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 ...

Rationale Age and coronary artery disease may negatively affect the function of human cardiac stem cells (hCSCs) and their potential therapeutic efficacy for autologous cell transplantation in the failing heart. Objective Insulin-like growth factor 1 (IGF-1) and 2 (IGF-2), and angiotensin II (Ang II) and their receptors, IGF-1R, IGF-2R and AT1R, were characterized in c-kit-positive-hCSCs to establish whether these systems would allow us to separate hCSC classes with different growth reserve in the aging and diseased myocardium. Methods and Results C-kit-positive-hCSCs were collected from myocardial samples obtained from 24 patients, 48 to 86 years of age, undergoing elective cardiac surgery for coronary artery disease. The expression of IGF-1R in hCSCs recognized a young cell phenotype defined by long telomeres, high telomerase activity, enhanced cell proliferation and attenuated apoptosis. In addition to IGF-1, IGF-1R-positive-hCSCs secreted IGF-2 that promoted myocyte differentiation. Conversely, the presence of IGF-2R and AT1R, in the absence of IGF-1R, identified senescent hCSCs with impaired growth reserve and increased susceptibility to apoptosis. The ability of IGF-1R-positive-hCSCs to regenerate infarcted myocardium was then compared with that of unselected c-kit-positive-hCSCs. IGF-1R-positive-hCSCs improved cardiomyogenesis and vasculogenesis. Pretreatment of IGF-1R-positive-hCSCs with IGF-2 resulted in the formation of more mature myocytes and superior recovery of ventricular structure. Conclusions hCSCs expressing only IGF-1R synthesize both IGF-1 and IGF-2, which are potent modulators of stem cell replication, commitment to the myocyte lineage and myocyte differentiation, pointing to this hCSC subset as the ideal candidate cell for the management of human heart failure.

Rationale: Physiological hypertrophy in the developing heart has been considered the product of an increase in volume of preexisting fetal cardiomyocytes in the absence of myocyte formation. Objective: In this study, we tested whether the mouse heart at birth has a pool of cardiac stem cells (CSCs) that differentiate into myocytes contributing to the postnatal expansion of the parenchymal cell compartment. Methods and Results: We have found that the newborn heart contains a population of c-kit-positive CSCs that are lineage negative, self-renewing, and multipotent. CSCs express the Notch1 receptor and show the nuclear localization of its active fragment, N1ICD. In 60% of cases, N1ICD was coupled with the presence of Nkx2.5, indicating that the commitment of CSCs to the myocyte lineage is regulated by Notch1. Importantly, overexpression of N1ICD in neonatal CSCs significantly expanded the proportion of transit-amplifying myocytes.To establish whether these in vitro findings had a functional counterpart in vivo, the Notch pathway was blocked in newborn mice by administration of a ␥-secretase inhibitor. This intervention resulted in the development of a dilated myopathy and high mortality rates. Ventricular decompensation was characterized by a 62% reduction in amplifying myocytes, which resulted in a 54% decrease in myocyte number. A t birth, the myocardium must accommodate the abrupt changes in the patterns of blood flow and circulatory resistance and the increasing demands of the rapidly growing organism. Physiological hypertrophy in the developing heart has been considered the result of an increase in volume of preexisting fetal cardiomyocytes, whereas the contribution of new myocytes to the postnatal expansion in ventricular mass was viewed as negligible at best. 1 However, the documentation of high levels of DNA synthesis in myocytes together with apoptotic cell death 2 indicate that the postnatal maturation of the heart is a complex process in which cardiomyogenesis may play a critical role in the acquisition of the adult heart phenotype.By viral tagging and clonal marking, we have shown that cardiomyogenesis in the adult mouse heart depends on the activation and differentiation of a pool of cardiac stem cells (CSCs) that express the c-kit antigen. 3 On the basis of these findings, the question can be raised whether primitive cells with similar properties are present in the neonatal heart and the generation of new myocytes is dictated by lineage commitment of these cells. According to the traditional model of growth in self-renewing organs, stem cells give rise to progenitorprecursor cells, which then become highly dividing amplifying cells that eventually reach terminal differentiation and growth arrest. 4 This hierarchically structured mechanism of parenchymal cell generation may account for the expansion in myocyte number and volume of the neonatal heart. CSC-derived amplifying myocytes replicate and concurrently differentiate, mimicking cellular hyperplasia and hypertrophy. 5 The possibility exists t...

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5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450 K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals at base resolution. Implementing this approach, termed “TAB-array”, we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular progenitors and neural precursor cells. With the ability to discriminate 5mC and 5hmC, we identified a large number of novel dynamically methylated genomic regions that are implicated in the development of these lineages. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease.

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The study of neurodegenerative diseases using pluripotent stem cells requires new methods to assess neurodevelopment and neurodegeneration of specific neuronal subtypes. The cholinergic system, characterized by its use of the neurotransmitter acetylcholine, is one of the first to degenerate in Alzheimer’s disease and is also affected in frontotemporal dementia. We developed a differentiation protocol to generate basal forebrain-like cholinergic neurons (BFCNs) from induced pluripotent stem cells (iPSCs) aided by the use of small molecule inhibitors and growth factors. Ten iPSC lines were successfully differentiated into BFCNs using this protocol. The neuronal cultures were characterised through RNA and protein expression, and functional analysis of neurons was confirmed by whole-cell patch clamp. We have developed a reliable protocol using only small molecule inhibitors and growth factors, while avoiding transfection or cell sorting methods, to achieve a BFCN culture that expresses the characteristic markers of cholinergic neurons.