Adult c-kitpos Cardiac Stem Cells Are Necessary and Sufficient for Functional Cardiac Regeneration and Repair

See related article, pages 89–97 Ischemic diseases and heart failure remain the major causes of morbidity and mortality in the industrialized world. Several new therapeutic modalities have been developed to interfere with the response to injury and to improve cardiac function after ischemia or cardiomyopathy. Experimental and clinical studies demonstrated that the transplantation of bone marrow–derived or tissue-resident stem/progenitor cells in diseased hearts improves neovascularization and functional recovery.1 However, the recent discovery of cardiac stem cells (CSCs) in the adult heart poses the question whether activation of the endogenous pool of these resident stem cells may compensate for the loss of cardiac tissue after injury and improve functional recovery. Indeed, a genetic fate-mapping study elegantly demonstrated that stem cells or precursor cells significantly contributed to the replacement of adult mammalian cardiomyocytes after injury.2 Although it remains unclear to what extent cardiac regeneration in this genetic model was mediated by CSCs or circulating progenitor cell populations being attracted to the heart after injury, mounting evidence suggests that injury-associated signals activate CSCs in situ, which subsequently contribute to the refreshment of the injured heart.3 Obviously, to play a significant role in endogenous cardiac regeneration, the relatively small number of endogenous CSCs needs to be expanded after injury before differentiating into cardiac myocytes. However, the molecular mechanisms underlying the activation, expansion, and recruitment of resident CSCs after injury remain unclear. In the current issue of Circulation Research , Siddiqi et al provide an intriguing concept, which might help to mechanistically explain how proliferation of CSCs is regulated in response to injury.4 The authors investigated the cardiac expression of stem cell specific genes, involved in self-renewal and proliferation, during cardiomyopathic injuries and found increased expression of the nucleolar protein nucleostemin—recently discovered in embryonic and adult stem cells as well …

See related article, pages 1090–1092 In the last few years we have witnessed one of most extraordinary revolutions in cardiovascular medicine, namely, an explosion of basic and clinical studies that support the notion that the diseased heart can be repaired by administration of stem cells, resulting in formation of functional new myocytes and vessels. Although the mechanism by which cell therapy improves cardiac function and anatomy remains uncertain, translation of basic findings to the clinical setting is proceeding at a feverish pace.1 A multitude of small, mostly nonrandomized clinical studies have reported improvement in cardiac perfusion and function after therapy with various cell types in patients with acute myocardial infarction or chronic ischemic cardiomyopathy.1 Larger, randomized, double-blinded studies will be reported soon; if they confirm the salubrious effects of cell-based therapies observed in the initial trials, our management of acute myocardial infarction and heart failure will change dramatically. One of the most important and unresolved issues in this scenario is the identity of the ideal cell for myocardial reconstitution. Although most of the clinical studies reported to date have used bone marrow– or skeletal muscle–derived cells, a host of other cells are being investigated in the experimental laboratory. Among these, resident cardiac stem cells (CSCs), discovered by Anversa’s group in 2003,2 hold great promise. In their initial report,2 Beltrami et al identified a lin−/c-kit+ population of primitive cells that can be clonally expanded, differentiates into cardiac myocytes, smooth muscle cells, and endothelial cells in vitro, and is able to reconstitute infarcted myocardium in vivo. It is of translational interest that these cells can effect cardiac repair when delivered via the intravascular route.3 CSCs have also been shown to be present in the human heart, where they give rise to new myocytes in patients with …

See related article, pages 663–673 Research on myocardial regeneration is an exciting and promising area, which challenges the dogma that the heart is a nonregenerating organ. Recently, several methods of stem cell therapy have been developed. One method is to transplant cells into the infarcted area of the myocardium. Currently, clinical trials of autologous skeletal myoblast transplantation into the failed heart are underway and have been reported to improve the cardiac function.1 However, the mechanism of its efficacy is unknown, and there are some questions about the safety because myoblasts do not transdifferentiate into cardiomyocytes and may induce lethal arrhythmia.1 In this point, embryonic stem (ES) cells that can differentiate into cardiomyocytes are thought to be more promising.2 For patients experiencing extensive myocardial infarction or dilated cardiomyopathy, however, the effectiveness of cell transplantation is questionable. Bone marrow-derived cells have been reported to transdifferentiate into various types of cells in situ. Indeed, bone marrow-derived stem cells were reported to prevent left ventricular remodeling after myocardial infarction and improve cardiac function by their differentiation into cardiomyocytes.3 However, recent accumulating evidence has indicated that very few bone marrow cells, if any, transdifferentiate into cardiomyocytes.4–6 Cytokine therapy using G-CSF strongly prevents ventricular remodeling after myocardial infarction by antiapoptotic and angiogenic effects, but not by recruitment of bone marrow cells.7 Over the past few years, adult hearts have been reported to contain the cardiac stem/progenitor cells such as c-kit+,8 Sca-1+,9,10 isl-1+,11 and side population cells.12 Because these cells have the ability to proliferate and …

