Resident human cardiac stem cells: role in cardiac cellular homeostasis and potential for myocardial regeneration

Current treatments for myocardial infarction have significantly reduced the acute mortality of ischemic cardiomyopathy. This reduction has resulted in the survival of a large cohort of patients left with a significant 'myocyte deficit'. Once this deficit leads to heart failure there is no available therapy to improve long-term cardiac function. Recent developments in stem cell biology have focused on the possibility of regenerating contractile myocardial tissue. Most of these approaches have entailed the transplantation of exogenous cardiac-regenerating cells. Recently, we and others have reported that the adult mammalian myocardium, including that in humans, contains a small pool of cardiac stem and progenitor cells (CSCs) that can replenish the cardiomyocyte population and, in some cases, the coronary microcirculation. The human CSCs (hCSCs) are involved in maintaining myocardial cell homeostasis throughout life and participate in remodeling in cardiac pathology. They can be isolated, propagated and cloned. The progeny of a single cell clone differentiates in vitro and in vivo into myocytes, smooth muscle and endothelial cells. Surprisingly, in response to different forms of stress, hCSCs acquire a senescent, dysfunctional phenotype. Strikingly, these nonfunctional CSCs constitute around 50% of the total CSC pool in older individuals-those most likely to be candidates for hCSC-based myocardial regeneration. Therefore, the challenge to develop clinically effective therapies of myocardial regeneration is twofold: to produce the activation of the hCSCs in situ in order to obviate the need for cell transplantation, and to elucidate the mechanisms responsible for hCSC senescence in order to prevent or reverse its development.     

Myocyte death and renewal: modern concepts of cardiac cellular homeostasis

The adult mammalian myocardium has a robust intrinsic regenerative capacity because of the presence of cardiac stem cells (CSCs). Despite being mainly composed of terminally differentiated myocytes that cannot re-enter the cell cycle, the heart is not a postmitotic organ and maintains some capacity to form new parenchymal cells during the lifespan of the organism. Myocyte death and formation of new myocytes by the CSCs are the two processes that enable this organ to maintain a proper and uninterrupted cardiac output from birth to adulthood and into old age. CSCs are activated in response to pathological or physiological stimuli, whereby they enter the cell cycle and differentiate into new myocytes (and vessels) that significantly contribute to changes in myocardial mass. The future of regenerative cardiovascular medicine is arguably dependent on our success in dissecting the biology and mechanisms regulating the number, growth, differentiation, and aging of CSCs. This information will generate the means to manipulate CSC growth, survival, and differentiation and, therefore, will provide the tools for the design of more physiologically relevant clinical regeneration protocols. In this article, we review the developments in cardiac cell biology that might, in our opinion, have a broad impact on cardiovascular medicine.     

Cardiac stem and progenitor cell biology for regenerative medicine

Stem cell therapy is a new and promising treatment of heart disease. However, the race is still on to find the "best" cell to reconstitute the myocardium and improve function after myocardial damage. The recent discovery in the adult mammalian myocardium of a small cell population with the phenotype, behavior, and regenerative potential of cardiac stem and progenitor cells has proposed these cells as the most appropriate for cell therapy. The existence of these cells has provided an explanation for the hitherto unexplained existence of a subpopulation of immature cycling myocytes in the adult myocardium. These findings have placed the heart squarely among other organs with regenerative potential despite the fact that the working myocardium is mainly constituted of terminally differentiated cells. Although CSCs (cardiac cells proven to have stem and/or progenitor characteristics) can be isolated and amplified in vitro or stimulated to differentiate in situ, it has become reasonable to exploit this endogenous regenerative potential to replace the lost muscle with autologous functional myocardium. Therefore, it is imperative to obtain a better understanding of the biology and regenerative potential of the endogenous CSCs. This will enable us to design better protocols for the regeneration of functional contractile mass after myocardial injury.     

