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

Abstract

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.

Elevated Serum SFRP5 Levels During Preeclampsia and Its Potential Association with Trophoblast Dysfunction via Wnt/β-Catenin Suppression

“…Canonical Wnt signalling, activated by the accumulation and nuclear translocation of βcatenin, promotes invasive trophoblast differentiation [44]. SFRP5 inactivates the Wnt/β-catenin signalling pathway by decreasing phosphorylated GSK3β and non-phosphorylated β-catenin (active β-catenin) and subsequent T-cell transcription factor 4 (TCF4)/lymphoid enhancer-binding factor 1 (LEF1)-mediated gene expression in cancer cells and pluripotent stem cells [56][57][58]. Since the invasive behaviour of trophoblasts in early placentation is similar to that of tumour cells, we hypothesised that SFPR5 might have a similar effect on their function [59,60].…”

Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing

“…Besides studies using scFvs raised against reporter proteins or oncogenes as used in the present study, one could envision a reporter cell system that allows a deeper understanding of cellular interactions in more complex cell aggregates or in developing embryos. For instance, during the generation of organoids from pluripotent stem cells, a variety of intrinsic and extrinsic signals steer the formation of complex multicellular structures [22][23][24]. In this scenario, the maturation of cells within a complex structure could be monitored if a membrane-bound epitope is expressed at a given developmental stage.…”

Activation of Wnt/β-catenin signalling via GSK3 inhibitors direct differentiation of human adipose stem cells into functional hepatocytes

“…Wnt signalling combined with fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signalling regulates foregut endoderm identity dependent on the graded activity of Wnt. A secreted frizzled-related protein 5, Wnt ligand and frizzled (Fzd) 7 interactions regulate differential thresholds of Wnt/β-catenin and Wnt/JNK signalling that coordinate endoderm fate, proliferation and morphogenesis69. Previously, we demonstrate that the high concentration (100 ng/mL) of activin A signalling, which mimics the Nodal pathway, induces definitive endoderm specific transcription factors, including HEX, GATA4, FOXA2, and SOX17, expression in hASCs10.…”