Abstract: Transplant InternationalVolume 20, Issue 9 p. 731-746 Free Access Stem cells and cardiac regeneration Alfred A. Kocher, Alfred A. Kocher Department of Cardiac Surgery, Innsbruck Medical University, Innsbruck, AustriaSearch for more papers by this authorBernhard Schlechta, Bernhard Schlechta Department of Cardiac Surgery, Vienna Medical University, Vienna, AustriaSearch for more papers by this authorAneta Gasparovicova, Aneta Gasparovicova Department of Cardiac Surgery, Vienna Medical University, Vienna, AustriaSearch for more papers by this authorErnst Wolner, Ernst Wolner Department of Cardiac Surgery, Vienna Medical University, Vienna, AustriaSearch for more papers by this authorNikolaos Bonaros, Nikolaos Bonaros Department of Cardiac Surgery, Innsbruck Medical University, Innsbruck, AustriaSearch for more papers by this authorGünther Laufer, Günther Laufer Department of Cardiac Surgery, Innsbruck Medical University, Innsbruck, AustriaSearch for more papers by this author Alfred A. Kocher, Alfred A. Kocher Department of Cardiac Surgery, Innsbruck Medical University, Innsbruck, AustriaSearch for more papers by this authorBernhard Schlechta, Bernhard Schlechta Department of Cardiac Surgery, Vienna Medical University, Vienna, AustriaSearch for more papers by this authorAneta Gasparovicova, Aneta Gasparovicova Department of Cardiac Surgery, Vienna Medical University, Vienna, AustriaSearch for more papers by this authorErnst Wolner, Ernst Wolner Department of Cardiac Surgery, Vienna Medical University, Vienna, AustriaSearch for more papers by this authorNikolaos Bonaros, Nikolaos Bonaros Department of Cardiac Surgery, Innsbruck Medical University, Innsbruck, AustriaSearch for more papers by this authorGünther Laufer, Günther Laufer Department of Cardiac Surgery, Innsbruck Medical University, Innsbruck, AustriaSearch for more papers by this author First published: 06 June 2007 https://doi.org/10.1111/j.1432-2277.2007.00493.xCitations: 25 Alfred A. Kocher MD, Department of Cardiac Surgery, Medical University of Innsbruck, Anichstrasse 35, 6020 Innsbruck, Austria. Tel.: ++43 512 504 23806; fax: ++43 512 504 22528;e-mail: [email protected] AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary Despite many advances in cardiovascular medicine, heart failure (HF) remains the leading cause of death in developed countries affecting at least 10 million people in Western Europe alone. The poor long-term prognosis of HF patients, and immense public health implications has fuelled interest in finding new therapeutic modalities. Recent observations of the beneficial effect of stem cells on the damaged heart in animal experiments have generated tremendous excitement and stimulated clinical studies suggesting that this approach is feasible, safe, and potentially effective in humans. Cell-based myocardial regeneration is currently explored for a wide range of cardiac disease states, including acute and chronic ischemic myocardial damage, cardiomyopathy and as biological heart pacemakers. The aim of the present manuscript is to review the work that has been done to establish the role of stem cells in cardiac repair, give an update on the clinical trials performed so far, as well as to discuss critically the controversies, challenges and future surrounding this novel therapeutic concept. Scope of the problem Heart disease, including coronary atherosclerosis and myocardial infarction were known in remote antiquity, with the oldest documentation in Egyptian mummies. Description of the clinical syndrome of angor pectoris can be found in the Ebers' papyrus, the oldest preserved medical document from about 1552 BC, and clinical and prognostic data regarding cases of angina, infarction and sudden death are documented in the Corpus Hippocraticum [1]. While there are no data on the incidence in those days, cardiovascular disease has become the biggest healthcare burden of our times. Nearly 5 million Americans have heart failure (HF) today, with an incidence approaching 10 per 1000 population among persons older than 65 years of age [2]. Cardiovascular disease is the leading cause of death in Europe, of which nearly half is attributable to coronary artery disease (CAD) [3]. This situation is expected to become worse, with a sharp increase in CVD in developing countries and a predicted 25 million CVD deaths worldwide by 2020. The aging world population faces a pandemic of CAD as projected by the WHO [4]. Therapeutic options Although there are a number of investigative therapeutic modalities for treating end-stage HF, the two primary treatment options available are pharmacologic therapy and cardiac transplantation [5]. Advances in medical therapy have had an important impact on symptom status and short-term survival of patients with moderate to severe HF. The mainstay life-saving drugs are angiotensin-converting enzyme (ACE) inhibitors