Stem cell-based therapies in ischemic heart diseases: a focus on aspects of microcirculation and inflammation

Stem cells possessing the potential to replace damaged myocardium with functional myocytes have drawn increasing attention in the past decade in treating ischemic heart diseases; these diseases are the leading cause of morbidity and mortality in the world. The adult heart has recently been shown to contain a few cardiac stem cells (CSCs) that, in theory, suggest cardiac repair following acute myocardial infarction is possible if the CSC titer could be increased. Stem cell-based therapies, including hematopoietic stem cells and mesenchymal stem cells, were proven to be marginal and transitional. Multiple factors and mechanisms, rather than direct cardiac regeneration are involved in stem cell-mediated cardiac functional improvement. This review will focus on (1) the interaction between inflammation and stem cells; (2) the fate of stem cells at the microcirculatory level, and their subsequent influences on stem cell-based therapies.     

Myocardial aging

This review questions the old paradigm that describes the heart as a post-mitotic organ and introduces the notion of the heart as a self-renewing organ regulated by a compartment of multipotent cardiac stem cells (CSCs) capable of regenerating myocytes and coronary vessels throughout life. Because of this dramatic change in cardiac biology, the objective is to provide an alternative perspective of the aging process of the heart and stimulate research in an area that pertains to all of us without exception. The recent explosion of the field of stem cell biology, with the recognition that the possibility exists for extrinsic and intrinsic regeneration of myocytes and coronary vessels, necessitates reevaluation of cardiac homeostasis and myocardial aging. From birth to senescence, the mammalian heart is composed of non-dividing and dividing cells. Loss of telomeric DNA is minimal in fetal and neonatal myocardium but rather significant in the senescent heart. Aging affects the growth and differentiation potential of CSCs interfering not only with their ability to sustain physiological cell turnover but also with their capacity to adapt to increases in pressure and volume loads. The recognition of factors enhancing the activation of the CSC pool, their mobilization, and translocation, however, suggests that the detrimental effects of aging on the heart might be prevented or reversed by local stimulation of CSCs or the intramyocardial delivery of CSCs following their expansion and rejuvenation in vitro. CSC therapy may become, perhaps, a novel strategy for the devastating problem of heart failure in the old population.     

内源性心脏干/祖细胞活化的研究进展

随着现代生活方式的改变和老龄化的问题,心血管疾病已成为威胁人类健康的第一杀手。然而当前医疗诊治技术仍难以彻底改善心肌梗死后缺血性心肌病及心力衰竭患者的预后。干细胞的出现为心肌再生带来了新的希望。与其他成体干细胞相比,内源性心脏干细胞(CSCs)具有分化成心肌细胞和血管内皮细胞的潜能,可对受损心肌起到明显的修复作用。研究发现,心肌梗死后内源性CSCs的激活与多种因素有关。最近几年,内源性CSCs活化介导的心肌修复引起了研究人员极大的兴趣。本文将对CSCs激活所涉及的主要途径进行论述,以深化对内源性CSCs功能的理解。     

Autologous cell transplantation for the treatment of damaged myocardium

Autologous cell transplantation for the treatment of damaged myocardium after myocardial infarction is becoming an increasingly promising strategy. This form of treatment can be divided into 2 treatment strategies: The first uses differentiated cell types to replace the scarred tissue with living cells, while the second strategy uses stem cells in an attempt to regenerate myocardium. Over the past decade, multiple cell types have been used in animal studies, and clinical trials to determine the safety of injecting and engrafting skeletal myoblasts into damaged myocardium are presently being conducted. Animals studies focused on using stem cells to regenerate damaged myocardium have shown a naturally occurring reparative process that consists of up-regulation of progenitor cell release from the bone marrow after myocardial infarction, homing of these cells to the injured tissue, and differentiation of these progenitor cells into vascular cells and cardiac myocytes within the infarcted tissue. Unfortunately, this process occurs with great infrequency. Strategies to regenerate myocardium with stem cells either extract stem cells from the bone marrow and inject these cells into the damaged area or they attempt to increase the efficiency of the natural reparative process by increasing the mobilization of bone marrow-derived stem cells after myocardial infarction. This review summarizes the field of cell transplantation over the past decade, discusses areas of controversy, and proposes an outline of advancements that need to be made in both the clinical and scientific arenas for autologous cell transplantation to fully reach its clinical potential.     

