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

Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme that plays roles in response to DNA damage, apoptosis, and genetic stability. Recent evidence has implicated PARP in transcription of eukaryotic genes. However, the existing paradigm tying PARP function to the presence of DNA strand breaks does not provide a mechanism by which it may be recruited to gene-regulating domains in the absence of DNA damage. Here we report that PARP can bind to the DNA secondary structures (hairpins) in heteroduplex DNA in a DNA end-independent fashion and that automodification of PARP in the presence of NAD ؉ inhibited its hairpin binding activity. Atomic force microscopic images show that in vitro PARP protein has a preference for the promoter region of the PARP gene in superhelical DNA where the dyad symmetry elements likely form hairpins according to DNase probing. Using a chromatin cross-linking and immunoprecipitation assay we show that PARP protein binds to the chromosomal PARP promoter in vivo. Reporter gene assays have revealed that the transcriptional activity of the PARP promoter is 4 -5-fold greater in PARP knockout cells than in wild type fibroblasts. Reintroduction of vectors expressing full-length PARP protein or its truncated mutant (DNA-binding domain retained but lacking catalytic activity) into PARP ؊/؊ cells has conferred transcriptional down-regulation of the PARP gene promoter. These data provide support for PARP protein as a potent regulator of transcription including down-regulation of its own promoter.Poly(ADP-ribose) polymerase (PARP, 1 EC 2.4.2.30) is a chromatin-associated enzyme that catalyzes the transfer of successive units of the ADP-ribose moiety from NAD ϩ to itself and other nuclear acceptor proteins (1). PARP is a zinc fingercontaining protein, which allows enzyme binding to either double or single strand DNA breaks without any apparent sequence preference (2, 3). The catalytic activity of PARP is strictly dependent on the presence of strand breaks in DNA and is modulated by the level of automodification (4,5). Data from many studies show that PARP is involved in numerous biological functions, all of which are associated with breaking and rejoining DNA strands, and it plays a pivotal role in DNA damage repair (2, 6 -8).Recent studies have implicated PARP in transcription of eukaryotic genes (9 -16). PARP-dependent gene regulation involves poly(ADP-ribosyl)ation of transcription factors, which, in turn, prevents their binding to specific promoter sequences (10). The basal transcription factors TFIIF and TEF-1 as well as transcription factors TATA box-binding protein, YY1, SP-1, cAMP-response element-binding protein, p53, and NFB are all highly specific substrates for poly(ADP-ribosyl)ation (10,11,14,16). PARP may also interact directly with gene promoters. For instance, recombinant full-length PARP bound the DNA sequences within the MCAT1 regulatory element (11) and to the DF4 protein binding site of the Pax-6 gene neuroretinaspecific enhancer (17). Furthermore, PARP involvement in the active...

Abstract-Recent studies suggest that rather than being terminally differentiated, the adult heart is a self-renewing organ with the capacity to generate new myocytes from cardiac stem/progenitor cells (CS/PCs). This study examined the hypotheses that new myocytes are generated during adolescent growth, to increase myocyte number, and these newly formed myocytes are initially small, mononucleated, proliferation competent, and have immature properties. Ventricular myocytes (VMs) and cKit ϩ (stem cell receptor) CS/PCs were isolated from 11-and 22-week feline hearts. Bromodeoxyuridine incorporation (in vivo) and p16INK4a immunostaining were measured to assess myocyte cell cycle activity and senescence, respectively. Telomerase activity, contractions, Ca 2ϩ transients, and electrophysiology were compared in small mononucleated (SMMs) and large binucleated (LBMs) myocytes. Heart mass increased by 101% during adolescent growth, but left ventricular myocyte volume only increased by 77%. Most VMs were binucleated (87% versus 12% mononucleated) and larger than mononucleated myocytes. A greater percentage of SMMs was bromodeoxyuridine positive (SMMs versus LBMs: 3.1% versus 0.8%; PϽ0.05), and p16INK4a negative and small myocytes had greater telomerase activity than large myocytes. Contractions and Ca 2ϩ transients were prolonged in SMMs versus LBMs and Ca 2ϩ release was disorganized in SMMs with reduced transient outward current and T-tubule density. The T-type Ca 2ϩ current, usually seen in fetal/neonatal VMs, was found exclusively in SMMs and in myocytes derived from CS/PC. Myocyte number increases during adolescent cardiac growth. These new myocytes are initially small and functionally immature, with patterns of ion channel expression normally found in the fetal/neonatal period (Circ Res. 2007;100:536-544.)