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OPEN ACCESS ARTICLE
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TRANSLATIONAL AND CLINICAL RESEARCH |
aCardiovascular Research Centre, Royal Adelaide Hospital, Adelaide, South Australia, Australia;
bCentre for Stem Cell Research, Department of Medicine, University of Adelaide, Adelaide, South Australia, Australia;
cBone and Cancer Laboratories, Division of Haematology, Institute of Medical and Veterinary Science and Hanson Institute, Adelaide, South Australia, Australia
Key Words. Cardiac diseases • Tissue engineering • Mesenchymal stromal cells • Myogenesis • Angiogenesis
Correspondence: Peter J. Psaltis, M.D., Cardiovascular Research Centre, Royal Adelaide Hospital and Department of Medicine, University of Adelaide, Adelaide, South Australia 5000, Australia. Telephone: 61-8-82223735; Fax: 61-8-82223162; e-mail: peter.psaltis{at}adelaide.edu.au
Received April 30, 2008;
accepted for publication June 25, 2008.
First published online in STEM CELLS EXPRESS July 3, 2008.
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ABSTRACT |
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Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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Cellular therapy has evolved quickly over the last decade both at the level of in vitro and in vivo preclinical research and more recently in clinical trials of myocardial infarction/ischemia and heart failure. A variety of embryonic and adult-derived cell types have been investigated for their capacity to mediate cardiac and vascular repair. This review will discuss the cardiac therapeutic potential of postnatal tissue-derived mesenchymal stromal cells (MSCs).
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OVERVIEW OF CELLULAR THERAPY FOR HEART DISEASE |
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Lessons from Studies Using Unfractionated Bone Marrow
Many cardiovascular studies have avoided the use of specific cell fractions, preferring to adopt a "blanket" treatment approach by administering unfractionated BMCs or BMMNCs. These cells have been investigated in preclinical models of ischemic and nonischemic cardiac disease, with perhaps their most notable benefits relating to improvements in myocardial vascularity [5–7].
Since 2001, numerous clinical studies have demonstrated the feasibility of administering autologous BMCs or BMMNCs to patients with recent myocardial infarction (MI) [8–12] and ischemic cardiomyopathy [13–16]. Meta-analyses have indicated the safety of this treatment, as well as moderate benefits in physiologic and anatomic cardiac parameters, above and beyond conventional therapy [17, 18]. Review of approximately 700 patients receiving intracoronary bone marrow (BM) therapy for acute MI demonstrated a modest but significant increase in left ventricular ejection fraction (EF) and reduction in infarct size and left ventricular end-systolic volume [18]. These improvements were greatest in patients with lower EF (<45%) and when cells were administered between days 5 and 7 after index MI. Although meta-analysis results have been encouraging, there has been a discrepancy between outcomes of individual trials [12, 19]. This may reflect the heterogeneous nature of autologous BM treatment, as the cell composition of unfractionated BMCs/BMMNCs is mixed, comprising only a small proportion of true progenitor/stem cells. The content and functionality of stem cells in BM may also be affected by (a) differences in isolation and storage techniques [20] and (b) patient comorbidity, including the presence of chronic disease and cardiovascular risk factors [21]. It is currently unclear whether the cardiac reparative potential of unfractionated BM is hampered by its low frequency of immature progenitor cells or strengthened by the pleiotropic properties of its more mature cells. In any case, the study of more homogeneous cell preparations, including MSCs, provides an important opportunity for researchers to clarify the cell type or types that are most beneficial to cardiovascular repair and the mechanisms by which they act.
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MESENCHYMAL STROMAL CELLS: BASIC PrINCIPLES |
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Traditional Isolation of MSCs
Traditional isolation of MSCs from BM has been based on simple density gradient separation of mononuclear cells and subsequent culture in tissue culture plastic. The mesenchymal subfraction is adherent to plastic and therefore easily separated from nonadherent hematopoietic cells [30, 31]. Under appropriate conditions, the adherent cells form colonies of varied morphologies (colony-forming units-fibroblast [CFU-F] cells) over 7–14 days, usually at a frequency of 1–10 colonies per 105 mononuclear cells plated [32]. These CFU-F cells are a mixture of clonogenic, mesenchymal stem, and progenitor cells. A proportion of CFU-F have extensive proliferative capacity in vitro, and under optimal conditions their progeny can be expanded for upwards of 50 population doublings, achieving the high numbers required for transplantation.