Regeneration of the infarcted heart with stem cells derived by nuclear transplantation

Nuclear transfer techniques have been proposed as a strategy for generating an unlimited supply of rejuvenated and histocompatible stem cells for the treatment of cardiac diseases. For this purpose, c-kit-positive fetal liver stem cells obtained from cloned embryos were injected in the border zone of infarcted mice to induce tissue reconstitution. Cloned embryos were derived from somatic cell fusion between nuclei of cultured LacZ-positive fibroblasts and enucleated oocytes of a different mouse strain. We report that regenerating myocardium replaced 38% of the scar at 1 month. The rebuilt tissue expressed LacZ and was composed of myocytes and vessels connected with the coronary circulation. Myocytes were functionally competent and expressed contractile proteins, desmin, connexin43, and N-cadherin. These structural characteristics indicated that the new myocytes were electrically and mechanically coupled. Similarly, the formed coronary arterioles and capillary structures contained blood and contributed, therefore, to tissue oxygenation. Cardiac replacement resulted in an improvement of ventricular hemodynamics and in a reduction of diastolic wall stress. These beneficial effects were obtained by stem cell transdifferentiation and commitment to the cardiac cell lineages. Myocardial growth was independent from fusion of the injected stem cells with preexisting partner cells. In conclusion, c-kit-positive stem cells derived by nuclear transfer cloning restore infarcted myocardium. Although problems currently plague nuclear transplantation, including the potential for epigenetic and imprinting abnormalities, stem cells derived from cloned embryos are sufficiently normal to repair damaged tissue in vivo. Importantly, the magnitude of myocardial regeneration obtained in this study is significantly superior to that achieved with adult bone marrow cells.     

Generation of new cardiomyocytes in the adult heart: Prospects of myocardial regeneration as an alternative to cardiac transplantation]

The classic dogma, still prevalent in cardiology, that the adult myocardium is a terminally differentiated tissue unable to produce new cardiomyocytes needs to be revised in light of recent results. In human and experimental animals there is now incontrovertible evidence that new myocytes are continuously generated throughout life in response to physiological and pathological stimuli. Moreover, the elucidation of mechanisms responsible for the hypertrophic response indicate similarity and overlap with the mechanisms involved in cell death by apoptosis as well as cell growth.During cardiac development, from birth to adulthood, there is a balance between the stimuli induce cell growth -by hypertrophy and hyperplasia- on one hand and those that induce programmed cell death on the other. In human and experimental animals it has been well documented that pathological conditions, such as diabetes and hypertension, can increase dramatically the rate of cell death. Moreover, high rates of cell death have been measured in normal adult human hearts and those of mice and rats. No surprisingly, these values increase significantly with age and high in senescence. By themselves, these high rates of normal cell death provide a very compelling argument in favor of cardiomyocyte regeneration. Without cell renewal, these rates of cell death would be incompatible with survival because the heart would disappear before early adulthood. As expected, direct measurement of rates of new cell formation in adult hearts demonstrate high rates of cell renewal that compensate for cell death. Thus, the heart is in continuous cellular turnover with new myocardial cells replacing the older ones.Experiments with fetal mouse cardiocytes shows that the retinoblastoma protein is responsible for the cardiocyte withdrawal from the cell cycle during development. The identification in the adult heart of a subpopulation of quiescent cells that have many of the characteristics of stem cells able to rapidly enter the cell cycle and generate new cardiocytes is yet another evidence that the heart continuously produces new cardiocytes to replace those that disappear either by apoptosis or necrosis.Surprisingly, stem cells other that those from the heart are able to produce new cardiocytes and repopulate the myocardium. We have used bone marrow stem cells injected into the border zone of post-coronary occlusion necrosis. Remarkably, these cells have proven to be very effective in generating new myocardium in the necrotic zone that is integrated to the rest of the muscle and irrigated by new vessels. These results demonstrate that stem cells provide a new avenue for the generation of new contractile tissue. This approach could prove useful in the treatment of chronic cardiac failure and post-ischemic necrosis.     