Cardiac stem cell therapy: Does a newborn infant's heart have infinite potential for stem cell therapy?

The view of the heart as a static organ implies that myocyte death and formation play a negligible role in cardiac homeostasis. Although stem cells have been unexpectedly identified in several organs, including the brain, kidney, lung, and skeletal muscle, the search for a cardiac stem cell (CSC) has been perceived as a futile effort, given the acknowledged lack of regenerative potential of the myocardium. Nevertheless, in the past several years, the demonstration of myocyte renewal in the normal and diseased heart has revealed a new, dynamic, and lively picture of this organ. Components of the cell cycle machinery and markers of cell replication have been detected in cardiomyocytes. The demonstration that karyokinesis and cytokinesis involve cells expressing contractile proteins has provided evidence that cardiomyocyte division occurs in the adult heart.1 More recently, pulse-chase assays with thymidine analogs,2 lineage tracing protocols,3 and 14C birth dating of cells4 have shown the existence of myocyte turnover, a process that has been found to differ in magnitude according to the methods used for its documentation and quantification. Article see p 2559 Over the years, the heart has provided us with the evidence that solves the critical problem of the origin of cardiomyocytes. At this moment in time, paraphrasing Eugenio Montale, we could say that the human heart is “on the verge of betraying [its] final secret offering the opportunity to uncover…the still point of the world, the link that won't hold, the thread to untangle that will finally lead to the heart of a truth.”5 Newly generated myocytes may derive from division of preexisting parenchymal cells or from activation and differentiation of resident CSCs. Discriminating between these two possibilities is not an easy task. Fate-mapping strategies, which are commonly used to track the origin of cells and …

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1–2% of total c-kitpos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

The recognition that myocyte mitosis occurs in the fetal, neonatal, adult, and hypertrophied heart and that a pool of primitive, undifferentiated cells is present in the myocardium has put forward a different view of the biology of the heart. The new paradigm suggests that myocyte formation is preserved during postnatal life, in adulthood or senescence, pointing to a remarkable growth reserve of the heart throughout the course of life of the organism. This article reviews a large body of novel information, which has been obtained in the last 2 decades, in favor of the notion that the mammalian heart has the inherent ability to continuously replace its parenchymal cells and that this unexpected characteristic has important implications in understanding myocardial homeostasis, cardiac aging, and tissue repair. The paradigm that the heart is a postmitotic organ incapable of regenerating parenchymal cells was established in the 1970s, and this dogma has profoundly conditioned basic and clinical research in cardiology for the last 3 decades. On the basis of this paradigm, cardiomyocytes undergo cellular hypertrophy1,2 but cannot be replaced either by entry into the cell cycle of a subpopulation of nonterminally differentiated myocytes or by activation of a pool of primitive cells that become committed to the myocyte lineage. The only response of cardiomyocytes to stress is hypertrophy and/or death. Therefore, a tremendous effort was made to identify the molecular mechanisms of myocyte hypertrophy and their genetic control. A sophisticated knowledge of various signaling pathways has been achieved, and our understanding of the biology of hypertrophic myocyte growth has advanced markedly.3 An array of new technologies has been introduced that has led to a scientific revolution in terms of questions, approaches, and interpretation of experimental results. Despite this enormous progress in our understanding of basic mechanisms of hypertrophy, however, very little …

