Figure 1.
EPIC generation and characterization.
A–C. Primary culture of E11.5 embryonic epicardium. A. Whole heart culture. B. Detail showing the outgrowth of epicardial cells from the explanted hearts. C. Epicardial cell halo growing on gelatin-coated coverslips. D,E. Epicardial cells normally express cytokeratin, a marker for epicardial cells. F-F′. The majority of EPICs display a mesenchymal phenotype (F, confluent culture; F′, subconfluent culture) and express Sox9, a known marker for epicardial mesenchymal cells. However, EPICs do not express Tcf21 (G). A few, small epithelial-like cell clones (H, dotted line) are found dispersed in the culture. Cells in these clones express the epithelial markers ZO-1 (I) and cadherins (J). K. EPIC growth dynamics. The graph shows the parameters defining EPIC cell growth in culture (lag time; population doubling time; plateau level; and saturation density). Scale bars: A,C,D = 100 µm; B,E,F,G = 50 µm; H = ; I,J = 20 µm.
Figure 2.
Differentiation potential along the proepicardium-epicardium transition.
Proepicardia cultured in vitro express differentiation markers for striated heart muscle (MF20, A, B), endothelial progenitors/cells (E, F), smooth muscle cells (I, J) and fibroblasts (M, N). E11.5 epicardial cells do not express myocardial (C, D) or endothelial markers (G, H), but continue to express smooth muscle (α-SMA, K, L) and fibroblastic ones (FSP-1, O, P). Scale bars: A,C,E,G,I,K,M = 100 µm; B,D,F,H,J,L,N,O = 50 µm; P = 25 µm.
Figure 3.
EPIC differentiation marker expression.
A–C. EPIC express α-SMA (red) and SM22 (green). D–F Treatment with TGFβ1,2 does not altere the number of cells expressing these two markers, but affects the phenotype of the cells which spread and elongate in culture. EPICs also express fibroblast protein markers like FSP-1 (G,H) and Collagen I (I; I′ shows the negative, non-inmune control for collagen I immunohistochemistry). J. sqPCR profiling. EPIC (left column), E9.5 proepicardium (middle columns) and E11.5 epicardium (right column). Scale bars: A,B,C,D,E,F,H,I′ = 65 µm; G = 100 µm; I = 10 µm.
Figure 4.
EPIC cell surface marker expression (FACS).
EPIC expression of cell surface markers was evaluated by flow cytometry. Additional FACS analyses on ephrin and Eph receptors can be found in Fig. S4.
Figure 5.
MMPs, ADAMs & TIMPs expression.
A. EPIC spheroids cultured on regular fibrin gels (treated or un-treated with soluble bFGF, Wnt3a or Wnt5a) or on transglutaminase-bound BMP2 or VEGF fibrin gels for 48 hours. Matrix degradation is indicated by an halo around the cell spheroids. B. qPCR study of MMP, ADAM and TIMP expression levels. (p<0.05). Scale bars: 100 µm.
Figure 6.
Evaluation of EPIC clones (cEP) proteolytic activity and sprouting.
A. Representative images are shown for the culture of EPIC clones (cEP1–8) in 3D fibrin gels. B. The phenotype of the clones is illustrated in the left table. Note that some cell spheroids (asterisks) preferentially degrade the fibrin (‘proteolytic’ clones), generating characteristic halo around the cells (arrowheads). Others (‘sprouting’ clones) attach to the fibrin and spread over it forming multicellular sprouts (arrowheads). The fibrin gel digested area was graphically represented for each clone (middle) and plotted against the respective sprouting area of each clone (µm2, right). C. qPCR analyses of MMP, ADAM and TIMP expression in three significant cEP (cEP4 for maximal proteolysis and cEP6,7 for maximal sprouting). (p<0.05). Scale bars: 100 µm.