The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.generation of human myocardium ͉ progenitor cells ͉ stem cell niches

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 labelretaining 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 lineagecommitted 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-lineagecommitted cells and supporting cells. The undifferentiated state of CSCs is coupled with the expression of ␣4-integrin, which colocalizes with the ␣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.

Abstract-Cardiac stem cells and early committed cells (CSCs-ECCs) express c-Met and insulin-like growth factor-1 (IGF-1) receptors and synthesize and secrete the corresponding ligands, hepatocyte growth factor (HGF) and IGF-1. HGF mobilizes CSCs-ECCs and IGF-1 promotes their survival and proliferation. Therefore, HGF and IGF-1 were injected in the hearts of infarcted mice to favor, respectively, the translocation of CSCs-ECCs from the surrounding myocardium to the dead tissue and the viability and growth of these cells within the damaged area. To facilitate migration and homing of CSCs-ECCs to the infarct, a growth factor gradient was introduced between the site of storage of primitive cells in the atria and the region bordering the infarct. The newly-formed myocardium contained arterioles, capillaries, and functionally competent myocytes that with time increased in size, improving ventricular performance at healing and long thereafter. The volume of regenerated myocytes was 2200 m 3 at 16 days after treatment and reached 5100 m 3 at 4 months. In this interval, nearly 20% of myocytes reached the adult phenotype, varying in size from 10 000 to 20 000 m 3 . Moreover, there were 43Ϯ13 arterioles and 155Ϯ48 capillaries/mm 2 myocardium at 16 days, and 31Ϯ6 arterioles and 390Ϯ56 capillaries at 4 months. Myocardial regeneration induced increased survival and rescued animals with infarcts that were up to 86% of the ventricle, which are commonly fatal. In conclusion, the heart has an endogenous reserve of CSCs-ECCs that can be activated to reconstitute dead myocardium and recover cardiac function. (Circ Res. 2005;97:663-673.)Key Words: cardiac progenitor cells Ⅲ myocardial regeneration Ⅲ mortality A dult cardiac stem cells (CSCs) and early committed cells (ECCs) express the stem cell antigens c-kit, MDR1, and Sca-1. 1-4 c-kit POS cells are self-renewing, clonogenic, and multipotent and give rise to myocytes, smooth muscle cells (SMCs), and endothelial cells (ECs) in vitro and in vivo. 1,5 A similar category of CSCs-ECCs has been found in the human heart, 6,7 suggesting that these undifferentiated cells participate in the normal turnover of cardiac cells and, under favorable conditions, have the ability to form myocytes, coronary arterioles, and capillary structures. 1,5,7 The presence of CSCs-ECCs raises the question of why they fail to respond to ischemic injury with regeneration of myocytes and coronary vessels and restoration of function. CSCs-ECCs distributed within the damaged area may die together with parenchymal cells, however, and SMCs and ECs in coronary vessels prevent myocardial repair. For this reason, we have explored the possibility that CSCs-ECCs, if properly activated, can translocate to sites of damage, survive the unfavorable environment, multiply, and differentiate, forming functionally competent myocardium.Hepatocyte growth factor (HGF) stimulates cell migration 8 by expression of metalloproteinases (MMPs) 9 that by breaking down the extracellular matrix favor cell locomotion, homing, and tissue reco...

