Figure 3.

Clonogenicity and differentiation potential of clonal CD45^negc-kit^pos CSCs. (a) Sub-cloning efficiency of a rat c-kit^pos CSC clone (Cl) at every tenth passage. Data are mean±S.D., n=6. (b) Serial sub-clones of c-kit^pos CSCs, generated at P50 of the respective parental clone, maintained similar expression of multipotent and cardiac transcription factor genes. Colour scale indicates change in Ct (threshold cycle) relative to the normalised GAPDH control. Higher delta Ct values correspond to lower relative gene expression, with every Ct decrease of 3.3 representing a twofold increase in relative expression. (c) Serial spherogenesis efficiency of a clone of c-kit^pos eCSCs (1, primary; 1.1, secondary; 1.1.1, tertiary CSs). Representative image of tertiary CSs are shown in the left panel, bar=50μm. (d) CSs generated from clonal c-kit^pos (green) CSCs plated in basal differentiation media spontaneously commit to the CM (cTnI, red), smooth muscle (SMA, red) and endothelial (vWF, red) cell lineages. Nuclei are stained in blue by DAPI. Bar=50 μm. (e) CFU formation of cloned versus freshly isolated (at passage 1, P1) c-kit^pos CSCs plated at different numbers in 1.2 ml of MethoCult medium; *P<0.05 versus cloned c-kit^pos CSCs. Data are mean±S.D., n=6. (f) Representative light microscopy images of a CFU from freshly isolated (P1) c-kit^pos CSCs (left panel) and cloned c-kit^pos CSCs (right panel), bar=50 μm. (g and h) Clonal-derived c-kit^pos CSCs show greater clonogenic (g) and CS formation (h) potential, when compared to freshly isolated (P1) c-kit^pos CSCs. *P<0.05 versus freshly isolated P1. Data are mean±S.D., n=6. (i) Clonal-derived c-kit^pos CSCs show greater differentiation potential into CMs (number of cTnI-positive cells), SMCs (number of SMA-positive cells) and ECs (number of vWF-positive cells), when compared to freshly isolated c-kit^pos CSCs. *P<0.05 versus freshly isolated P1. Data are mean±S.D., n=6