Decreased cardiac differentiation of miPSC-derived EBs formed after blocking of collagen/β
integrin interaction. (A) Schematic protocol of cardiac differentiation from miPSCs. Vc, CIS or β1 integrin blocking antibody was administered prior to EB formation. (B) Time course quantification of spontaneous beating activity of differentiated cardiomyocytes was expressed as the percentage of beating EBs. Error bars indicate SEM. (C) Immunostaining for cardiac troponin-T (cTnT) of miPSC-derived cardiomyocytes on day 21. Nuclei were counterstained with DAPI (blue). Scale bars = 20 μm. (D, E) Gene expression profiles of cardiac-related transcription factors (GATA-4, Mef2c, Nkx2.5) and cardiac-specific structural genes (α-MHC, MLC2a, MLC2v) were analyzed by semiquantitative RT-PCR (D) and real-time PCR (E). Treatment with β1 integrin blocking antibody decreased the expression of these factors and genes. Expression levels of each gene were normalized to β-actin or GAPDH. Mean fold change relative to control and SD from triplicate experiments are shown. *p < 0.05 relative to control.