Abstract-Recent studies in mice have challenged the ability of bone marrow cells (BMCs) to differentiate into myocytes and coronary vessels. The claim has also been made that BMCs acquire a cell phenotype different from the blood lineages only by fusing with resident cells. Technical problems exist in the induction of myocardial infarction and the successful injection of BMCs in the mouse heart. Similarly, the accurate analysis of the cell populations implicated in the regeneration of the dead tissue is complex and these factors together may account for the negative findings. In this study, we have implemented a simple protocol that can easily be reproduced and have reevaluated whether injection of BMCs restores the infarcted myocardium in mice and whether cell fusion is involved in tissue reconstitution. For this purpose, c-kit-positive BMCs were obtained from male transgenic mice expressing enhanced green fluorescence protein (EGFP). EGFP and the Y-chromosome were used as markers of the progeny of the transplanted cells in the recipient heart. By this approach, we have demonstrated that BMCs, when properly administrated in the infarcted heart, efficiently differentiate into myocytes and coronary vessels with no detectable differentiation into hemopoietic lineages. However, BMCs have no apparent paracrine effect on the growth behavior of the surviving myocardium. Within the infarct, in 10 days, nearly 4.5 million biochemically and morphologically differentiated myocytes together with coronary arterioles and capillary structures were generated independently of cell fusion. In conclusion, BMCs adopt the cardiac cell lineages and have an important therapeutic impact on ischemic heart failure. Key Words: transdifferentiation Ⅲ myocardial regeneration Ⅲ cell fusion S everal studies have suggested that adult bone marrow cells (BMCs) can differentiate into cell lineages distinct from the organ in which they reside. 1 The recognition that BMCs maintain some of the growth potential of younger cells has promoted a heated debate about stem cell plasticity and the utilization of BMCs in the treatment of ischemic heart failure. 2 The efficacy of BMCs for myocardial regeneration after infarction was documented 3 years ago, 3 and this protocol was rapidly applied clinically. 4 Nine clinical trials have been completed and several are ongoing and, with the exception of one, 5 all other show positive results. 4,6 -12 Because of the difficulty to demonstrate myocardial regeneration in humans in the absence of cardiac biopsies, three possibilities have been raised in the interpretation of the improvement of cardiac function in patients. They include the development of coronary vessels that rescue hibernating myocardium, 11,12 de novo formation of myocytes 8,10 and vascular structures 4,8,9,12 or the activation and growth of resident progenitor cells via a paracrine effect 12 mediated by BMCs. These are important biological and clinical questions that can be addressed experimentally to acquire a better understanding of the re...

The myocardium behaves like a sophisticated orchestra that expresses its true potential only if each member performs the correct task harmonically. Recapitulating its complexity within engineered 3D functional constructs with tailored biological and mechanical properties, is one of the current scientific priorities in the field of regenerative medicine and tissue engineering. In this study, driven by the necessity of fabricating advanced model of cardiac tissue, we present an innovative approach consisting of heterogeneous, multi-cellular constructs composed of Human Umbilical Vein Endothelial Cells (HUVECs) and induced pluripotent cell-derived cardiomyocytes (iPSC-CMs). Cells were encapsulated within hydrogel strands containing alginate and PEG-Fibrinogen (PF) and extruded through a custom microfluidic printing head (MPH) that allows to precisely tailor their 3D spatial deposition, guaranteeing a high printing fidelity and resolution. We obtained a 3D cardiac tissue compose of iPSC-derived CMs with a high orientation index imposed by the different defined geometries and blood vessel-like shapes generated by HUVECs which, as demonstrated by in vivo grafting, better support the integration of the engineered cardiac tissue with host’s vasculature.

