FIG. 1.

Characterization of CPCs after three passages in proliferation medium. (A) Experimental cell isolation schema. (B) Bright-field image of human CPCs in culture in proliferation condition. (C) RT-PCR of early cardiac markers (Gata4, Nkx2.5, Isl1, Mef2c), late cardiac markers (α-MHC, β-MHC, cardiac troponin I), Cx-43, and GAPDH as a loading control in the human fetal heart at 12 weeks of gestation (fetal heart) and in the isolated cells (CPCs in proliferation). (D) Quantitative real-time RT-PCR of the cardiac markers α-MHC, β-MHC, and cardiac troponin I of the isolated cells relative to the human fetal heart at 12 weeks of gestation. White bars: fetal heart; black bars: CPCs. (E) Staining of the cells. Upper rows represent costaining for Nkx2.5 (green) and Ki67 (red). Middle rows stained for Nkx2.5 (red) and KDR (green). Lower rows correspond to Nkx2.5 (red) and Isl1 (green); white arrowhead indicates double-stained cells. Nuclei were stained with DAPI (blue). Scale bars: 25 μm. The bars in D represent means±SD. Asterisks denote significant difference compared with expansion medium (**p<0.01; ***p<0.001). CPCs, cardiac precursor cells; RT-PCR, reverse transcriptase–polymerase chain reaction; MHC, myosin heavy chain; Cx, connexin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; SD, standard deviation; DAPI, 4′,6′-diamidino-2′-phenylindole.