干细胞移植治疗心肌梗死

近年来,干细胞包括骨骼肌成肌细胞、胚胎干细胞和骨髓干细胞已被应用于心肌再生.心肌梗死动物模型的实验研究表明,干细胞移植改善了梗死心肌的功能,但其作用机制尚不十分清楚.本文对干细胞移植治疗心肌梗死研究中取得的成就、目前的研究焦点和有待解决的问题以及临床应用的潜力做一个总的评述.     

改善心肌原位干细胞治疗心肌梗死疗效的策略

虽然药物治疗、心脏介入手术及心脏搭桥手术等治疗心肌梗死（MI）的方法在不断改进,但MI仍然是全球范围内人类的主要死因。干细胞治疗作为一种可以替代梗死心肌细胞的治疗方法,已经进行了临床试验,但其改善心功能的效果十分有限。目前关于改善心肌原位干细胞（CSCs）治疗MI疗效的策略主要有：①选择更合适的CSCs;②用基因工程的方法处理CSCs;③采用不同导入CSCs的方法。本文就以上3种主要策略的最新研究进展、以CSCs治疗MI的机制以及目前存在的问题作一综述。     

Current issues and innovations in stem cell based therapy

It is well established that stem cells can differentiate into cell types of the organ in which these are transplanted.However,the process is very slow due to lack of understanding of signals important for their survival and differentiation,most optimal stem cells and their plasticity.Limitations and advantages of various cell subtypes will be described. The rate of stem cells mobilization and their survival in the ischemic environment are major obstacles in engraftment and differentiation of stem cells for meaningful repair of the infarcted myocardium. Manipulation of stem cells with ischemic preconditioning,combined gene and cell therapy together with simultaneous activation of diverse signaling pathways for massive stem cell mobilization & regeneration has significant impact on the repair process by stem cells.These and other difficulties encountered in efficient use of various stem cells have resulted in invention of induced pluripotent stem cells which could revolutionize the stem cell based therapy and their applications for understanding of human disease and drug screening in the near future. Reprogramming of adult cells into iPS cells without the use of viral vectors is a major challenge towards getting iPS cells without viral integration into cells.To meet this challenge we have repro-grammed skeletal myoblasts into iPS cells with high efficiency using epigenetic modifiers.Transplantation of iPS cells derived pure cardiac progenitors into infarcted myocardium led to extensive repopulation of scar area with fully developed myocytes without tumor formation and resulting in marked improvement in cardiac function.Reprogramming with pure chemical means will make therapeutic use of these cells more safer.Targeting the induced pluripotent stem cells towards cardiac progenitors and their application towards transplantation is a major step forward in enhancing the myocardial repair capacity by these cells.     

Cardiovascular regenerative medicine at the crossroads. Clinical trials of cellular therapy must now be based on reliable experimental data from animals with characteristics similar to human's

