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

Coronary artery disease is the most common cause of cardiac failure in the Western world, and to date there is no alternative to bypass surgery for severe coronary atherosclerosis. We report that c-kitpositive cardiac progenitor cells (CPCs) activated with insulin-like growth factor 1 and hepatocyte growth factor before their injection in proximity of the site of occlusion of the left coronary artery in rats, engrafted within the host myocardium forming temporary niches. Subsequently, CPCs divided and differentiated into endothelial cells and smooth muscle cells and, to a lesser extent, into cardiomyocytes. The acquisition of vascular lineages appeared to be mediated by the up-regulation of hypoxia-inducible factor 1␣, which promoted the synthesis and secretion of stromal-derived factor 1 from hypoxic coronary vessels. Stromal-derived factor 1 was critical in the conversion of CPCs to the vascular fate. CPCs formed conductive and intermediate-sized coronary arteries together with resistance arterioles and capillaries. The new vessels were connected with the primary coronary circulation, and this increase in vascularization more than doubled myocardial blood flow in the infarcted myocardium. This beneficial effect, together with myocardial regeneration attenuated postinfarction dilated myopathy, reduced infarct size and improved function. In conclusion, locally delivered activated CPCs generate de novo coronary vasculature and may be implemented clinically for restoration of blood supply to the ischemic myocardium.coronary blood flow ͉ infarct size ͉ myocardial regeneration ͉ stem cells ͉ vasculogenesis

During bacterial infection, the bone marrow hematopoietic activity shifts toward granulocyte production, which is critical for host defenses. Along with this enhancement of granulopoiesis, the bone marrow also increases its release of hematopoi-

Alcohol abuse predisposes the host to bacterial infections. In response to bacterial infection, the bone marrow hematopoietic activity shifts toward granulocyte production, which is critical for enhancing host defense. This study investigated the hematopoietic precursor cell response to bacteremia and how alcohol affects this response. Acute alcohol intoxication was induced in BALB/c mice 30 min before initiation of Escherichia coli bacteremia. Bacteremia caused a significant increase in the number of bone marrow lineage (lin−)-c-kit+Sca-1+ cells. Marrow lin−c-kit+Sca-1+ cells isolated from bacteremic mice showed an increase in CFU-granulocyte/macrophage activity compared with controls. In addition to enhanced proliferation of lin−c-kit+Sca-1+ cells as reflected by BrdU incorporation, phenotypic inversion of lin−c-kit+Sca-1+Sca-1− cells primarily accounted for the rapid increase in marrow lin−c-kit+Sca-1+ cells following bacteremia. Bacteremia increased plasma concentration of TNF-α. Culture of marrow lin−c-kit+Sca-1+Sca-1− cells with murine rTNF-α for 24 h caused a dose-dependent increase in conversion of these cells to lin−c-kit+Sca-1+ cells. Sca-1 mRNA expression by the cultured cells was also up-regulated following TNF-α stimulation. Acute alcohol intoxication inhibited the increase in the number of lin−c-kit+Sca-1+ cells in the bone marrow after E. coli infection. Alcohol impeded the increase in BrdU incorporation into marrow lin−c-kit+Sca-1+ cells in response to bacteremia. Alcohol also suppressed the plasma TNF-α response to bacteremia and inhibited TNF-α-induced phenotypic inversion of lin−c-kit+Sca-1+Sca-1− cells in vitro. These data show that alcohol inhibits the hematopoietic precursor cell response to bacteremia, which may serve as one mechanism underlying the impaired host defense in alcohol abusers with severe bacterial infections.