and β-blockers. Additional benefits are obtained when angiotensin-receptor blockers or aldosterone antagonists are added [6]. Existing pharmacologic agents have met with only moderate success in patients with class IV HF, and the 1-year survival rate is only 40–50% [7]. Heart transplantation remains the treatment modality with the best outcome with 5-year survival rates of around 65%, however, due to the current serious shortage of donor organs only 3000 cardiac transplants are performed each year worldwide with 15 000 patients on waiting lists for transplants [8]. The success of cardiac transplantation remains further limited by the complications of long-term immunosuppression and the development of allograft CAD. Since the inception of the artificial heart program at the National Institutes of Health in 1964, a variety of circulatory support devices have been developed and are being used as bridge to transplant and more recently for destination therapy in patients experiencing end-stage HF who are ineligible for transplantation. The randomized evaluation of mechanical assistance for treatment of congestive HF (REMATCH) trial explored the use of left ventricular (LV) assist devices (LVADs) as permanent implants and showed they can increase median survival by 7.4 months and improve functional status in comparison with medical management in end-stage HF patients. The clinically meaningful survival and quality-of-life benefit for patients with LVADs comes at a high device failure rate and numerous complications, mainly infections and bleeding as a result of necessary anticoagulant therapy, while raising the cost of end-of-life care considerably [9, 10]. Cellular therapy Functional restoration of the damaged heart presents a formidable challenge with none of the current treatment modalities leading to a reduction in scare size after myocardial infarction or significant improvement of an impaired cardiac pumping ability in HF. Conversely, cell-based cardiac repair offers the promise of regenerating damaged myocardium by rebuilding the injured heart from its component parts. Ideally, transplanted cells would mimic the lost myocytes morphologically and functionally, with the ability to contract and to establish electrical connectivity with the native myocardial cells. Exploration of stem cell transplantation as a potential means of treating patients with cardiac disorders has attracted immense scientific and public interest only recently. The concept of stem cells, however, is old and originates from attempts to understand the mechanisms of tissue homeostasis and renewal in adults, particularly in the hematopoietic system. The existence of a blood stem cell was proposed as long ago as 1909 [11]. The legend of Prometheus, who transgressed the law of the ancient gods and stole fire for humankind and was chained to the Caucasus for punishment, where a vulture preyed daily on his liver, which was renewed as quickly as it was devoured indicates that the remarkable potential of the body to rebuild itself – a key feature of stemness – was known in the distant past [12]. Meanwhile, stem cells have been identified in many adult human organs and tissues, not only in those that undergo frequent renewal, but also in others like the nervous system, which until recently was believed to be incapable of renewal during adult life [13]. The dogma of the heart as a postmitotic organ that is terminally differentiated with approximately 5 billion cells at birth which would only decrease with age was established in the 1970s and preserved for almost three decades. In the early 1990s, Anversa et al. described that cardiomyocytes undergo apoptosis at a significant rate and hereby the traditional view of the heart as an organ incapable of regeneration has been challenged [14]. Seminal studies and observations by the very same researchers have led to a paradigm shift in cardiac biology. They demonstrated that myocyte mitosis occurs not only in the fetal but also the adult heart and that myocyte turnover is markedly enhanced in pathologic states, such as HF and myocardial infarction [15]. Cell proliferation contributes to the homeostasis of the normal myocardium and the increase in muscle mass after myocardial infarction. The same group of investigators were the first to show dividing cells of extracardiac origin in sex-mismatched heart transplant patients, indicating that extracardiac progenitor cells are capable of repopulating most major cell types in the transplanted human heart including not only cardiomyocytes, but also endothelial, smooth muscle and Schwann cells [16]. And finally it was Anversa again who succeeded in identifying and characterizing lineage committed cardiac stem cells (CSCs) residing in the myocardium, which ultimately give rise to small developing myocytes that further evolve into the adult phenotype [17]. Thus a new