs need to be activated by exposing them to IFN-g to prevent GVHD.74 The immunomodulatory properties of MSCs have seen them actively evaluated in the management of SR-GVHD. A number of studies have been published (Table 1). Approaches differ in source of MSCs, culture conditions, dose, frequency of administration and definition of endpoints. On the whole the populations reported represent patients with advanced aGVHD who have failed multiple lines of immunosuppression. The first description of successful use of MSCs in the treatment of aGVHD was from the Karolinska group. A 9-year old-boy with severe refractory aGVHD was given two infusions of maternal haploidentical MSCs with complete resolution of symptoms. Following cessation of immunosuppression, GVHD

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recurred, and this episode responded to another MSC infusion.99 Subsequently, results of MSC infusion in eight patients were reported. Six patients achieved a complete remission, and five remain alive between 2 months and 3 years after transplantation, a superior outcome compared to historical controls.100 These results led to the establishment of a Phase II multicentre study of MSC infusion in SR-GVHD. In this study, an overall response of 71% in 55 patients treated with bone marrow derived MSCs was reported. The majority of MSCs were derived from third party unrelated donors. The MSCs were cultured in different laboratories but a common batch of FCS was used to ensure comparability of the final MSC product. Patients who achieved a complete remission had a 2 year survival of 52%. There was a suggestion of improved response in children.101 Other studies reported have assessed MSCs derived from different tissues or cultured in different media. Fang et al. reported that five of six patients with grade III–IV GVHD achieved complete remission after one or two infusions of MSCs which had been derived from adipose tissue.102 Because of concerns surrounding the use of cellular products cultured in FCS, MSCs cultured in conditions containing platelet lysate have also been assessed. von Bonin reported results of 13 adult patients who received between one and five infusions of MSCs. The majority of patients had grade IV aGVHD. An overall response of 58% was reported, with an 8 month overall survival of 31%. Patients with skin GVHD showed a better response.103 Lucchini et al. also evaluated bone marrow derived MSCs cultured in platelet lysate in a compassionate use program in a paediatric population. Eleven patients with either acute or chronic GVHD (cGVHD) received between one and four infusions of MSCs, with an overall response of 71% and a 10 month overall survival of 63%. This group also reported a better response in patients with skin aGVHD.104 As third party unrelated MSCs appear as efficacious as other donor sources, they lend themselves to ‘mass’ production to generate an ‘off the shelf’ cellular therapy product. The biotechnology company Osiris (USA) has facilitated a number of studies utilising its pre-manufactured universal donor MSC product (Prochymal). Kebriaei et al. reported results of Prochymal infusion in 32 adult patients with refractory aGVHD. The study was randomised between two doses of MSCs, 2  106/kg and 8  106/kg. All patients received two infusions. A high response rate was reported, with a complete remission rate of 77% and partial response of 16%. This is possibly due to a larger proportion of patients having grade II GVHD compared to other studies, rather than the higher cell dose recipients received. No difference was noted between the MSC dose of 2 or 8  106 cells/kg received.105 Prasad et al. infused 2 or 8  106/kg MSCs in a compassionate use program in a paediatric population. Infusions were administered twice a week for 4 weeks with responding patients receiving ongoing treatment, with one patient receiving 21 infusions. A complete remission rate of 58% was reported, with high response rates for patients with gut GVHD.106 In a large Phase III randomised study, Martin et al. reported, in abstract, results of 163 patients administered 8–12 infusions of Prochymal compared to 81 patients administered placebo. There was no difference in response rates between Prochymal and placebo treated patients. Post-hoc analysis showed benefit for Prochymal in patients with gut or hepatic GVHD.107 Kurtzberg et al. have also reported results of Prochymal

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38%

Survival

OS 64% (40 m)

CR 83%

OS 52% (2 y) (if CR)

OS 71% (d 90)

CR 77% PR 16%

2 (16) or 8 (15) 2

68% 23% 10% Prochymal

10% 45% 45% BM FCS 1.4 (0.4–9) 1 (27) 2 (22) 3–5 (6) CR 55% PR 16%

OS 31% (d 257)

CR 8% OR 54%

15% 85% BM Platelet lysate 0.9 (0.6–1.1) 2 (1–5)

13 58 (21–69)