Evidence for the multipotency of plastic adherent-derived MSCs has often been claimed on the basis of their capacity for in vitro differentiation into osteogenic, chondrogenic, and adipogenic cells, whereas cell purity has been inferred from their expression of certain surface antigens [33]. This is despite the fact that these antigens are neither specific nor definitive markers of cells with bona fide stem cell properties. The colonies derived by plastic adherence are quite heterogeneous with respect to size, morphology, and degree of contamination with more mature mesenchymal cells (e.g., osteoblasts, fibroblasts, fat cells) and nonmesenchymal cells (e.g., macrophages and endothelial cells) [32, 34]. As these colonies are expanded ex vivo over several passages, their progeny exhibit evidence of senescence, losing their capacity for proliferation and differentiation. The historical description of these cells as mesenchymal/stromal/somatic stem cells is therefore misrepresentative, and the preferred nomenclature, now universally adopted, is "multipotent mesenchymal stromal cells" [35, 36]. The minimal immunophenotypic profile used to ensure consistent characterization of MSCs is presented in Table 1. The term "mesenchymal stem cell" (also abbreviated MSC) should be reserved for cells fulfilling specified stem cell criteria.
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Immunoselection describes the isolation of highly purified mesenchymal precursor cells based on their reaction with specific monoclonal antibodies that have been developed by immunizing mice with human mesenchymal lineage precursors. Combinations of different monoclonal antibodies to antigens such as stromal precursor antigen-1 (STRO-1), CD49a/CD29 (VLA-1, 
1β1 integrin), CD106 (vascular cell adhesion molecule 1), CD146 (MUC-18, S-endo), low-affinity nerve growth factor receptor, platelet-derived growth factor receptor, epidermal growth factor receptor, insulin-like growth factor receptor, and bone-specific alkaline phosphatase (STRO-3) have been used to enrich for MSCs from BM and other tissues (Table 1) [22, 24, 26, 32, 37–40]. Immunoselected cells are devoid of contaminating hematopoietic, endothelial, and fibroblast lineages and possess many hallmark characteristics of immature stem cells [32]. Advocates of prospective immunoselection argue that this technique maximizes functional reproducibility by initiating mesenchymal cell culture with homogeneous and pure populations that also share similar phenotypic properties with previously described multipotent adult progenitor cells [41, 42].
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MESENCHYMAL STROMAL CELLS IN CARDIOVASCULAR REPAIR |
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The vast majority of in vivo studies have used the nonselective, plastic-adherent approach to MSC preparation. Animal studies of MSC transplantation in MI [43–45] and ischemic cardiomyopathy [6] have reported wide-reaching biological and functional benefits that include attenuation of myocardial scar and infarct size, improved regional and global ventricular function, restoration of myocardial mechano-energetics, and increased vascular density and myocardial perfusion. There has also been evidence of benefit in models of nonischemic, dilated cardiomyopathy [46–48], arrhythmia [49], and valvular disease [50, 51].
The provascular properties of MSCs are consistent with growing speculation that mesenchymal lineage precursors have an important role in normal vascular development and may be derived from a subset of smooth muscle cells or pericytes [24, 26, 40, 52]. In an athymic nude rat model of acute MI, cardiac injection of human STRO-1Bright mesenchymal progenitors resulted in dose-dependent enhancement of vascular density and neovascularization, with accompanying improvement in myocardial ejection fraction [53].