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Enhancement of stem cell antigen-1 (Sca-1) expression by myeloid precursors promotes the granulopoietic response to bacterial infection. However, the underlying mechanisms remain unclear. Extracellular-regulated kinase (ERK) pathway activation strongly enhances proliferation of hematopoietic progenitor cells. Here, we investigated the role of Sca-1 in promoting ERK-dependent myeloid lineage proliferation and the effects of alcohol on this process. Thirty minutes after intraperitoneal injection of alcohol, mice received intravenous challenge with 5 × 107 E.coli for 8 or 24 h. A subset of mice received intravenous BrdU injection 20 h post challenge. Bacteremia increased Sca-1 expression, ERK activation, and proliferation of myeloid and granulopoietic precursors. Alcohol administration suppressed this response and impaired granulocyte production. Sca-1 expression positively correlated with ERK activation and cell cycling, but negatively correlated with myeloperoxidase content in granulopoietic precursors. Alcohol intoxication suppressed ERK activation in granulopoietic precursors and proliferation of these cells during bacteremia. Granulopoietic precursors in Sca-1−/− mice failed to activate ERK signaling and could not increase CFU-GM activity following bacteremia. These data indicate that Sca-1 expression promotes ERK-dependent myeloid cell proliferation during bacteremia. Suppression of this response could represent an underlying mechanism for developing myelosuppression in alcohol abusing hosts with severe bacterial infection.

Enhanced granulopoietic activity is crucial for host defense against bacterial pneumonia. Alcohol impairs this response. The underlying mechanisms remain obscure. Granulocyte colony-stimulating factor (G-CSF) produced by infected lung tissue plays a key role in stimulating bone marrow granulopoiesis. This study investigated the effects of alcohol on G-CSF signaling in the regulation of marrow myeloid progenitor cell proliferation in mice with Streptococcus pneumoniae pneumonia. Chronic alcohol consumption plus acute alcohol intoxication suppressed the increase in blood granulocyte counts following intrapulmonary challenge with S. pneumoniae. This suppression was associated with a significant decrease in bone marrow granulopoietic progenitor cell proliferation. Alcohol treatment significantly enhanced STAT3 phosphorylation in bone marrow cells of animals challenged with S. pneumoniae. In vitro experiments showed that G-CSF-induced activation of STAT3-p27Kip1 pathway in murine myeloid progenitor cell line 32D-G-CSFR cells was markedly enhanced by alcohol exposure. Alcohol dose-dependently inhibited G-CSF-stimulated 32D-G-CSFR cell proliferation. This impairment of myeloid progenitor cell proliferation was not attenuated by inhibition of alcohol metabolism through either the alcohol dehydrogenase pathway or the CYP450 system. These data suggest that alcohol enhances G-CSF-associated STAT3-p27Kip1 signaling, which impairs granulopoietic progenitor cell proliferation by inducing cell cycling arrest and facilitating their terminal differentiation during the granulopoietic response to pulmonary infection.

Background Alcohol abuse is associated with an increased incidence and severity of pneumonia. In both the general population and in individuals consuming excess alcohol, Streptococcus pneumoniae is the most frequent lung infection pathogen. Alcoholic patients with pneumonia frequently present with granulocytopenia, which is predictive of increased mortality. The mechanisms underlying this impaired granulopoietic response to pneumococcal pneumonia have yet to be elucidated. Methods Acute alcohol intoxication was induced in mice 30 minutes before intrapulmonary infection with Streptococcus pneumoniae. Bone marrow and blood samples were collected. Bone marrow cells were also isolated from naïve mice and treated in vitro with plasma from mice infected with S.pneumoniae. Results Alcohol intoxication impaired the pneumococcal-induced increase in granulocyte recruitment into the alveolar space, decreased bacterial clearance from the lung, and increased mortality. Pneumococcal pneumonia significantly increased bone marrow lineage−c-Kit+Sca-1+ (LKS) cell number and colony forming unit – granulocytes and monocyte (CFU-GM) activity of these cells. Both enhanced proliferation of LKS cells and re-expression of Sca-1 surface protein on downstream progenitor cells bearing lineage−c-Kit+Sca-1− surface markers accounted for the expansion of marrow LSK cells during pneumonia. Alcohol intoxication impaired these two mechanisms of LKS cell population expansion and was associated with a relative granulocytopenia during pneumococcal lung infection. Conclusions Alcohol inhibits the hematopoietic precursor cell response to pneumonia which may serve as a mechanism underlying the granulocytopenia and impaired host defense in alcohol abusers with bacterial pneumonia.