Single centre, case series

Phase II, randomised, multicentre 31 34–67

von Bonin et al.103

Kebriaei et al.105

55 0.5–64

Phase II, multicentre

LeBlanc et al.101

DCR Skin Gut Liver

35% 78% 82%* 76%* NR

8–12

22% 51% 27% Prochymal

163 NR

Multicentre, Phase III randomised

Martin et al.107

30% 77% 68% 47% NR

8–12

26% 58% 16% Placebo

81 NR

OS 62% (d 100)

OR 64%

2 8–12

10% 33% 57% Prochymal

59 8 (median)

Expanded access

Kurtzberg et al.108

OS 63% (10 m)

CR 38% OR 71%

BM Platelet lysate 1.2 (0.7–3.7) 1–4

38% 12% 50%

8 4–15

Compassionate use, multicentre

Lucchini et al.104

OS 42% (d 611)

CR 58% OR 75%

2 (10) or 8 (2) 8 (2–21)

42% 58% Prochymal

12 0.4–15

Compassionate use, multicentre

Prasad et al.106

* p < 0.05. BM, bone marrow; CR, complete remission; d, days; DCR, durable complete response; FCS, fetal calf serum; GVHD, graft versus host disease; m, months; NR, not recorded; OR, overall response; OS, overall survival; PR, partial response; y, years.

OR 75%

33% 67% Adipose tissue FCS 1.0 1 (4) 2 (2)

75% 25% BM FCS 1 (0.7–9) 1 (6) 2 (2)

Response rates

6 22–49

Single arm, single centre

Single arm, single centre

8 8–61

Fang et al.102

Ringden et al.100

Summary of clinical experience of MSC infusion for the treatment of steroid refractory aGVHD

n Age GVHD grade I II III IV MSC source Media MSC dose (106/kg) Infusion number

Study

Table 1

POTENTIAL THERAPEUTIC APPLICATIONS OF MESENCHYMAL STROMAL CELLS

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infusion in a paediatric population (n ¼ 59) with SR-GVHD. An overall response of 62% was seen.108 MSC infusion has also been evaluated for the prevention of aGVHD. In a single arm study, 46 patients received 1–5  106 MSCs/kg 4 hours prior to infusion of HLA matched sibling peripheral blood or bone marrow HCT. No adverse events were noted. Reported rates of aGVHD were possibly reduced at 28%, with 61% of recipients developing cGVHD and 24% relapsing.47 In a recently reported randomised study, 10 patients were infused with 0.03–1.5  106 MSCs/kg and compared with 15 patients who did not receive MSCs. The MSC treated group had a lower incidence of aGVHD, 11.1% compared to the control group with a rate of 53.3%. However, the MSC treated group did have a higher relapse rate of 60% compared to 20% in the control group, although the MSC group had a higher proportion of high risk malignancies.109 cGVHD is a significant complication of allogeneic HCT contributing to morbidity and mortality. It is often refractory to various therapies and remains a therapeutic challenge. Experience in the use of MSCs for the treatment of cGVHD is less than in aGVHD. Promising results have been reported in a study of 19 patients with refractory cGVHD treated with a single dose (median 0.6  106/kg) of unrelated donor MSCs. There were five deaths, but in the surviving patients, there were four complete remissions and 10 partial remissions, and five patients were able to discontinue systemic immunosuppression.110 Another, smaller study showed clinical improvement in four patients with sclerodermatous GVHD following MSC infusion.111 What conclusions can be drawn from this group of disparate studies? It is apparent that a number of significant differences exist between the studies to limit our conclusions. The differences in MSC source, MSC culture conditions, MSC dose, and MSC infusion number may contribute to the results reported. Furthermore, the definition of response and the differences in follow-up also restrict firm conclusions being made. It is apparent that patients with SR-GVHD do respond to MSC infusion and responding patients have a superior survival. MSC infusion is well tolerated and there have been no reports of ectopic tissue formation. Whether MSC infusion represents an advance in the therapy of SR-GVHD requires further well designed randomised studies. Improved understanding of the mechanisms of MSC action and homing may lead to development of optimal culture conditions to produce an MSC product tailored for the treatment of aGVHD. MSC therapy for autoimmune disorders Autoimmune diseases, characterised by a dysregulated immune system, tissue inflammation and destruction, are an attractive target for MSC based therapies. Current treatment for autoimmune diseases is based on non-selective immunosuppression with recent development of targeted therapies toward cellular effectors and cytokines. These newer therapies are a significant advancement in the management of autoimmune diseases, but they have limitations. Cell based therapy, in the form of HCT, has been utilised in the treatment of some autoimmune diseases including rheumatoid arthritis, scleroderma, Crohn’s disease and multiple sclerosis. This approach is based on the rationale that ablative chemotherapy combined with re-infusion of HSCs will result in a re-programming of the immune system inducing a remission. Even though the majority of patients undergoing HCT have