The long-term safety of cardiac MSC transplantation remains, as it does with other cell types, a critical yet unresolved issue. Animal studies have not commonly documented adverse effects at myocardial sites of MSC injection, although there are some notable exceptions. Breitbach et al. recently reported that MSC transplantation in a murine model of cryo-infarction resulted in a high frequency of encapsulated areas containing ossifications and/or calcifications (approximately 50% of the injection sites) [54]. In an earlier study of cell transplantation in rats with acute MI, intramyocardial calcification was observed 2 weeks after injection of unselected BM cells (28.5% of rats), but not after treatment with selected multipotent progenitor cells [55]. The potential for proarrhythmic effects of MSC therapy has also been raised, due possibly to the sprouting of new cardiac sympathetic nerves [56–58]. The prevalence and severity of complications such as abnormal tissue development and arrhythmia are not clear and may have been under-reported. Studies should include rigorous investigation for evidence of conversion of MSCs to unwanted lineage cells in vivo. This will necessitate sufficient follow-up after cell transplantation, incorporating sensitive and accurate tissue analysis by myocardial imaging and histopathology, as well as comprehensive electrophysiologic evaluation.
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MECHANISMS OF CARDIOVASCULAR REPAIR |
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In vitro differentiation of MSCs into cells resembling cardiomyocytes prompted early expectation of their capacity to regenerate these cells in vivo. Although not universally successful [59], exposure of MSCs to the DNA demethylating chemical 5-azacytidine has been the commonest strategy for inducing their cardiac differentiation in vitro. Under this condition, immortalized stromal cell lines and primary MSCs, from different species and different tissue sources, have been reported to modify their phenotype, with adoption of myotube morphology, expression of immature action potentials, and a variety of cardiac-specific genes (e.g., myocyte enhancer factor (MEF)-2A and MEF-2D) and peptides (e.g., myosin, desmin, actinin, and atrial and brain natriuretic peptides) [60–64]. Functional differentiation has been indicated by the formation of intercellular connections via intercalated discs and by evidence of spontaneous cell contractility. Typically, these changes occur within 2–4 weeks of exposure to 5-azacytidine. Because of the potential genotoxicity of this chemical, other in vitro approaches to cardiomyocyte transdifferentiation have included culture in medium enriched with dexamethasone and ascorbic acid [65], bone morphogenetic protein-2, and fibroblast growth factor-4 [66] and coculture with cardiomyocytes [67]. It is unknown whether in vitro differentiation of MSCs into cardiomyocytes will enhance the reparative effects of these cells once they are transplanted in vivo [68].
Mesenchymal stromal cells can also produce cardiac connexin-43 and in theory, unlike skeletal myoblasts, can electromechanically couple to host cardiomyocytes in vivo [69]. Evidence for in vivo differentiation has come from immunohistological analysis in small animal studies of xenogeneic MSC transplantation [43, 53] and large animal studies of autologous [70] or allogeneic [6, 44] cell therapy. Generally, authentication of differentiation has been incomplete, as engrafted MSCs have de novo expression of some myocyte (e.g., desmin, troponin T, phospholamban) or vascular (e.g., factor VIII, -smooth muscle actin) markers, without acquiring the full phenotypic complement [6, 44, 70–72]. Adding to skepticism about the differentiative capacity of transplanted cells, have been confounders of traditional immunofluorescent and immunohistochemical analysis, including tissue artifact and cellular fusion. Several groups have reported that engrafted BM-derived cells actually undergo fusion with endogenous cardiac cells, rather than true differentiation [73, 74]. However, this phenomenon has also been contradicted by other reports confirming the diploid chromosomal content of new cardiomyocytes generated after cell transplantation [75, 76]. Sophisticated new cell labeling techniques, such as direct immunofluorescence with quantum dots, should significantly improve the assessment of cellular differentiation in vivo and have recently been applied to demonstrate cardiomyocyte differentiation of c-kit-positive BMCs in a murine study [77].