conceptual framework of the heart has emerged. The heart is now viewed as a self-renewing organ in which myocyte regeneration occurs throughout the organism lifespan with CSCs preserving organ homeostasis and cell turnover. In the circumstance of a devastating, acute muscle cell death from myocardial infarction myocyte replacement by endogenous repair mechanisms to offset the extent of tissue loss is insufficient, although it could be sufficient to repair subclinical lesions after the blockage of small capillaries. As a result, the concept of cardiac regeneration by exogenous cellular elements has gained increasing attention. Initially the goal was to replace lost myocardial tissue by contractile elements. Therefore cell-based cardiac repair began with the transplantation of autologous skeletal satellite cells, progenitor cells that normally mediate regeneration of skeletal muscle [18]. However, in addition to myocardial loss, cardiomyocytes in the immediate vicinity of the scar tissue become hibernating because of insufficient myocardial perfusion. Hence, promotion of blood vessel formation is another important pillar of cardiac regeneration by stem cells. Stem cells are reported to differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells, and partially restore cardiac function suggesting new avenues for treatment of heart disease [19]. Stem cell definition Stem cells are undifferentiated tissue progenitor cells that can proliferate and are defined by their ability to self-renew and to form one or more differentiated cell types [20-22]. They can be categorized anatomically, functionally, or by cell surface markers, transcription factors, and the proteins they express. Different populations of stem cells are distinguished by the types of specialized cells that they generate. One clear division of the stem cell family is between those isolated from the embryo, known as embryonic stem cells (ESCs), and those in adult somatic tissue known as adult stem cells. Within these categories, stem cells can be further divided according to the number of differentiated cell types they can produce. Totipotent stem cells are able to form all fully differentiated cells of the body and trophoblastic cells of the placenta. The embryo, zygote, and the immediate descendants of the first two cell divisions are the only cells considered to be totipotent [23]. Pluripotent cells can differentiate into almost all cells that arise from the three germ layers, but are unable to give rise to the placenta and supporting structures. At around 5 days after fertilization, ESCs that form the inner cell mass of the blastocyst are considered pluripotent. Multipotent stem cells are capable of producing a small range of differentiated cell lineages appropriate to their location and are usually found in adult tissues. However, the use of the term multipotent might be somewhat redundant, since some adult stem cells, once removed from their usual location seem to transdifferentiate into cells that reflect their new environment. Stem cells with the least potential for differentiation are termed unipotent; for example, the epidermal stem cell found in the basal skin layer that only produces keratinized squames [23]. Potential donor cell types Conceptually, a variety of stem and progenitor cell populations could be used for cardiac repair. Each cell type has its own profile of advantages, limitations, and practicability issues in specific clinical settings. Studies comparing distinct cell types are scarce. The first clinically relevant cells to be proposed as a surrogate for cardiomyocytes were skeletal muscle myoblasts. Bone marrow which is easily accessible is, at present, the most frequent source of cells used for clinical cardiac repair [24]. It contains a complex assortment of progenitor cells, including hematopoietic stem cells (HSCs), so-called side population (SP) cells, which account for most long-term self renewal [25] (of hematopoietic lineages after single-cell grafting [26]; mesenchymal stem cells (MSCs) or stromal cells [27]; and multipotential adult progenitor cells (MAPCs), a subset of MSCs [28]. Peripheral blood-derived progenitor cells are isolated from mononuclear blood cells and selected ex vivo by culturing in 'endothelium-specific' medium prior to reinjection into the heart [24, 29, 30]. Further progenitor/stem cell populations investigated include: fat tissue-derived multipotent stem cells [31] multipotential cells from bone marrow or skeletal muscle [32] somatic stem cells from placental cord blood [33], amniotic fluid-derived stem (AFS) cells [34], and cardiac-resident progenitor cells that have a heightened predisposition to adopt the cardiac muscle fate [35, 36]. In each of these newer cases, techniques to isolate and purify the numerically minor population of potent cells will need to be optimized for clinical use. Most