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advanced disease, often refractory to multiple lines of therapy, encouraging results have been reported. Randomised studies in scleroderma, multiple sclerosis and Crohn’s disease are currently being conducted and the results of these are awaited. The potent in vitro immunomodulatory effects of MSCs and their homing properties to sites of inflammation have led to preliminary studies in autoimmune diseases. Potential advantages over HCT based therapies include the avoidance of potentially toxic pre-transplant chemotherapy, the ability to administer multiple doses and the targeted delivery of cells to the inflamed environment. Although third party allogeneic MSCs have been delivered to recipients with aGVHD, concern remains about the potential for long-term adverse sequelae, such as ectopic tissue or tumour formation. However, the ability to source an ‘off the shelf’ universal donor product is an advantage. Autologous MSCs may be preferred for the treatment of autoimmune disease to abrogate the risks of tumour formation but as they may contribute to the pathogenesis of the underlying disease this may be deleterious. In vitro studies of MSCs derived from patients with Crohn’s disease and multiple sclerosis show the cells have similar properties to MSCs isolated from healthy donors.112 –114 Clinical studies of MSC administration are limited. A Phase I study of autologous MSC infusion in Crohn’s disease demonstrated improvement in three of 10 patients and worsening in three of 10 patients.113 The group from Nanjing University treated 31 patients with refractory SLE with a single infusion of 1  106 allogeneic MSCs/kg. The MSCs were bone marrow derived in 15 cases and umbilical cord derived in 16 cases. All patients showed improvement in disease activity scores and serological markers of disease activity, although some patients received high dose cyclophosphamide pre-infusion and post-infusion, potentially confounding the results.115 A smaller study in two patients with SLE who received a single dose of 1  106 autologous bone marrow-derived MSCs/kg, while demonstrating increased numbers of Treg cells, showed no effect on disease activity.116 Studies are ongoing in autoimmune disease including Crohn’s, SLE and multiple sclerosis but further advancement requires well conducted randomised studies using a common protocol of MSC source, culture and infusion regimen.

MSC THERAPY FOR TISSUE REPAIR Ever since Caplan demonstrated that MSCs have differentiation potential in the early 1990s,117 there has been a huge interest in the therapeutic use of these cells in tissue repair and regenerative medicine. Three areas of research are bone and cartilage, cardiac and neurological repair. Bone and cartilage repair The main area of interest in the use of MSCs therapeutically in orthopaedics is in the repair of cartilage damage, especially in patients with osteoarthritis. Articular cartilage defects have a poor capacity to undergo self-repair, owing to the low mitotic potential of chondrocytes in vivo. Since articular cartilage defects can progress to osteoarthritis in some patients, they need to be repaired, even though their exact natural course remains obscure. MSCs, with chondrogenic differentiation capacity, are an attractive alternative to chondrocytes, which must be surgically harvested from a very limited supply of nonweight-bearing articular cartilage. MSCs have been used in numerous animal and human pilot studies, however there are

