Biphasic modulation of Wnt signaling supports efficient foregut endoderm formation from human pluripotent stem cells.

Pluripotent stem cells (embryonic stem cells and induced pluripotent stem cells) are of great promise in regenerative medicine, including molecular studies of disease mechanisms, if the affected cell type can be authentically generated during in vitro differentiation. Most existing protocols aim to mimic embryonic development steps by the supplementation of specific cytokines and small molecules, but the involved signaling pathways need further exploration. In this study, we investigated enhanced initial activation of Wnt signaling for definitive endoderm formation and subsequent rapid shutdown of Wnt signaling for proper foregut endoderm specification using 3 μM CHIR99021 and 0.5 μg/mL of secreted frizzled-related protein 5 (sFRP-5) for biphasic modulation of the Wnt pathway. The definitive endoderm and foregut endoderm differentiation capabilities of Wnt pathway-modulated cells were determined based on the expression levels of the endodermal transcription factors SOX17 and FOXA2 and those of the transcription activator GATA4 and the α-fetoprotein (AFP) gene, respectively. Furthermore, the resulting biphasic Wnt pathway modulation was investigated at the protein level by analyzing phosphorylation of glycogen synthase kinase 3 beta (GSK3β) and β-catenin. Finally, Wnt target gene expression was determined using an improved lentiviral reporter construct that enabled robust T-cell transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1)-mediated luciferase expression in differentiating pluripotent stem cells. In conclusion, we demonstrated robust, homogeneous, and efficient derivation of foregut endodermal cells by inducing a biphasic modulation of the Wnt signaling pathway.