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 ...

Rationale Embryonic and fetal myocardial growth is characterized by a dramatic increase in myocyte number, but whether the expansion of the myocyte compartment is dictated by activation and commitment of resident cardiac stem cells (CSCs), division of immature myocytes or both is currently unknown. Objectives In this study, we tested whether prenatal cardiac development is controlled by activation and differentiation of CSCs and whether division of c-kit-positive CSCs in the mouse heart is triggered by spontaneous Ca2+ oscillations. Results We report that embryonic-fetal c-kit-positive CSCs are self-renewing, clonogenic and multipotent in vitro and in vivo. The growth and commitment of c-kit-positive CSCs is responsible for the generation of the myocyte progeny of the developing heart. The close correspondence between values computed by mathematical modeling and direct measurements of myocyte number at E9, E14, E19 and one day after birth strongly suggests that the organogenesis of the embryonic heart is dependent on a hierarchical model of cell differentiation regulated by resident CSCs. The growth promoting effects of c-kit-positive CSCs are triggered by spontaneous oscillations in intracellular Ca2+, mediated by IP3 receptor activation, which condition asymmetric stem cell division and myocyte lineage specification. Conclusions Myocyte formation derived from CSC differentiation is the major determinant of cardiac growth during development. Division of c-kit-positive CSCs in the mouse is promoted by spontaneous Ca2+ spikes, which dictate the pattern of stem cell replication and the generation of a myocyte progeny at all phases of prenatal life and up to one day after birth.

Bone marrow (BM) has long been considered a potential stem cell source for cardiac repair due to its abundance and accessibility. Although previous investigations have generated cardiomyocytes from BM, yields have been low, and far less than produced from ES or induced pluripotent stem cells (iPSCs). Since differentiation of pluripotent cells is difficult to control, we investigated whether BM cardiac competency could be enhanced without making cells pluripotent. From screens of various molecules that have been shown to assist iPSC production or maintain the ES cell phenotype, we identified the G9a histone methyltransferase inhibitor BIX01294 as a potential reprogramming agent for converting BM cells to a cardiac-competent phenotype. BM cells exposed to BIX01294 displayed significantly elevated expression of brachyury, Mesp1, and islet1, which are genes associated with embryonic cardiac progenitors. In contrast, BIX01294 treatment minimally affected ectodermal, endodermal, and pluripotency gene expression by BM cells. Expression of cardiac-associated genes Nkx2.5, GATA4, Hand1, Hand2, Tbx5, myocardin, and titin was enhanced 114,76, 276,46, 635, 123, and 5-fold in response to the cardiogenic stimulator Wnt11 when BM cells were pretreated with BIX01294. Immunofluorescent analysis demonstrated that BIX01294 exposure allowed for the subsequent display of various muscle proteins within the cells. The effect of BIX01294 on the BM cell phenotype and differentiation potential corresponded to an overall decrease in methylation of histone H3 at lysine9, which is the primary target of G9a histone methyltransferase. In summary, these data suggest that BIX01294 inhibition of chromatin methylation reprograms BM cells to a cardiac-competent progenitor phenotype.

Objective-The vascular competence of human-derived hematopoietic progenitors for postnatal vascularization is still poorly characterized. It is unclear whether, in the absence of ischemia, hematopoietic progenitors participate in neovascularization and whether they play a role in new blood vessel formation by incorporating into developing vessels or by a paracrine action. Methods and Results-In the present study, human cord blood-derived CD34 ϩ (hCD34 ϩ ) cells were transplanted into preand postgastrulation zebrafish embryos and in an adult vascular regeneration model induced by caudal fin amputation. When injected before gastrulation, hCD34 ϩ cells cosegregated with the presumptive zebrafish hemangioblasts, characterized by Scl and Gata2 expression, in the anterior and posterior lateral mesoderm and were involved in early development of the embryonic vasculature. These morphogenetic events occurred without apparent lineage reprogramming, as shown by CD45 expression. When transplanted postgastrulation, hCD34 ϩ cells were recruited into developing vessels, where they exhibited a potent paracrine proangiogenic action. Finally, hCD34ϩ cells rescued vascular defects induced by Vegf-c in vivo targeting and enhanced vascular repair in the zebrafish fin amputation model. Key Words: endothelium Ⅲ vascular biology Ⅲ angiogenesis Ⅲ embryology Ⅲ stem cells I t has long been supposed that cells present in the bone marrow, peripheral blood (PB), and cord blood (CB), which copurify with hematopoietic stem cells (HSCs), also give rise to endothelial cells. This speculation was supported by the notion that CD34 ϩ and CD133 ϩ cells differentiate, in culture and in vivo, into cells that express mature endothelial cell markers. These cells, called endothelial progenitor cells (EPCs), [1][2][3] have been the subject of numerous basic and translational studies showing their participation in neovascularization. Recent studies have revealed possible separation between hematopoietic-and nonhematopoietic-derived EPCs. 4 -8 These 2 cell types have been called early and late EPCs, 9 or colony-forming unit-endothelial cells (CFU-ECs) and endothelial colony-forming cells (ECFCs). 7 They are fundamentally distinguished on the basis of hematopoietic marker expression (eg, CD45) and the ability to proliferate or to differentiate into endothelial cells. 10 Separation between hematopoietic and the endothelial lineages during early mouse 11 and zebrafish 12 development is established during by asymmetrical division of primitive cells located in the dorsal aorta endothelium, through a novel differentiation event called endothelial-hematopoietic transition (EHT). [13][14][15][16] More uncertain is whether cells with a similar potency of generating HSCs or endothelial cells are present at postnatal stages 17 ; recently, however, derivation of endothelial cells from CD34 ϩ /CD38 ϩ /CD45 ϩ /CD133 ϩ CB progenitors was demonstrated, 18 suggesting the existence of similar progenitors at least during fetal life. Conclusion-TheseThe zebrafish (Danio rerio...