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POTENTIAL THERAPEUTIC APPLICATIONS OF MESENCHYMAL STROMAL CELLS

currently no randomised placebo-controlled clinical trials. Most of these trials have utilised the combination of MSCs and a supportive matrix, and more recently the addition of osteoinductive growth factors. There have been great advances in technology in the field of biomaterials, that have led to a transition from non-porous, biologically inert materials, to more porous, osteoconductive biomaterials. Wakitani et al. were the first to describe the use of MSCs in a collagen scaffold to repair full thickness cartilage defects in animals.118 They then took this to humans and in 2002 reported on their use in patients with osteoarthritis.119 Twelve patients undergoing high tibial osteotomy had transplantation of MSCs embedded in collagen gel into the articular cartilage defect in the medial femoral condyle and covered with autologous periosteum. Another 12 subjects served as cell-free controls. Whilst there was no statistically significant clinical improvement, the arthroscopic and histological grading score was better in the cell-transplanted group than in the cell-free control group. Porous ceramic scaffolds loaded with autologous MSCs were successfully implanted into three patients with large bone defects.120 Kurodo et al. reported on the use of autologous MSCs, which were embedded within a collagen gel scaffold, to repair a full-thickness articular cartilage defect in the medial femoral condyle of a 31-year-old male judo player suffering from pain in the right knee.121 The implant was covered with an autologous periosteal flap. Seven months after surgery, arthroscopy revealed the defect to be covered with smooth tissues and one year after surgery, the clinical symptoms had improved significantly. Horwitz et al. demonstrated the feasibility of combined allogeneic bone marrow transplantation and MSC infusion for children with severe osteogenesis imperfecta, a brittle bone disease that affects all of the skeletal tissues in which type I collagen is synthesised.122 Gene marked, donor marrowderived MSCs were administered twice by infusion to six children with this disease. Five of six patients showed engraftment in one or more sites, including bone, skin, and marrow stroma, and had an acceleration of growth velocity during the first 6 months post-infusion. Interestingly, the one patient who did not respond showed a 150-fold increase in antibody titre against fetal bovine serum proteins after the second infusion, compared with his lack of detectable antibodies on the pre-infusion assay. The other five patients did not have detectable antibodies. There was no clinically significant toxicity except for an urticarial rash in one patient just after the second infusion. Cardiac repair There is evidence that the adult heart contains stem and progenitor cells that have a regenerative potential.123 Likewise, circulating bone marrow-derived progenitor cells can home to injured myocardium to participate in cardiac repair.124 However, acute myocardial infarction is associated with a massive loss of myocardial cells and endogenous regenerative mechanisms are simply inadequate. In a seminal paper, Orlic et al. in 2001 reported that adult mouse bone marrow-derived stem cells can transdifferentiate into cardiomyocytes to regenerate infarcted myocardium in vivo.125 This report has stimulated a significant amount of interest in this field over the last 10 years. The first Phase I study of bone marrow stem cell-based therapy for myocardial repair was reported only 2 years later.126 Subsequent studies

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over the next few years adopted a strategy using autologous unfractionated mononuclear bone marrow cells, rather than selected cell components. These studies generally demonstrated that this was a safe procedure, however efficacy was mixed.127,128 The use of unfractionated bone marrow has distinct disadvantages, including the fact that the specific cell population responsible for any cardiac improvement has not been identified. Therefore, investigators have switched to using specific bone marrow-derived populations, including MSCs. Chen et al. were one of the first groups to report on the use of autologous bone marrow MSCs to treat 69 patients who presented with an acute myocardial infarction.129 MSCs significantly increased the left ventricular ejection fraction 3 months after transplantation compared with the control group and there were no adverse events. Since then, a number of clinical trials have been published; however, the numbers of patients have been small, there is considerable heterogeneity in the timing of cell transplantation and delivery routes (intravenous, intracoronary and intramyocardial), follow-up has been short and overall results have been inconclusive.130–132 The use of an ‘off-the-shelf’ MSC product (Prochymal) in 53 patients was published in a Phase I, randomised, double-blind, placebo-controlled, dose-escalation study of intravenous allogeneic adult MSCs in patients with an acute myocardial infarction.133 MSCs for this study were derived from a single healthy donor. Patients had suffered a first acute myocardial infarction in the previous 10 days and were randomised to receive one of three intravenous doses of Prochymal or placebo, without immunosuppression. No procedure-related complications were observed, no patients developed tumours, and the 6 month adverse event was similar in the two groups. Although the study did not primarily aim to assess clinical efficacy, there was a trend towards improved clinical symptoms and increased left ventricular ejection fraction at 3 months, but not at 6 months, in the cell-treated group. A Phase II study is in progress. Mesoblast (Australia) has recently reported on the endoventricular injection of an allogeneic MSC product (Revascor) along the infarct border zone in 60 patients with congestive cardiac failure.134 A total 22 patients were found to have reduced myocardial blood flow at baseline by SPECT perfusion scan, indicating the presence of ischaemic heart muscle. Of these, 17 were randomised to receive treatment with Revascor while five were randomised as controls. Six months after treatment with a single injection of Revascor there was significant improvement in blood flow to the ischaemic heart muscle, with a 51% reduction in myocardial ischaemia ( p ¼ 0.01). In contrast, no change in blood flow to the ischaemic heart muscle was seen at 6 months in the controls. On the basis of these results, Mesoblast will now proceed with Phase 2b trials of Revascor for the treatment of vascular conditions including chronic refractory angina and acute myocardial infarction. Neurological repair The ability to promote functional recovery after traumatic injuries, ischaemic insults, or the onset of neurodegenerative diseases in the brain and spinal cord remains very limited. As a result there is considerable scientific, as well as media interest, in the potential for cellular therapy (including the use of MSCs) in these conditions. There has been a paradigm shift in the understanding of how MSCs effect tissue repair. MSCs initially

