Figure 2. Endothelin-1 permits long-term clonal expansion of ISL1⁺ CVPs.

(a) Growth profile of ISL1⁺ CVPs cultured over five passages in basal medium supplemented with 100 ng ml⁻¹ WNT3A, 100 ng ml⁻¹ EDN1 or both. The cells were passaged once they became confluent and continued to be cultured in their respective culture conditions. n=6 experiments. The P values between the experimental groups are as shown, evaluated by Student's t-test. (b,c) Immunoblot showing activation of Akt pathway in ISL1⁺ CVPs when cultured in the presence of EDN1. Refer to Supplementary Fig. 8 for full version of blots. Cell lysates were probed with antibodies against phospho-AKT, total AKT and β-actin (loading control). Densitometry of band intensities was performed using ImageJ software. **P<0.001, evaluated by Student's t-test. (d) Relative gene expression of pluripotent markers (OCT4, SOX2 and NANOG) and cardiac mesoderm markers (KDR, T-BRACHYURY, MESP1, ISL1, NKX2.5, GATA4, MEF2C and TBX5) were assessed after ISL1⁺ CVP was cultured in defined medium supplemented with EDN1 and WNT3A for over 20 passages. n=3 experiments. *P<0.05 and **P=0.01, evaluated by Student's t-test. w/o, without.