The modest evidence for in vivo differentiation observed with MSC therapy to date may partly be due to the impure, heterogeneous nature of cells obtained through plastic-adherence isolation and the underwhelming retention of multipotent stem cells after ex vivo expansion. Compounding this are the extremely modest rates of intramyocardial retention, engraftment, and survival of cells following their administration by current delivery techniques [78]. Cell survival may be especially compromised in the presence of unresolved myocardial ischemia or inflammation, and there is also evidence from research with skeletal myoblasts that host rejection may be further accentuated if cells have been cultured in media containing xenogeneic ingredients such as fetal bovine serum [79]. A significant challenge for this field of research is the refinement of cellular biology and delivery technology to maximize the engraftment, survival, and function of cells in vivo.
Cardiac Repair: Paracrine Mechanisms
The variable observations relating to cell transdifferentiation have prompted a rethinking of the mechanisms that account for the functional benefits observed in studies of cardiac cell therapy. Increasingly, it is believed that current cell therapies assist the heart predominantly by facilitating endogenous repair processes, rather than through actual regeneration of lost cardiac and vascular cells. Paracrine actions may underlie much of this reparative potential, including the capacity for cell transplantation to induce neovascularization, reduce infarct size and scar formation, and improve myocardial contractility [80, 81] (Fig. 1). Mesenchymal stromal cells are able to influence other cells in their vicinity by direct cell-to-cell interaction and by release of a wide array of soluble growth factors and cytokines [80]. These soluble factors are influenced by the developmental status and properties of the MSCs themselves and by the local milieu in which they find themselves. Relevant signaling and growth factors identified from both in vitro and in vivo experiments include stromal cell-derived factor-1 (SDF-1/CXCL12), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor, hypoxia inducible factor-1 (HIF-1), vascular endothelial growth factor (VEGF), angiopoeitin-1, monocyte chemoattractant protein-1, interleukins-1 and -6, placental growth factor, plasminogen activator, and tumor necrosis factor- [80–82]. Of these factors, HIF-1 is a transcription factor that is closely linked with cellular expression of VEGF and is activated and stabilized under hypoxic conditions [83]. Recent results indicate that in addition to its role in angiogenesis, it may also help mediate the protective effects that MSCs exert on cardiomyocytes during in vitro hypoxic culture [84].
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OPTIMIZING THE REPARATIVE BIOLOGY OF MSCs |
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Another promising area of investigation has been the in vitro manipulation of cells prior to transplantation, in an attempt to maximize their biologic and functional properties. Examples include the preparation of cells in special bioscaffolds [94], preconditioning of cells in culture, and genetic transfection. Various culture strategies have been trialed to improve MSC viability, such as exposing cells to hypoxia [95, 96], specific growth factors [97], or low doses of chemical agents (e.g., cyclosporine A, hydrogen peroxide) [98]. In small animal studies this has generally translated into better engraftment of cells in vivo, accompanied by greater benefit to cardiac contractility and vascularity. Investigators have also demonstrated the effectiveness of genetic engineering by transfecting MSCs with genes encoding the antiapoptotic factor Akt [99], SDF-1/CXCL12 [100], fibroblast growth factor-2 [29], angiopoietin [101], VEGF [102], and hypoxia-regulated heme oxygenase-1 [103]. Overexpression of Akt has enhanced the reparative effects of MSC therapy in animal models of infarction, improving both global left ventricular function and size of scar [99, 104]. These outcomes have been quite durable [105] and may reflect greater resistance of these cells to in vivo apoptosis and stronger paracrine actions on the host myocardium [106].
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PROGRESSING TO CLINICAL APPLICATION |
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Mesenchymal stromal cells possess immune properties that make them a potential candidate for safe allogeneic therapy in immunocompetent patients. Specifically, they have a relatively immunoprivileged phenotype, enabling them to exist in inflammatory environments without activating immune cells [108]. This is partly due to their hypoimmunogenic state, including a lack of surface expression of major histocompatibility complex class II molecules in the resting state and also their lack of expression of costimulatory molecules for T cell induction (CD40, CD40 ligand, and the B7 molecules CD80 and CD86) [109]. Mesenchymal stromal cells downregulate lymphocyte proliferation [110] and suppress the maturation and function of various other immune cells, even after they have been differentiated to bone, cartilage, or adipocytes [109, 111]. This is mediated both by direct cell contact with the immune cells and by the release of immune-modifying, soluble factors, such as transforming growth factor β1, HGF, interleukin-10, and prostaglandin E-2 [108, 112]. These immune-modifying effects have resulted in the clinical investigation of MSC therapy for inflammatory diseases such as graft-versus-host disease [113].