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attracted interest for their presumed ability to home to injured tissues and differentiate into multiple cellular phenotypes in vivo. However, recently this has been challenged with observations of low levels of tissue engraftment together with in vitro studies showing that significant biological effects on target cells could be achieved without the need of cell contact.135 This has led to the hypothesis that the therapeutic benefit of MSCs relies greatly on the paracrine release of molecules rather than transdifferentiation. This property of MSCs, along with their immunomodulating properties, has resulted in interest in using these cells to treat disorders such as multiple sclerosis, spinal cord injury, multiple system atrophy, amyotrophic lateral sclerosis, Parkinson’s disease, and even stroke. Experimental autoimmune encephalomyelitis (EAE) is a murine model for human multiple sclerosis. Several groups have demonstrated that the intravenous infusion of MSCs improved the clinical course of EAE.136,137 It has been demonstrated in these experiments that following administration of MSCs in mice with EAE, there is an effect on oligodendrocytes enhancing remyelination possibly through the release of neurotrophins such as brain derived neurotrophic factor. In a pilot study in 10 unresponsive multiple sclerosis patients, the intrathecal injections of autologous MSCs were reported to improve the sensory, pyramidal and cerebellar functions in six patients, although no firm conclusions were able to be made.138 In spinal cord injuries, pilot studies have been performed with the intrathecal injection of MSCs. These studies have been small with no conclusions able to be made on efficacy, however feasibility and safety was established.139 Multisystem atrophy is a rare condition that causes symptoms similar to Parkinson’s disease. Lee et al. reported that intravenous and intra-arterial MSC therapy in patients with multisystem atrophy was safe and delayed the progression of neurological deficits with achievement of functional improvement.140 Autologous MSCs from seven patients with amyotrophic lateral sclerosis were directly transplanted into the spinal cord. After 36 months, four of the seven patients showed a significant reduction in the linear decline of lung function and amyotrophic lateral sclerosis functional rating scale.141 Likewise, in seven patients with Parkinson’s disease, autologous MSCs were transplanted directly into the sublateral ventricular zone by stereotaxic surgery, resulting in a subjective improvement in symptoms, and two patients were able to significantly reduce the doses of their Parkinson’s medication.142 Studies have also demonstrated that intravenous infusion of autologous MSCs to stroke patients is a feasible and safe therapy, although again efficacy is not clear.143,144

UNRESOLVED ISSUES The field of MSC therapeutics has clearly generated a huge amount of interest amongst scientists and physicians with the potential to treat a broad range of medical conditions. As at June 2011, there were 110 open clinical trials registered at clinicaltrials.gov, with some being Phase II and Phase III. However, it is still early days and there are many issues that need to be addressed to take this field forward. There are a lot of question marks about efficacy with a wide range of discrepancies reported by different researchers. Although the reasons for the discrepancies are multifactorial, the lack of standard procedures for the isolation, culture, expansion and administration of MSCs makes the comparison and

