Fig. 5

Wnt/β-catenin signaling activation contributes to the 3D PCL nanofibrous scaffold-induced cardiac-directed differentiation of iPSCs. a Schematic diagram of the time windows for DKK-1/CH treatment during the differentiation periods. The miPSCs cultured in TCPs or on 3D PCL nanofibrous scaffolds were treated with the canonical Wnt/β-catenin signaling modulators DDK-1 (100 ng/ml) or CH (10 μmol/l) for different periods: D0-3, D4-7 and D0-7. Untreated groups were used as controls. b–f Quantitative RT-PCR analysis of gene expression of non-treated miPSCs in the PCL and TCP groups at different time points: D0, D1, D3, and D7. b β-CATENIN and (c) AXIN2 mRNA expression in the TCP group. d β-CATENIN and (e) AXIN2 mRNA expression in the PCL group. The expression levels of each gene were normalized to the endogenous control GAPDH. **Significantly different versus D0, ^##significantly different between D3 and D1, **^## P < 0.01. n = 3 samples. f AXIN2 expression in cells differentiated in TCPs and on 3D PCL nanofibrous scaffolds with or without CH/DKK-1 treatment for 3 days. GAPDH was used as endogenous control. The fold changes relative to TCP Control D3 from triplicate experiments are presented. **Significantly different versus the TCP Control D3 group, ^##significantly different versus the PCL Control D3 group, **^## P < 0.01. At the end of the 15 days of differentiation, the cTnT protein levels were determined. Western blot analysis of cTnT expression levels in miPSC-derived cells (g) in TCPs treated with CH, h on 3D PCL nanofibrous scaffolds treated with DDK-1, and (i) on 3D PCL nanofibrous scaffolds treated with CH. **Significantly different between the DKK-1/CH treated groups versus the control groups, ^##significantly different compared with the D0-3 group, **^## P < 0.01. n = 5-7. All results are presented as the mean ± SE. DDK-1: Dickkopf-1, a selective Wnt/β-catenin signaling inhibitor; CH: CHIR99021, a selective Gsk3 inhibitor that activates Wnt/β-catenin signaling