Although there have been cautionary reports of the immunogenicity of allogeneic and xenogeneic MSCs in immunocompetent mice [114, 115] and rats [116], other groups have reported safe administration of allo-MSCs to the heart in immunocompetent pigs [44, 57] and dogs [117], providing the "green light" for the commencement of clinical trials in humans [118].
Myocardial Delivery of Cells
The approach to delivery of exogenous cells to the heart is of major importance to clinical application, with the main objective being to safely ensure sufficient initial retention of cells within the target myocardium and adequate local engraftment and function at this site. The basic strategies for directing cells to the heart are (a) systemic therapy, either by peripheral venous infusion or growth factor mobilization; (b) regional coronary vascular infusion; and (c) local direct myocardial injection (Fig. 2).
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Systemic Delivery. It is well known that hematopoietic cells and endothelial progenitor cells can be mobilized from BM by growth factors [119]. Although there is recent evidence in rats to show the mobilization of BM MSCs into the peripheral circulation after hypoxic insult [120], it is not yet clear whether human MSCs respond significantly to growth factor administration.
In animal studies, peripheral venous injection of MSCs has achieved modest cardiac repair after MI, but at the expense of noncardiac entrapment of cells [121–123]. Intravenous therapy with allogeneic BM MSCs (Provacel; Osiris Therapeutics, Baltimore, http://www.osiristx.com) has been the subject of a randomized, multicenter, dose escalation trial enrolling 53 patients with acute MI in the U.S. [118]. In this study, amplification of the cellular product yielded approximately 5,000 off-the-shelf "doses" from each single donor. Cells were administered as a single peripheral infusion within the first 10 days of acute MI, after establishing patency of the culprit coronary artery. At 6-month follow-up, MSC therapy was associated with improved cardiac function in patients presenting with anterior MI (7%–8% absolute increase in EF from baseline) (presented at the American College of Cardiology Meeting, New Orleans, March 2007). Importantly, systemic delivery of MSCs did not result in increased ectopic tissue formation on whole-body computed tomography scanning, whereas arrhythmias were observed to be less frequent than in the placebo group.
Intracoronary Arterial Infusion. Selective intracoronary administration of cells achieves higher first-pass delivery to the heart than systemic therapy. The intracoronary route has been used safely and effectively for the infusion of unfractionated BM and peripheral blood-derived cells in clinical trials of acute MI [8–10, 124, 125], post-MI cardiomyopathy [14, 16], chronic ischemic heart disease [126], and nonischemic cardiomyopathy [127]. Cells are typically injected through the central lumen of an over-the-wire balloon catheter during transient balloon inflations to stop coronary blood flow and increase cell exposure to the microcirculation and myocardium.
In the context of MSC transplantation, preclinical studies have not been able to guarantee the safety of intracoronary injection because of observations that these adherent cells may aggregate within the microvessels and impair blood flow to the heart [78, 128]. Mesenchymal stromal cells (
20 µm) are larger than capillaries (8–10 µm) and are also less deformable than BMMNCs (8–12 µm). Despite these safety concerns, two clinical studies have used the coronary route for autologous MSC therapy after MI [124, 129]. Chen et al. administered very high doses of MSCs (48–60 x 109 cells) after a mean interval of 18 days from index presentation [124]. Six-month results were reported favorably (absolute increase in mean EF from 49%–67% in the MSC group); however, the investigators did not provide detailed analysis of safety outcomes or sufficient description of how such high numbers of cells were achieved from only 2–3 weeks of culture.