of the cells undergoing clinical evaluation are used in an autologous way, so that tissue rejection is obviated. MSCs are thought to be immune-privileged, have been successfully transplanted experimentally in an allogeneic setting without immunosuppression and are currently evaluated in a clinical trial allogeneicly [24]. Modes of cell delivery Cells for cardiac repair can be administered in various ways. The goal of any cell delivery strategy is to transplant sufficient numbers of cells into the myocardial region of interest and to achieve maximum retention of cells within that area. The success of cell delivery is further determined by the local milieu, as it will influence short-term cell survival and cell properties with regard to cell adhesion, transmigration through the vascular wall, and tissue invasion. The three most frequently used routes are intracoronary, percutaneous endocardial or direct intramyocardial injection during surgery. Intracoronary infusion requires migration through the vessel wall into the damaged tissue. Some cell type like bone marrow-derived and blood-derived progenitor cells are known to extravasate and migrate to ischemic areas [37], whereas skeletal myoblasts do not. Satellite cells and mesenchymal cells have been shown to even obstruct the microcirculation after intra-arterial administration, leading to embolic myocardial damage [38]. By contrast, direct delivery of progenitor cells into scar tissue or areas of hibernating myocardium by catheter-based needle injection, direct injection during open-heart surgery, and minimally invasive thoracoscopic procedures are not limited by cell uptake from the circulation or by embolic risk. An offsetting consideration is the risk of ventricular perforation, which may limit the use of direct needle injection into freshly infarcted hearts. In addition, it is hard to envisage that progenitor cells injected into uniformly necrotic tissue – lacking the syncytium of live muscle cells that may furnish instructive signals and lacking blood flow for the delivery of oxygen and nutrients – would receive the necessary cues and environment to engraft and differentiate. Most cells, if injected directly, simply die [39]. Finally, in diffuse diseases such as dilated nonischemic cardiomyopathy, focal deposits of directly injected cells might be poorly matched to the underlying anatomy and physiology. In experimental models, intravenous delivery of endothelial progenitor cells (EPCs) has been shown to improve cardiac function after AMI [40, 41]. However, homing of cells to noncardiac organs limits the clinical applicability of this approach. Thus, it already appears likely that patients' individual pathobiology – the specific underpinnings of their HF – will ultimately influence, if not dictate, the source and route chosen among potential progenitor cell therapies. Given such variations in the underlying clinical context, it is not yet possible on the basis of existing pilot clinical trials, whose design and findings are detailed below, to assert an 'optimal' cell type or 'best' mode of delivery [24]. Possible mechanisms of action The mechanisms by which stem cells repair damaged myocardium or lead to improvement in cardiac function are largely unknown, however, the two fundamental activities of stem cells are, respectively (i) the use of cell therapies to directly or indirectly improve neovascularization, i.e., vasculogenesis, angiogenesis and arteriogenesis; and (ii) differentiation into cardiomyocytes and formation of myocardial tissue. Functional benefits may also be mediated through paracrine secretion of growth factors or cytokines which could indirectly promote survival of cardiomyocytes by inhibition of cardiac apoptosis, may lead to mobilization of endogenous progenitor cells and affect remodeling. Stem cells may also fuse with the native dysfunctional myocytes to augment function [42, 43]. A wide range of cell population have been tested and almost all appear to confer benefit which hints at a possible involvement of various mechanisms. The extent to which these different mechanisms are active may critically depend on the cell type and setting, such as acute or chronic injury. Because fundamentally different pathophysiologic processes are targeted and yield some improvement in both experimental and clinical trials, the mechanisms of action are not predetermined but depend also on the host environment. For example, in patients with acute myocardial infarction (AMI), progenitor cell transplantation is predicted to significantly modify postinfarction LV remodeling through enhanced neovascularization and reduced cardiomyocyte apoptosis, irrespective of long-term engraftment and transdifferentiation. Conversely, these mechanisms may have little or no benefit in patients with long-established scars, apart from the functional rescue of hibernating