Self-renewal and differentiation of cardiovascular progenitor cells

Herz-Kreislauf-Erkrankungen sind weltweit die häufigste Todesursache. Das adulte Herz wurde lange Zeit für ein post-mitotisches Organ ohne regenerativer Kapazität gehalten. Innerhalb der letzten Jahre wurde das Vorhandensein von Herzvorläuferzellen von verschiedenen Arbeitsgruppen gezeigt. Eine in-vivo Aktivierung und Differenzierung dieser Zellen würde neue Therapiemöglichkeiten für Herzinfarkt-Patienten eröffnen. In dieser Arbeit berichten wir über die Isolierung einer stabilen, euploiden, murinen Herz-Vorläufer-Zelllinie (cardiovascular progenitor cell (CVPC) line). Wir konnten diese Zellen über 60 Passagen kultivieren ohne einen Verlust der Selbsterneuerungskapazität oder einer Minderung der Potenz festzustellen. CVPCs exprimieren die Stammzellmarker Oct3/4, Nanog und Sox2 gleichzeitig mit den Herzzellmarker Nkx2.5, Mef2c und GATA4. Letzeres steht im Gegensatz zu murinen embryonalen Stammzellen (mESCs). CVPCs differenzieren ausschließlich zu Zellen des Herzens, nämlich Kardiomyozyten, glatter Muskelzellen und Endothelzellen. Sowohl die Expression von Herzellmarkern im undifferenzierten Zustand als auch die Limitierung des Differenzierungspotentials sind Hinweise darauf, dass CVPCs bereits auf die Differenzierung zu Zellen des Herzens eingeschränkt sind. Die Differenzierung von CVPCs wird durch den Entzug von Leukemia Inhibitory Factor (LIF) eingeleitet und ist unabhängig von anderen induzierenden Faktoren. LIF ist nötig und hinreichend um die Selbsterneuerung von CVPCs zu erhalten und hat einen negativen Effekt auf die Differenzierung von CVPCs. Eine zeitlich begrenzte Inhibierung der LIF-Signalgebung unterstützt die Differenzierung von CVPCs und Bone Morphogentic Protein 2 erhöht die Differenzierung zu Kardiomyocyten im Cardiac Body System signifikant. Weitere Untersuchungen von CVPCs werden zu einem besseren Verständnis der Selbsterneuerungskapazität des murinen Herzens führen.Cardiovascular Diseases are the leading cause of death globally and account for almost one out of two deaths in Austria. Recently, the existence of cardiovascular progenitor cells in the adult heart was demonstrated by various groups. The activation and differentiation of these cells in vivo would provide a new therapy option for MI patients. Here we report the isolation of a stable, euploid murine cardiovascular progenitor cell (CVPC) line. We successfully cultured these cells for over 60 passages without a loss of self-renewal capacity or a decrease in potency. CVPCs express stemness factors Oct3/4, Nanog, and Sox2 simultaneously with cardiac markers Nkx2.5, Mef2c, and GATA4, the latter stands in contrast to murine embryonic stem cells (mESCs). The expression of cardiac markers in the undifferentiated state of CVPCs goes hand in hand with the potency-limitations of these cells compared to mESCs. CVPCs are restricted to differentiate to cell types of the cardiac lineage, namely cardiomyocytes, smooth muscle cells, and endothelium. The expression of cardiac markers in the undifferentiated state and the limitation of the differentiation potential indicate that CVPCs are already committed to give rise to cells of the cardiac lineage. The differentiation of CVPCs is induced upon Leukemia Inhibitory Factor (LIF) deprivation and is not dependent on other inductive signals. Furthermore, LIF is necessary and sufficient to maintain self-renewal of CVPCs and has a negative effect on differentiation. Inhibition of LIF signaling aids differentiation of CVPCs and Bone Morphogenetic Protein2 significantly enhances differentiation to cardiomyocytes in the Cardiac Body system

Supplementary Material for: Embryonic Stem Cells Facilitate the Isolation of Persistent Clonal Cardiovascular Progenitor Cell Lines and Leukemia Inhibitor Factor Maintains Their Self-Renewal and Myocardial Differentiation Potential in vitro

Compelling evidence for the existence of somatic stem cells in the heart of different mammalian species has been provided by numerous groups; however, so far it has not been possible to maintain these cells as self-renewing and phenotypically stable clonal cell lines in vitro. Thus, we sought to identify a surrogate stem cell niche for the isolation and persistent maintenance of stable clonal cardiovascular progenitor cell lines, enabling us to study the mechanism of self-renewal and differentiation in these cells. Using postnatal murine hearts with a selectable marker as the stem cell source and embryonic stem cells and leukemia inhibitory factor (LIF)-secreting fibroblasts as a surrogate niche, we succeeded in the isolation of stable clonal cardiovascular progenitor cell lines. These cell lines self-renew in an LIF-dependent manner. They express both stemness transcription factors Oct4, Sox2, and Nanog and early myocardial transcription factors Nkx2.5, GATA4, and Isl-1 at the same time. Upon LIF deprivation, they exclusively differentiate to functional cardiomyocytes and endothelial and smooth muscle cells, suggesting that these cells are mesodermal intermediates already committed to the cardiogenic lineage. Cardiovascular progenitor cell lines can be maintained for at least 149 passages over 7 years without phenotypic changes, in the presence of LIF-secreting fibroblasts. Isolation of wild-type cardiovascular progenitor cell lines from adolescent and old mice has finally demonstrated the general feasibility of this strategy for the isolation of phenotypically stable somatic stem cell lines