In contrast to this trial, others have found from their experience with BMC infusion that cell therapy is probably most effective when given between 4 and 7 days after MI [11, 130]. Indeed, the issue of optimal timing for cell delivery is still contentious. Earlier infusion of cells may cause excessive obstruction and dysfunction of the microvascular bed, whereas longer delays allow more time for cardiomyocyte apoptosis and scar development. The latter may partly account for the lack of benefit achieved by intracoronary administration of low-dose MSCs and EPCs in a small study of "old" anteroseptal MI [129].
Intramyocardial Injection. Unlike intravascular infusion, direct intramyocardial injection targets specific regions of myocardium without relying on the upregulation of inflammatory signals to assist transvascular cell migration and tissue invasion. Preclinical results indicate that direct MSC injection may result in less noncardiac entrapment of cells than intracoronary and i.v. infusion [78]. Moreover, myocardial cell retention may also be higher, culminating in greater benefit to cardiac function [117]. This delivery approach appears well suited to larger and adherent cell types (e.g., MSCs), as well as to chronic myocardial disease, such as chronic ischemia or scarred myocardium from old infarction. Direct injection can either be (a) transepicardial during open chest surgery or via the coronary venous system or (b) transendocardial by percutaneous catheter-based techniques. The invasiveness of open transepicardial injection restricts its clinical use to patients undergoing sternotomy for other cardiac surgery, such as coronary artery bypass surgery.
Various multicomponent systems are available for percutaneous catheter-based delivery and are usually used in conjunction with imaging/mapping of the myocardium to help guide transendocardial injection. Examples of such imaging modalities are electromechanical mapping (NOGA XP Cardiac Mapping System, Biologics Delivery Systems Group, Cordis Corporation, Diamond Bar, CA, http://www.cordisbds.com), which has been incorporated into the Myostar delivery system (Cordis Corporation) [131], conventional x-ray fluoroscopy [132], and real-time MRI [133].
Current Status of Clinical Trials
Human studies with MSCs have lagged well behind trials using autologous, unfractionated BM. The translation of MSC therapy to clinical cardiology requires that current and future studies be carefully designed and rigorously conducted to address key issues, such as comparison to other cell types, optimal cell dose, timing and mode of delivery, suitable patient cohorts, and demonstration of long-term safety and efficacy outcomes. Inherently critical in this is the application of standardized, robust end points that are clinically relevant (e.g., rates of major adverse cardiac events, reinfarction, hospitalization), in addition to standard surrogate measures (e.g., ejection fraction, myocardial perfusion, infarct size).
Of the many ongoing trials investigating cell therapy for cardiovascular disease (ClinicalTrials.gov [http://www.clinicaltrials.gov]), a minority are devoted to MSCs (Table 2). With the exception of the Provacel trial described above, the studies are administering MSC therapy by intramyocardial injection. Angioblast Systems, Inc. (New York, http://www.angioblast.com), is sponsoring a multicenter, randomized trial that will evaluate the safety of NOGA-guided transendocardial injection of cells early after MI. This study is also notable for its dose comparison design and the provision of allogeneic, prospectively immunoselected cells called mesenchymal precursor cells. Other investigators in Scandinavia and the U.S. are applying MSC therapy to patients with chronic ischemic heart disease and ischemic cardiomyopathy, two patient groups that can be especially challenging for clinicians, when refractory to conventional treatment. Although all of these studies are restricted in patient number (phase 1/2 design), they promise to shed some light on the safety and utility of MSC therapy in the clinical context of cardiac disease and will provide an invaluable early step in determining the role that these cells play in future treatment strategies.
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CONCLUSION |
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Substantial challenges are still to be overcome before MSC therapy can fulfill its promise in wider clinical practice (Table 3). Basic research will continue to optimize the techniques by which these cells are isolated, cultured, and manipulated in vitro to improve their engraftment, survival, and function following transplantation into the heart. Uptake into clinical research is still in its infancy for this cell type, but will be equally critical in addressing unresolved issues that are best studied in the human context. Although the task ahead seems considerable, the potential gain of developing a new and effective therapy for patients with refractory cardiovascular disease is enormous.
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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Abstract
Introduction
