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IDE1 enhances the definitive endoderm (DE) differentiation of human-induced pluripotent stem cells (hiPSCs) with Activin A/Wnt3a being significantly more potent in both 2D and 3D cultures compared to IDE1. IDE1 could efficiently induces DE differentiation through various protocols in vitro. Treatment of the hiPSCs-derived EBs with IDE-1 shows minor increase (p<0.01) of DE-markers cells compared to Activin A/Wnt3a treatment. IDE1 possess several advantages over other inducing factors including high permeability, influence, diversity, low cost, and easy to use and for the first time, Melton’s team showed that Activin A can be substituted by two cell-permeable small molecules, IDE1 and IDE2. IDE1 could induce phosphorylation of Smad2 after incubation for 24 h or more at levels comparable to those induced by Activin A treatment. Treatment of hiPSCs with IDE1 (2 mM) also leads to endodermal differentiation but with a significantly lower efficiency than Activin A/Wnt3a.