Abstract 3486: Distinct Roles of Wnt/β-catenin and TGF-β1/Smad2 Signaling in Adult Cardiac Stem Cell Expansion and Specification in vitro
Wnt/β-catenin and TGF-β/SMADs signaling pathways have distinct functions in mammalian cardiogenesis. We have tested through gain and loss of function experiments the effects of these two signaling pathways on adult rat c-kitpos cardiac stem cell (CSC) fate. To promote Wnt/β-catenin pathway, we have used soluble Wnt-3a, Wnt-3a conditioned medium or 6-bromoindirubin-3′-oxime (BIO, a specific inhibitor of GSK-3, stabilizing β-catenin). To disrupt Wnt/β-catenin, rat c-kitpos CSCs were treated with Dickkopf-1 (Dkk-1), an inhibitor of canonical Wnts or transfected with a specific short hairpin RNA for β-catenin (β-catshRNA). Wnt-3a, Wnt-3a-conditioned medium and BIO were able to foster c-kitpos CSC expansion, clonogenicity and cardiosphere formation vs. control treatment in vitro. In contrast, inhibiting canonical Wnt signaling with Dkk-1 or β-catshRNA resulted in a decrease of the three in vitro stemness properties. When c-kitpos CSCs were placed in differentiation medium, Wnt-3a, Wnt-3a-conditioned medium and BIO reduced the transcription of myogenic lineage markers and the number of myocyte differentiating cells. On the other hand, Dkk-1 increased CSC myocyte specification. In gain of function experiments to evaluate TGF-β/SMAD signaling, TGF-β1 was added to c-kitpos CSCs in vitro. In loss of function experiments, we disrupted TGF-β1-dependent SMAD signaling by Smad2shRNA. Neither TGF-β1 supplementation nor Smad2 knock-down affected c-kitpos CSC expansion, clonogenicity and cardiosphere formation vs. control treatment. When c-kitpos CSCs were placed in differentiation medium, TGF-β1 significantly induced the transcription of myogenic lineage markers and the number of myocyte differentiating cells. Accordingly, Smad2shRNA reduced CSC myocyte specification and completely prevented TGF-β1-dependent myogenic effects. Importantly, the combination of TGF-β1, Bmp2/4 and Dkk-1 induced full myocyte differentiation of CSCs with rhythmic beating. These data show that canonical Wnt pathway not only promotes but is required for CSC expansion while its antagonism drives CSC myogenic specification. On the other hand, TGF-β1/Smad2 pathway is dispensable for CSC expansion while its activation drives CSC myogenic specification.