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reproduction of the results difficult. Likewise, further investigations are necessary to determine the safety, optimal cell dose, timing and route of administration, and course of treatment for each condition. New medical therapies are always associated with risks and the potential medical benefits must be weighed against these risks. A concern with the use of any cellular therapy, including MSCs, is the risk of malignant transformation. It has been established that there is risk with embryonic stem cells and induced pluripotent cells.145,146 Whilst the risk is lower for adult cells, it does increase if cells are expanded in culture, as is done with most clinical MSC protocols. To generate sufficient cells for clinical utility, MSCs are usually passaged four to six times when 60–120 mL of bone marrow is aspirated, which means MSCs will be cultured for about 40 days before being used. There have been three reports describing escape from senescence and generation of malignant cells when MSCs were expanded in culture.147–149 The Madrid group has since reported that their observation was in fact explained by contamination of their cultures with a small number of malignant cells.150 A group of leaders in the field of MSC research have published a paper to try to define the risks of MSC therapy.151 They make the important point that many researchers use the term ‘passage number’ rather than ‘population doublings’ to define proliferative capacity. The use of the former can be misleading and lead to a large under-estimation of the replicative lifespan of the cells. The probability of cells becoming immortal and tumorigenic is extremely low if MSCs are serially passaged well before confluency and replated at low or medium densities because the cells can be expanded through more than 30 population doublings before they enter senescence. An additional issue when using cells that are at high population doubling is that phenotypically they are very different from those at earlier population doubling. MSCs lose their differentiation potential,152,153 their morphology changes,153 there is evidence of an altered gene expression profile,154 and there are likely to be more restricted self-renewing progenitors.155 In addition, there is evidence that homing receptors such as CXCR4 might diminish, resulting in less efficient homing.156,157 Other factors during in vitro cell expansion can affect the final quality of MSCs, such as the use of fetal bovine serum, type of medium, glucose concentration, stable glutamine, and quality of the plastic surface. The source of MSCs, route, dose and timing of administration will clearly differ depending on the disease being addressed. The most common source of adult MSCs has been bone marrow, which although quite straightforward to acquire, requires an invasive procedure. Adipose tissue is also a source with abundant MSCs, which can be obtained from cosmetic liposuctions in large quantities, or alternatively it may be desirable to use fetal MSCs derived from umbilical cord blood, cord matrix, amniotic fluid, Wharton’s jelly or placenta, which are all discarded after delivery. The timing of administration of MSCs is clearly very important. Current studies have indicated that the early phase of injury or disease may be the optimal time window for therapy, suggesting that this would be the optimum time of administration. However, there has been a paradigm shift in the understanding of how MSCs have a beneficial effect on injury in that it is now believed the secretion of cytokines, growth factors or other humoral effects plus immunomodulation are more important than their differentiation capacity. Therefore, the aim

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POTENTIAL THERAPEUTIC APPLICATIONS OF MESENCHYMAL STROMAL CELLS

will be to give MSCs when cytokines are needed, not when the cells should rebuild tissue. Therefore, clinical researchers should focus on protocols that produce high levels of cytokine expression.

CONCLUSION Since their first description more than 40 years ago, our understanding of the basic biology of MSCs and their potential for use therapeutically has grown exponentially. Whilst it is appreciated that that MSCs have differentiation capacity, it is especially their immunomodulatory and paracrine release of molecule properties that have led to MSC therapies to treat conditions such as GVHD and autoimmune disorders like inflammatory bowel disease. Likewise, the field of regenerative medicine is exploring the use of MSCs for orthopaedic, cardiac and neurological tissue repair applications. However, there is tremendous heterogeneity in the methods used to derive and culture MSCs, as well as in the timing and route of administration, which is likely to explain the differences in efficacy achieved by different investigators. There is a strong need for more multi-centre randomised clinical studies to find the optimal therapeutic time window, cell dose, and route of administration in different applications. In addition, more emphasis is required on the standardisation of the isolation and culture expansion method of MSCs in these clinical studies. Also, to ensure the high quality of MSCs for therapeutic purposes, their production must adhere to good manufacturing practices, so as to ensure the delivery of a product that is safe, reproducible, and efficient. It is hoped that with the outcome of clinical studies in progress, and with the development of future randomised clinical trials, MSC therapeutics will play a significant role in the treatment of patients with a broad range of conditions. Address for correspondence: Dr I. D. Lewis, Haematology, SA Pathology, PO Box 14 Rundle Mall, SA 5000, Australia. E-mail: [email protected]

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