It is now over 4 years since early reports of murine models raised high expectations that bone marrow cell transplantation to the postischemic myocardium could produce physiologically significant myocardial regeneration. In quick succession, a flurry of publications documented the capacity of a variety of other types of adult cell to produce similar results. These publications were all controversial from the start because none addressed the mechanisms involved in the differentiation of transplanted cells. In addition, each report raised at least as many questions as it answered. Despite these obvious weaknesses, the first phase-I clinical trials were started immediately without any further animal experimentation. Today the results of more than a dozen trials are already in the public domain but we still do not have a single piece of solid data documenting whether any of the approaches used is capable of regenerating contractile cells in the human myocardium. This is one of the main reasons why the controversy over the effectiveness of this therapeutic approach is becoming increasingly heated. Moreover, skepticism about the efficacy, and even the feasibility, of inducing clinically relevant myocardial regeneration has increased to the point where it threatens the future of this nascent field. The present situation in myocardial generation contrasts sharply with that in neural regeneration. Although there is a solid and extensive body of knowledge on the origin, phenotype, and regulatory mechanisms of neural stem cells, the first clinical trials have only recently been started. To move this field forward it is necessary to distinguish between the procedures needed to establish proof-of-concept and those that have the potential for widespread clinical application. In addition, the technique must be implemented in such a way that it continues to add to existing knowledge. It is our belief that, if the necessary information is to be acquired, we need: a) significantly more extensive experimental data from animals whose anatomical and physiological characteristics are similar to human's, including data on, for example, dose-effect relationships, the best form of administration, and the duration of therapeutic responses; and b) better understanding of the molecular mechanisms that determine whether cardiac stem cells and transplanted cells will either remain as stem cells or differentiate. In summary, if we are to progress systematically in this area, we need better understanding of myocardial biology. Without it, we run the risk of holding back the field for decades, as happened with the first human heart transplants and with trials of gene therapy.     

Stem cell niches in the adult mouse heart

Cardiac stem cells (CSCs) have been identified in the adult heart, but the microenvironment that protects the slow-cycling, undifferentiated, and self-renewing CSCs remains to be determined. We report that the myocardium possesses interstitial structures with the architectural organization of stem cell niches that harbor long-term BrdU-retaining cells. The recognition of long-term label-retaining cells provides functional evidence of resident CSCs in the myocardium, indicating that the heart is an organ regulated by a stem cell compartment. Cardiac niches contain CSCs and lineage-committed cells, which are connected to supporting cells represented by myocytes and fibroblasts. Connexins and cadherins form gap and adherens junctions at the interface of CSCs-lineage-committed cells and supporting cells. The undifferentiated state of CSCs is coupled with the expression of alpha(4)-integrin, which colocalizes with the alpha(2)-chain of laminin and fibronectin. CSCs divide symmetrically and asymmetrically, but asymmetric division predominates, and the replicating CSC gives rise to one daughter CSC and one daughter committed cell. By this mechanism of growth kinetics, the pool of primitive CSCs is preserved, and a myocyte progeny is generated together with endothelial and smooth muscle cells. Thus, CSCs regulate myocyte turnover that is heterogeneous across the heart, faster at the apex and atria, and slower at the base-midregion of the ventricle.     

Hepatocyte growth factor/Met gene transfer in cardiac stem cells—potential for cardiac repair

The adult heart has been recently recognized as a self-renewing organ that contains a pool of committed resident cardiac stem cells (CSCs) and cardiac progenitor cells (CPCs). These adult CSCs and CPCs can be induced by cytokines and growth factors to migrate, differentiate, and proliferate in situ and potentially replace lost cardiomyocytes. Ligand-receptor systems, such as the tyrosine kinase receptor mesenchymal–epithelial transition factor (Met) and its ligand hepatocyte growth factor (HGF), are potential candidates for boosting migration, engraftment and commitment of CSCs. Here, we discuss the possible application of HGF/Met gene therapy to enhance the ability of CSCs to promote myocardial regeneration.     

Implantation of bone marrow stem cells reduces the infarction and fibrosis in ischemic mouse heart

Myocardial infarction may cause sudden cardiac death and heart failure. Adult cardiac myocytes do not replicate due to lack of a substantive pool of precursor, stem, or reserve cells in an adult heart. Ventricular myocytes following myocardial infarction are replaced by fibrous tissue and this leads to congestive heart failure in severe cases. Anversa et al. described that resident cardiac stem cells are present in the heart, and can repair the damaged mycardium by myocyte regeneration. Recent findings suggest the feasibility of cardiac repair using cell transplantation. However, it remains controversial which cell types are the best for cell transplantation in the ischemic heart. In this study, we demonstrate that cultured bone marrow stromal cells (MSCs) and Lin(-) bone marrow cells upon transplantation differentiate into myocytes and endothelial cells in the ischemic heart, eventually reducing both infarct size and fibrosis.     