The possibility that adult bone marrow cells (BMCs) retain a remarkable degree of developmental plasticity and acquire the cardiomyocyte lineage after infarction has been challenged, and the notion of BMC transdifferentiation has been questioned. The center of the controversy is the lack of unequivocal evidence in favor of myocardial regeneration by the injection of BMCs in the infarcted heart. Because of the interest in cell-based therapy for heart failure, several approaches including gene reporter assay, genetic tagging, cell genotyping, PCR-based detection of donor genes, and direct immunofluorescence with quantum dots were used to prove or disprove BMC transdifferentiation. Our results indicate that BMCs engraft, survive, and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes. BMCs and myocytes express at their interface connexin 43 and N-cadherin, and this interaction may be critical for BMCs to adopt the cardiomyogenic fate. With time, a large number of myocytes and coronary vessels are generated. Myocytes show a diploid DNA content and carry, at most, two sex chromosomes. Old and new myocytes show synchronicity in calcium transients, providing strong evidence in favor of the functional coupling of these two cell populations. Thus, BMCs transdifferentiate and acquire the cardiomyogenic and vascular phenotypes restoring the infarcted heart. Together, our studies reveal that locally delivered BMCs generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels. This process occurs independently of cell fusion and ameliorates structurally and functionally the outcome of the heart after infarction. myocardial infarction ͉ myocardial regeneration ͉ stem cells ͉ transdifferentiation T o date, the hematopoietic stem cell appears to be the most versatile stem cell in crossing lineage boundaries and the most prone to break the law of tissue fidelity (1). Early studies on c-kit-positive bone marrow cell (BMC) differentiation into myocardium have generated great enthusiasm (2, 3), but other observations have rejected the initial results (4-6) and promoted a wave of skepticism about the therapeutic potential of BMCs for the injured heart. The major criticisms include: (i) lack of utilization of genetic markers for the recognition of donor BMCs and their progeny; (ii) inaccurate interpretation of the original data due to autofluorescence artifacts; and (iii) the possibility that myocyte regeneration is mediated by fusion of BMCs with resident myocytes rather than BMC transdifferentiation. To address these important questions and demonstrate reproducibility of results, four laboratories with complementary expertise have undertaken a series of joined experiments to acquire information on the plasticity of BMCs and their therapeutic potential for the infarcted heart.In this effort, BMCs for myocardial regeneration were obtained from three transgenic mice. In the first, EGFP was driven by the ubiquitous ␤-actin promoter; in the second, EGFP was ...

Abstract-Heart failure is the leading cause of death in the elderly, but whether this is the result of a primary aging myopathy dictated by depletion of the cardiac progenitor cell (CPC) pool is unknown. Similarly, whether current lifespan reflects the ineluctable genetic clock or heart failure interferes with the genetically determined fate of the organ and organism is an important question. We have identified that chronological age leads to telomeric shortening in CPCs, which by necessity generate a differentiated progeny that rapidly acquires the senescent phenotype conditioning organ aging. CPC aging is mediated by attenuation of the insulin-like growth factor-1/insulin-like growth factor-1 receptor and hepatocyte growth factor/c-Met systems, which do not counteract any longer the CPC renin-angiotensin system, resulting in cellular senescence, growth arrest, and apoptosis. However, pulse-chase 5-bromodeoxyuridine-labeling assay revealed that the senescent heart contains functionally competent CPCs that have the properties of stem cells. This subset of telomerasecompetent CPCs have long telomeres and, following activation, migrate to the regions of damage, where they generate a population of young cardiomyocytes, reversing partly the aging myopathy. The senescent heart phenotype and heart failure are corrected to some extent, leading to prolongation of maximum lifespan. (Circ Res. 2008;102:597-606.)

Primitive cells capable of generating small resistance arterioles and capillary structures in the injured myocardium have been identified repeatedly. However, these cells do not form large conductive coronary arteries that would have important implications in the management of the ischemic heart. In the current study, we determined whether the human heart possesses a class of progenitor cells that regulates the growth of endothelial cells (ECs) and smooth muscle cells (SMCs) and vasculogenesis. The expression of vascular endothelial growth-factor receptor 2 (KDR) was used, together with the stem cell antigen c-kit, to isolate and expand a resident coronary vascular progenitor cell (VPC) from human myocardial samples. Structurally, vascular niches composed of c-kit-KDR-positive VPCs were identified within the walls of coronary vessels. The VPCs were connected by gap junctions to ECs, SMCs, and fibroblasts that operate as supporting cells. In vitro, VPCs were self-renewing and clonogenic and differentiated predominantly into ECs and SMCs and partly into cardiomyocytes. To establish the functional import of VPCs, a critical stenosis was created in immunosuppressed dogs, and tagged human VPCs were injected in proximity to the constricted artery. One month later, there was an increase in coronary blood flow (CBF) distal to the stenotic artery, resulting in functional improvement of the ischemic myocardium. Regenerated large, intermediate, and small human coronary arteries and capillaries were found. In conclusion, the human heart contains a pool of VPCs that can be implemented clinically to form functionally competent coronary vessels and improve CBF in patients with ischemic cardiomyopathy.