1. Ellison GM, Vicinanza C, Smith AJ, Aquila I, Leone A, Waring CD, et al. Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair. Cell . 2013;154:827–842. 2. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider F, Walsh S, et al. Evidence for cardiomyocyte renewal in humans. Science. 2009;324:98–102. 3. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003;114:763–776. 4. Senyo SE, Steinhauser ML, Pizzimenti CL, Yang VK, Cai L, Wang M, et al. Mammalian heart renewal by pre-existing cardiomyocytes. Nature. 2013;493:433–436. 5. Nelson TJ, Faustino RS, Chiriac A, Crespo-Diaz R, Behfar A, Terzic A. CXCR4+/FLK-1+ biomarkers select a cardiopoietic lineage from embryonic stem cells. Stem Cells. 2008;26:1464–1473. It is now accepted that the heart is a self-renewing organ,2 although its regenerative capacity is insufficient to restore healthy homeostasis after injury in human adults. Despite advances in characterization and attempts at modulating regeneration of the heart, the origin of newly formed cardiomyocytes is highly debated, with evidence independently supporting the existence of a critical stem cell compartment that supplies newly formed cardiomyocyte after injury3 or suggesting that division of mononucleated cardiomyocytes can replace lost cardiac cells.4 In this study, Ellison et al1 hypothesize that c-kit–positive endogenous cardiac stem cells (eCSCs) are the main source for new functional cardiomyocytes responsible for anatomic and functional recovery in the setting of acute heart failure caused by diffuse injury. Ellison et al1 applied a method of severe diffused myocardial damage caused by a single high dose of isoproterenol (ISO), originally described in adult rats, and translated it to a mouse model to allow lineage tracing experiments. The resulting injury was spontaneously reverted after 4 weeks, creating a system to study …

The cardiac stem cell compartment is indispensable for myocardial cell homeostasis, repair and regeneration in the adult

Endothelial progenitor cells: Neovascularization or more?

The identification of cardiac cells with stem cell properties changed the paradigm of the heart as a post mitotic organ. These cells proliferate and differentiate into cardiomyocytes, endothelial and vascular smooth muscle cells, providing for cardiac cell homeostasis and regeneration. microRNAs are master switches controlling proliferation and differentiation, in particular regulating stem cell biology and cardiac development. Modulation of microRNAs -regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnologic and therapeutic applications. To obtain insights into the regulatory networks active in cardiac stem cells, we characterized the expression profile of 95 microRNAs with reported functions in stem cell and tissue differentiation in mouse cardiac stem cells, and compared it to that of mouse embryonic heart and mesenchymal stem cells. The most highly expressed microRNAs identified in cardiac stem cells are known to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation. We report a subset of differentially expressed microRNAs that are proposed to act as regulators of differentiation and proliferation of adult cardiac stem cells, providing novel insights into active gene expression networks regulating their biological properties.

Background: Adult cardiac stem cells (CSCs) capable of self-renewal and differentiation into cardiac, smooth muscle and endothelial lineages have been shown to promote tissue repair after ischemic injury and improve heart function. Despite their potential role in cardiac regeneration, evidence suggests that resident CSCs are impaired in conditions such as myocardial infarction, limiting their regenerative capacity. Methods and Results: The growth rate of CSCs is significantly reduced by 40% (p<0.05) under hypoxic stress (0.5% O 2 ). Using an extracellular-matrix (ECM) and adhesion-focused PCR array, we found that incubation of CSCs for 18 hours under hypoxia leads to down-regulation of several genes, including osteopontin, thrombospondin-1 and integrins. Due to their critical role in communicating extracellular signals regulating stem cell mobilization, proliferation, survival, migration and differentiation we decided to investigate the role of integrins in CSCs self-renewal and differentiation. Our gene expression studies showed that integrin-β1 (Itgb1) is the most abundant integrin expressed in CSCs. Therefore, we knocked down (KD) Itgb1 in CSCs and evaluated these cells regarding adhesion, growth and differentiation properties. Our results showed that reduction in Itgb1 levels in CSCs significantly decreased their interaction with extracellular matrix components fibronectin, laminin and vitronectin by 32%, 20% and 24% (p<0.05), respectively. KD of Itgb1 in CSCs reduced growth by 40% (p<0.05), similar to what we observed by with wild-type cells in hypoxia. Assessment of endothelial differentiation showed that KD cells have a higher angiogenic potential vs. NS-control, as demonstrated by upregulation of vWF (3-fold), VEGF (2.5-fold), Pecam1 (2.3-fold) and Nos3 (1.9-fold) (p<0.05). Interestingly, Itgb1 KD per se induced upregulation of these vascular markers in CSCs. Conclusions: Our results suggest that Itgb1 plays an important role in CSCs self-renewal, differentiation and cell adhesion. It is possible that loss of self-renewal is a consequence of changes in CSCs fate as knockdown of Itgb1 leads to upregulation of endothelial lineage markers in these cells. This would limit the expansion of CSCs and their regenerative capacity.