myocytes. Those distinctions might not matter, since patients benefited from many established therapies – including aspirin – before the underlying mechanisms were elucidated. The ultimate success of cell therapy will rest on its ability to show clinical efficacy rather than on the imputed mechanism [24]. Tissue regeneration – goal of cellular transplantation A promethean goal of medicine is to repair damaged organs. Regeneration is an essential function of the human body, with the relatively long-life span predicated on processes that mend damaged muscles, repair broken bones, replenish blood, and restructure vessels. Most human tissues can rebuild themselves, recapturing their original shape over and over. The heart is less well equipped to deal with injury. An alternative strategy to repair a damaged heart is to stimulate it to regenerate or heal itself. While the sudden interruption of the blood supply caused by occlusion of an artery in mammals and amphibians typically leads quickly to cell death, loss of tissue, and fibrous scar formation, Poss et al. [44] demonstrated that zebrafish fully regenerate hearts within 2 months of 20% ventricular resection (Fig. 1). Regeneration occurs through robust proliferation of cardiomyocytes localized at the leading epicardial edge of the new myocardium. They went on to demonstrate that inhibition of regeneration would lead to scarring, since the hearts of zebrafish with mutations in the Mps1 mitotic checkpoint kinase, a critical cell cycle regulator, failed to regenerate and formed scars. Thus, injury-induced cardiomyocyte proliferation in zebrafish can overcome scar formation, allowing cardiac muscle regeneration. Zebrafish will be useful for genetically dissecting the molecular mechanisms of cardiac regeneration and might guide the way for cardiac regeneration in humans [44]. Figure 1Open in figure viewerPowerPoint Regeneration of ventricular myocardium in the resected zebrafish heart. Hematoxylin and eosin stain of the intact zebrafish heart before (a) and after about 20% ventricular resection (b). (c) An intact ventricular apex at higher magnification, indicating the approximate amputation plane (dashed line). All images display longitudinal ventricular sections of the amputation plane. (d) The large clot is filled with nucleated erythrocytes (arrowheads). (e) The heart section is stained for the presence of myosin heavy chain to identify cardiac muscle (brown) and with aniline blue to identify fibrin (blue). The apex is sealed with a large amount of mature fibrin. (f) The fibrin has diminished, and the heart muscle has reconstituted. (g) A new cardiac wall has been created, and only a small amount of internal fibrin remains (arrowhead). (h) This ventricle shows no sign of injury. (i) Quantification of healing at 0, 30, and 60 dpa. Values represent the size of the largest ventricular section (mean ± SEM; *P < 0.05); parentheses indicate the number of hearts examined. Scale bars, 100 μm. Figure adapted from Poss KD, Wilson LG, Keating MT. Heart regeneration in zebrafish. Science 2002: 298: 2188. Reprinted with permission from AAAS. Indications for stem cells and the heart Cellular cardiomyoplasty is intriguing, novel and complex. While its full potential remains to be seen, attempts to treat a variety of different cardiac pathologies have been reported with encouraging results. Stem cells have been shown not only to improve myocardial function after infarction, but also to yield beneficial effects in chronic ischemia [45], dilated cardiomyopathy, [46-49] rhythm disturbances, and more recently in acute myocarditis [50]. Stem cell therapy in nonischemic dilated cardiomyopathy The role of stem cell therapy in nonischemic dilated cardiomyopathy has only been explored in single patients [48] pilot studies [47] and clinical trials published in Chinese with no abstract in English available. While stem cell injection in those patients resulted in significant improved NYHA functional class and increased ejection fraction further studies are required to elucidate possible mechanisms and the true potential of this treatment approach in this setting. Stem cells as biological heart pacemakers Abnormalities in the pacemaker function of the heart or in cardiac impulse conduction may result in the appearance of a slow heart rate, traditionally requiring the implantation of a permanent electronic pacemaker [51]. Grafting stem cells as peacemaking cells, either derived directly during the differentiation of human ESCs (hESCs) or engineered from MSCs in an attempt to generate biological alternatives to implantable devices into the myocardium has been reported by several groups. Back in 1993, Maltsev et al. [52] reported of ESCs differentiated in vitro, into cardiomyocytes (CMs) representing phenotypes corresponding to sinus node, atrium or ventricle of the heart. Their action potential revealed