Epithelial-mesenchymal transition of epicardial cells requires specific signals from microenvironment and constitutes the source of cardiac primitive cells in the adult human heart

During heart morphogenesis a subset of epicardial cells undergoes an epithelial-mesenchymal transition and invades myocardium contributing to the formation of myofibroblasts, vascular endothelium and smooth muscle cells. Recently, the presence of resident stem cells able to give rise to the cells of cardiac lineages was observed in the adult human heart, raising questions concerning their origin and biology. The scope of the present study was to investigate whether the epithelial-mesenchymal transition takes place in the adult human heart, generating the population of cardiac stem cells, and which molecular factors are involved in its initiation and progression. Since the subepicardial space is rich in extracellular matrix components and cytokines, constituting the specific environment for epicardially derived cells, cardiac fibroblasts obtained from the subepicardium layer of adult human atrium were cultured to create the most appropriate conditions for the in vitro study. After the non-enzymatic removal of cells, the presence of extracellular matrix proteins - fibronectin, collagen III and IV, tenascin and laminin - was confirmed by immunofluorescence. This substrate was used to obtain and culture the sheets of epithelial cells from the epicardium of normal adult human atria. The epithelial phenotype of the cells was confirmed by the positive immunolabeling of E-cadherin and β-catenin at the intercellular junctions and cytokeratin in the cytoplasm. While in the presence of basic fibroblast growth factor (10 ng/ml) or platelet-derived growth factor-BB (40 ng/ml) the epithelial sheets remained intact, the addition of epidermal growth factor (40 ng/ml), hepatocyte growth factor (40 ng/ml) or transforming growth factor beta (0,5 ng/ml) resulted in the change of cell characteristics. The intercellular contacts were lost and the cells acquired spindle-like shape and vimentin expression. When induced to differentiate into endothelial, smooth muscle cells or cardiomyocytes, these cells expressed factor VIII, smooth muscle actin or myosin heavy chain α/β, respectively. The results of our study indicate that in the specific conditions epithelial-mesenchymal transition takes place in the adult human epicardial cells. We suggest that epicardially derived cells enrich the pool of cardiac primitive cells and contribute to the regenerative properties of the heart. A better understanding of complex interactions of the extracellular matrix proteins and growth factors with their specific receptors on the cell surface will be pivotal for progressing therapeutic cardiac regeneration.