Figure 1. Cardiac progenitor cell differentiation is concomitant with a downregulation of Wnt signaling and upregulation of Wnt inhibitors and is potentiated by inhibition of Wnt/β-catenin.

Cultured cardiac progenitor cells (CPCs) were incubated or not (control cells [Ctl]) with 5-azacytidine (AZA) and TGF-β (DIFF) for 5, 8, 11, or 14 days. Gene expression (relative to respective time Ctl) was analyzed using RT-qPCR and normalized to GAPDH. (A) Relative expression of Wnt/β-catenin pathway target genes (Axin2, Wnt4) and Wnt activators (Lef1 and Snai2). Bounds of boxes represent minimum and maximum values, inner line represent means. *P < 0.05 vs. CTL; n = 3 different preparations; Kruskal-Wallis with Bonferroni correction. Expression of β-catenin protein levels in DIFF, relative to Ctl at each time point. *P < 0.05 vs. Ctl; n ≥ 3 different preparations; Mann-Whitney test. (B) CPCs were cultured in Ctl or DIFF medium for 8 days and their supernatant was incubated with HEK cells expressing the TOPflash reporter construct, indicative of Wnt morphogen production by and Wnt activity in donor CPCs. TOPflash signal was normalized for transfection efficiency (cotransfected TKRenilla). *P < 0.05 vs. Ctl; n = 4 different cultures; Mann-Whitney. (C) Relative (to respective time Ctl) expression of Wnt repressors Wif1 and Hbp1. *P < 0.05 vs. Ctl; n = 3 different preparations; Kruskal-Wallis with Bonferroni correction. (D) Representative images of CPCs treated or not with AZA and either TGF-β or the pharmacologic inhibitor of Wnt signaling response (IWR1, 10 μM) for 26 days. Immunocytochemistry was performed using an antibody against cardiac troponin T. Relative gene expression of Nkx2.5 in CPCs treated with IWR1 in absence of AZA for 11 days. *P < 0.05, n ≥ 4 experiments; Mann-Whitney test. Scale bar: 20 μm. (E) Wif1 expression modulates CPC differentiation in coculture. Expression of Wif1 in CPCs transfected with 50 nM siRNA targeting Wif1 (or siRNA scramble) for 48 hours. CPCs transfected with siRNA-Wif1 (si-Wif1) or scramble control (si-Scr) were cocultured with cardiomyocytes and their differentiation monitored by expression of α-sr-actinin. Results are reported as relative to differentiation in si-Scr–transfected CPCs (set at 100%). CPC differentiation is significantly decreased upon Wif1 inhibition. *P < 0.05; n = 4 different preparations; Mann-Whitney test.