shapes, pharmacological characteristics and hormonal regulation inherent to adult sinus nodal, atrial or ventricular cells. Xue et al. [53] demonstrated that electrically active, donor CMs derived from hESCs could be stably genetically engineered by a recombinant lentivirus to functionally integrate with otherwise-quiescent, recipient, ventricular CMs to induce rhythmic electrical and contractile activities in vitro. The integrated syncytium was responsive to the β-adrenergic agonist isoproterenol as well as to other pharmacologic agents, such as lidocaine. Similarly, a functional hESC-derived pacemaker could be implanted in the left ventricle in vivo. Detailed optical mapping of the epicardial surface of guinea pig hearts transplanted with hESC-derived CMs confirmed the successful spread of membrane depolarization from the site of injection to the surrounding myocardium. Co-workers generated CM cell grafts from hESCs in vitro, using the embryoid body differentiating system [54, 55]. This tissue formed structural and electromechanical connections with cultured rat CMs. After transplantation into the hearts of swine with complete atrioventricular block these hESC-derived CMs showed integration and pacing function, as assessed by detailed three-dimensional electrophysiologic mapping and histopathologic examination. Still the field is very much in its infancy. Little is known of the longevity of the constructs used and the challenges with regard to teratogenicity of hESCs and rejection. Stem cells in ischemic heart disease Ischemic heart disease is by far the most prevalent pathology of the heart, the most important field of stem cell research for the heart and thus the focus of the current review. Despite medical therapy most patients will develop HF or major LV systolic dysfunction at some time after an MI. While mortality may be decreasing, the morbidity associated with coronary heart disease is increasing as more people survive acute MI and grow old. Therefore, a fundamental shift in the underlying etiology of HF is becoming evident, in which the most common cause is no longer hypertension or valvular disease, but long-term survival after AMI. The molecular, cellular, biochemical, and structural changes occurring in the myocardium, often referred to as remodeling, have been studied extensively in patients with HF [56]. After MI, a series of progressive adverse effects takes place, including: (i) noncontractile and potentially expanding scar tissue forming in the infarcted zone; (ii) the volume load induced by such expansion; and (iii) the pressure load induced by the increased volume load. The mixed pressure and volume load [57, 58] leads to a remodeling of the entire left ventricle in proportion to infarct size [59] with a fall in ejection fraction. However, the increase in the left-ventricular volume augments the stroke volume by the Starling mechanism so that cardiac output is relatively normal. Figure 2 summarizes the three patterns of remodeling. Early postinfarct remodeling could be beneficial and promote survival, but with deleterious hemodynamic consequences in the long-term. The increase in wall stress in the scar area results in lengthening of the remaining contractile tissue, and can occur up to 2 years postinfarct with increased cardiovascular death. In the postinfarct period, enhanced activity of metalloproteinases breaks down the existing collagen while promoting the formation of new collagen that is poorly cross-linked, resulting in a side-to-side slippage of myocytes that contributes to ventricular remodeling [60, 61] with thinning of the left-ventricular wall. Some manifestations of remodeling can occasionally be reversed by load reduction aiming to lessen the distending or deforming forces. ACE-inhibitor therapy helps to attenuate the increase in wall stress and to reduce dilation of the left-ventricle. If left-ventricular dilation is avoided, then the pure hypertrophic response of surviving myocytes gives hemodynamic benefit. Such early recovery could be explained by postreperfusion stunning or intrinsic repair of the surviving left-ventricular myocytes. β-blockade also reduces the afterload, and hence the intracavity systolic pressure, increases the ejection fraction while reducing end-diastolic left-ventricular volumes. There is also evidence that prolonged, near-complete unloading of the left ventricle with the use of a LVAD is associated with structural reverse remodeling accompanied by functional improvement [60, 61]. Figure 2Open in figure viewerPowerPoint Postinfarct left-ventricular remodeling patterns. (a) Simplified overall pattern based on animal models. There is potential for substantial remodeling of infarct zone and increased volume of non-infarcted zone. Endocardial wall motion of human hearts in (b) early postinfarct phase and (c) late postinfarct phase derived from contrast v