Cardiac regeneration

The role and even the existence of new myocyte formation in the adult heart remain controversial. Documentation of cell cycle regulators, deoxyribonucleic acid synthesis, and mitotic images has only in part modified the view that myocardial growth can be accomplished exclusively from hypertrophy of an irreplaceable population of differentiated myocytes. However, myocyte regeneration and death occur physiologically, and these cellular processes are enhanced in pathologic states. These observations have challenged the view of the heart as a postmitotic organ and have proposed a new paradigm in which parenchymal and non-parenchymal cells are continuously replaced by newly formed younger populations of myocytes as well as by vascular smooth muscle and endothelial cells. Heart homeostasis is regulated by a stem cell compartment characterized by multipotent cardiac stem cells that possess the ability to acquire the distinct cell lineages of the myocardium. Similarly, adult bone marrow cells are able to differentiate into cells beyond their own tissue boundary and create cardiomyocytes and coronary vessels. This process has been termed developmental plasticity or transdifferentiation. Because of these properties, bone marrow cells and cardiac stem cells have been employed experimentally in the reconstitution of dead myocardium after infarction. These cell classes hold promise for the treatment of heart failure in humans.     

Cardiac regeneration.

The role and even the existence of new myocyte formation in the adult heart remain controversial. Documentation of cell cycle regulators, deoxyribonucleic acid synthesis, and mitotic images has only in part modified the view that myocardial growth can be accomplished exclusively from hypertrophy of an irreplaceable population of differentiated myocytes. However, myocyte regeneration and death occur physiologically, and these cellular processes are enhanced in pathologic states. These observations have challenged the view of the heart as a postmitotic organ and have proposed a new paradigm in which parenchymal and non-parenchymal cells are continuously replaced by newly formed younger populations of myocytes as well as by vascular smooth muscle and endothelial cells. Heart homeostasis is regulated by a stem cell compartment characterized by multipotent cardiac stem cells that possess the ability to acquire the distinct cell lineages of the myocardium. Similarly, adult bone marrow cells are able to differentiate into cells beyond their own tissue boundary and create cardiomyocytes and coronary vessels. This process has been termed developmental plasticity or transdifferentiation. Because of these properties, bone marrow cells and cardiac stem cells have been employed experimentally in the reconstitution of dead myocardium after infarction. These cell classes hold promise for the treatment of heart failure in humans.     

Granulocyte Macrophage-Colony Stimulating Factor receptor expression on human cardiomyocytes from end-stage heart failure patients

BACKGROUND: In remodelling ventricles, the progression of heart failure is associated with structural changes involving the extra-cellular matrix (ECM) and the cytoskeleton of cardiomyocytes, associated with fibrosis, cellular damage and death. The role of some cytokines and haematopoietic growth factors in the mechanism of both damage and regeneration of cardiac tissue during acute myocardial infarction has been demonstrated. Following heart damage, the development of scarred tissue was considered to be the only outcome, since myocytes were considered to be terminally differentiated cells. However, recent studies in animal models and adult human hearts have shown that myocytes can proliferate under the modulation of several factors. AIMS: To assess Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) receptor expression in healthy and diseased human hearts, and to evaluate the possible role of GM-CSF and its receptor in the regeneration of cardiac tissue in chronic cardiomyopathy. METHODS AND RESULTS: GM-CSFR expression in human cardiac tissue from explanted hearts of ten patients with end-stage heart failure and in cardiac biopsies from eight normal human hearts was studied by immunohistochemistry, and cellular and molecular biology assays. Our results demonstrated an increase in GM-CSFR in cardiomyocytes from end-stage heart failure tissues as compared to normal control tissues. CONCLUSIONS: We hypothesize that GM-CSF plays a role in apoptotic and/or ECM deposition processes as well as in cytoskeleton modification in the myocardium.