In the last few years, several experimental studies have used stem cells of different sources to reconstitute damaged tissues.1 The brain and the heart have been the most investigated organs because of the long-standing view of the lack of regenerating potential of neurons and myocytes.2,3 Bone marrow stem cells (BMSCs) have been reported capable of transdifferentiating in various cell lineages distinct from the site of origin4 and, because of this property, they may constitute a new form of cellular therapy. Neuronal and myocardial growth mediated by bone marrow cells (BMCs) has been demonstrated, but these results have been challenged5,6 and the issue of BMSC transdifferentiation has become highly controversial. Heated debates at scientific meetings, letters in high-profile journals, and reports with contradicting observations have raised questions on the plasticity of BMSCs.5–7 If negative results would be more cautiously interpreted instead of being blown out of context, it is likely that the actual role that adult stem cells play in the repair of tissues and organs would be better understood and appreciated. This is particularly relevant when negative data are dropped as “valid” statements from the podium and are quoted before they undergo peer review and publication.6 A good example of the opposite approach is found in the study of Chen and colleagues8 in this issue of Circulation Research . The authors have utilized, among other sophisticated techniques, confocal microscopy to identify and characterize an important new function of human pluripotent adult mesenchymal BMSCs. In this report, an unequivocal demonstration was obtained on the ability of these cells to deliver vascular endothelial growth factor (VEGF) to an ischemic region of the brain. VEGF accumulation coupled with endogenous activation of endothelial cells and VEGF synthesis promoted vessel formation after stroke. BMSCs via VEGF secretion acted …

The goal of "Recent Developments" is to provide a concise but comprehensive overview of new advances in cardiovascular research, which we hope will keep our readers abreast of recent scientific discoveries and facilitate discussion, interpretation, and integration of the findings.This will enable readers who are not experts in a particular field to grasp the significance and effect of work performed in other fields.It is our hope and expectation that these "Recent Development" articles will help readers to gain a broader awareness and a deeper understanding of the status of research across the vast landscape of cardiovascular research-The Editors.

### Adult Cardiac-Resident MSC-Like Stem Cells With a Proepicardial Origin Chong et al Cell Stem Cell . 2011;9:527–540. The epicardium is derived from the proepicardial organ, a source of multipotent progenitor cells. Epicardium contribution to the developing coronary vasculature and to cardiac interstitial cells has been established. Studies over the past several years have suggested that epicardium-derived cells can adopt cardiomyocyte and vascular smooth muscle fates and can contribute to cardiac repair when activated by injury.1,2 Recently, Chong et al3 have provided a detailed characterization of a population of epicardium-derived multipotent cardiac progenitor cells (cCFU-Fs). These cells, which do not arise from the bone marrow, neural crest, or myocardium, resemble mesenchymal stem cells (MSCs) and may participate in cardiac development, homeostasis, and repair.3 During early cardiac development, cells derived from the proepicardial organ (a cluster of cells located dorsal and adjacent to the looped heart tube) migrate over the myocardium to form the epicardium. Subsequently, epicardium-derived progenitor cells undergo epithelial-to-mesenchymal transition, invade the underlying myocardium, and differentiate into various cardiac lineages.4,5 Signals and cellular contributions from the epicardium have been shown to be indispensable for the establishment of normal coronary vasculature and myocardial architecture.6 Cardiac interstitial cells arise from epicardium, and recent studies have suggested that reactivation of the epicardium after injury could contribute to scarring or myocardial repair after injury.1,2,6,7 In this context, the recent report from Chong et al3 is particularly relevant, because they used rigorous gene expression, culture, and fate lineage analysis to characterize a population of multipotent MSC-like cells resident in the heart that they called cardiac colony-forming units–fibroblast (cCFU-Fs). These cells derive from the proepicardium, not from cardiac myocytes, neural crest, or bone marrow, and they are able to differentiate into endoderm (eg, colonic epithelium), mesoderm (eg, smooth muscle, cardiac muscle, and adipose tissue), and neurectoderm …