Endogenous Epicardial Regenerative Response Figure 6. Expression of stem cell markers. The different stem cell markers, CD34 (A–C), cKit (D–H) and PDGFRa (I–L) are shown in green, cTnI in red, and the nuclei in blue. In the healthy heart CD34-positive cells are found to be scattered through the epicardium covering the atria (A). After the induction of the ischemia CD34 is up-regulated and becomes highly expressed in the epicardium covering the infarct and its border zone (B,C), as well as in mesenchymal cells in the subepicardial space in the border zone (B). cKit is expressed in the epicardium and individual adjacent mesenchymal cells of the atria in control hearts (D). cKit becomes evident in scattered cells of the epicardium overlaying the border zone of the infarct at 3 days post-MI (E). At one week post-MI cKit is expressed throughout the epicardium overlaying the infarct and its border zone, as well as in a subset of immediately adjacent mesenchymal cells (G, H). Moreover, at this time point cKit is also expressed in the epicardium and a subset of adjacent subepicardial mesenchymal cells in the atrioventricular sulcus (F). In control heart PDGFRa is expressed in a small portion of the mesenchyme of the atrioventricular sulcus (I). At three days post-MI (J) and one week post-MI (L) PDGFRa is expressed in the epicardium overlaying the infarction. In the border zone PDGFRa is also expressed in mesenchymal cells within the subepicardial space (L). The dotted line highlights the border between infarcted tissue and healthy myocardium. The double arrow highlights the width of the subepicardial space. Abbreviations: LA: left atrium, LV: left ventricle, Epi: epicardium, AVS: atrioventricular sulcus. The bar is 100 mm. doi:10.1371/journal.pone.0044692.g006 The results obtained by using in situ hybridization, immuno- fluorescence and classic histological analyses are summarized in a schematics (Figure 7). At one day post-MI most of the epicardium overlaying the ischemic region is disrupted. The epicardial layer immediately adjacent to the ischemic zone, i.e. the border zone, starts to express embryonic epicardial genes, starts to proliferate, and the space between the epicardium and myocardium widens. Within the epicardium of the border zone the expression of Wt1 mRNA and protein, as well as Cre is induced. At three days post- MI the ischemic myocardium is again covered with an epicardial layer that is separated from the underlying myocardium by a wide gap. This epicardium is expressing Wt1, Cre and bGal. Taken together these observations suggest that the new epicardial layer overlaying the infarct is formed from the epicardium overlaying the border zone. In line with this interpretation the epicardial cells overlaying the border zone express genes that are also expressed during formation of epicardium during development. The initially a-cellular gap between the newly formed epicar- dium and infarct becomes populated by mesenchymal cells. Tracing the Wt1-lineage showed that these mesenchymal cells are bGal-positive. This observation indicates that the mesenchymal cells are descendants of cells that express or have expressed Wt1. Therefore, these mesenchymal cells are derived from the overlaying epicardium or have invaded from a remote location. The following observation suggests that the mesenchymal cells are derived from the overlaying epicardium. The BrdU labeling experiments showed incorporation of this proliferation marker in cells of the epicardium overlaying the infarct and in adjacent mesenchymal cells. Though one could conclude that both cell populations are proliferating, one could also regard the incorpo- rated label as a short term lineage tracer, because the mice were exposed to BrdU 4 hours prior to sacrifice. As such the finding of BrdU labeling in both the epicardium and subepicardial mesen- chyme mesenchymal cells suggests that the mesenchymal cells are derived from the overlaying epicardium by a process of EMT. This conclusion is underscored by the observed expression of the EMT markers Snai1 and aSMA. Nevertheless, we cannot exclude that descendants of Wt1-expressing cells have invaded the infarct PLOS ONE | www.plosone.org 11 September 2012 | Volume 7 | Issue 9 | e44692