Background Cardiac stem cells (CSCs) delivered to the infarcted heart generate a large number of small fetal-neonatal cardiomyocytes which fail to acquire the differentiated phenotype. However, the interaction of CSCs with post-mitotic myocytes results in the formation of cells with adult characteristics. Methods and Results Based on in vitro and in vivo assays, we report that the commitment of human CSCs (hCSCs) to the myocyte lineage and the generation of mature working cardiomyocytes are influenced by microRNA-499 (miR-499) which is barely detectable in hCSCs, but is highly expressed in post-mitotic human cardiomyocytes. miR-499 traverses gap junction channels and translocates to structurally coupled hCSCs favoring their differentiation into functionally-competent cells. Expression of miR-499 in hCSCs represses the miR-499 target genes Sox6 and Rod1, enhancing cardiomyogenesis in vitro and after infarction in vivo. Although cardiac repair was detected in all cell-treated infarcted hearts, the aggregate volume of the regenerated myocyte mass and myocyte cell volume were greater in animals injected with hCSCs overexpressing miR-499. Treatment with hCSCs resulted in an improvement in ventricular function, consisting of a better preservation of developed pressure, and positive and negative dP/dt after infarction. An additional positive effect on cardiac performance occurred with miR-499, pointing to enhanced myocyte differentiation/hypertrophy as the mechanism by which miR-499 potentiated the restoration of myocardial mass and function in the infarcted heart. Conclusions The recognition that miR-499 promotes the differentiation of hCSCs into mechanically integrated cardiomyocytes has important clinical implications for the treatment of human heart failure.

An analysis of the clonality of cardiac progenitor cells (CPCs) and myocyte turnover in vivo requires genetic tagging of the undifferentiated cells so that the clonal marker of individual mother cells is traced in the specialized progeny. CPC niches in the atria and apex of the mouse heart were infected with a lentivirus carrying EGFP, and the destiny of the tagged cells was determined 1-5 months later. A common integration site was identified in isolated CPCs, cardiomyocytes, endothelial cells (ECs), and fibroblasts, documenting CPC self-renewal and multipotentiality and the clonal origin of the differentiated cell populations. Subsequently, the degree of EGFP-lentiviral infection of CPCs was evaluated 2-4 days after injection, and the number of myocytes expressing the reporter gene was measured 6 months later. A BrdU pulse-chasing protocol was also introduced as an additional assay for the analysis of myocyte turnover. Over a period of 6 months, each EGFP-positive CPC divided approximately eight times generating 230 cardiomyocytes; this value was consistent with the number of newly formed cells labeled by BrdU. To determine whether, human CPCs (hCPCs) are self-renewing and multipotent, these cells were transduced with the EGFP-lentivirus and injected after acute myocardial infarction in immunosuppressed rats. hCPCs, myocytes, ECs, and fibroblasts collected from the regenerated myocardium showed common viral integration sites in the human genome. Thus, our results indicate that the adult heart contains a pool of resident stem cells that regulate cardiac homeostasis and repair.F ate mapping protocols establish a lineage relationship between ancestors carrying the reporter gene and their descendents (1, 2), but do not provide information on the self-renewing property and clonogenicity of progenitor cells or clonal origin of daughter cells in vivo (3). Because of these limitations, viral gene-tagging remains the most accurate strategy for the analysis of stem cell growth (3-8). The semi-random insertion of retroviral and lentiviral vectors represents an effective tool for genetic marking, enabling the identification of the progeny generated by stem cell differentiation. Retroviruses and lentiviruses integrate permanently in the genome of the host cells; the insertion site of the viral genome is inherited by the population derived from the parental cell (6) and can be amplified by PCR. Thus, the detection of the sites of integration constitutes a unique approach for the documentation of self-renewal, clonogenicity, and multipotentiality of stem cells in vivo. So far, this methodology has been applied to the bone marrow (4-6) and the brain (3, 7, 8) and has not been used to characterize the mechanisms regulating cardiac homeostasis and pathology.The implementation of this technique in the adult heart is relevant for the incontrovertible demonstration of resident cardiac stem cells and the ability of the myocardium to undergo spontaneous regeneration. Moreover, the notion that cardiomyocytes have a long lifespan and ...