title: 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.
authors: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.
authors: Jeannine
authors: J
authors: Hoepfner
authors: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.
authors: Mandy
authors: M
authors: Kleinsorge
authors: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.
authors: Oliver
authors: O
authors: Papp
authors: iPSC Based Gene Therapy, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.
authors: Mania
authors: M
authors: Ackermann
authors: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.
authors: Susanne
authors: S
authors: Alfken
authors: Institute of Technical Chemistry, Leibniz University Hannover, Hannover, Germany.
authors: Ursula
authors: U
authors: Rinas
authors: Institute of Organic Chemistry, Leibniz University Hannover, Hannover, Germany.
authors: Wladimir
authors: W
authors: Solodenko
authors: Institute of Organic Chemistry, Leibniz University Hannover, Hannover, Germany.
authors: Andreas
authors: A
authors: Kirschning
authors: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.
authors: Malte
authors: M
authors: Sgodda
authors: Translational Hepatology and Stem Cell Biology, REBIRTH Cluster of Excellence, Hannover Medical School, Hannover, Germany.|Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, Hannover, Germany.|Cell and Developmental Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany.
authors: Tobias
authors: T
authors: Cantz
mesh_terms: D028341:Activins / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D053595:Embryonic Stem Cells / Q000166:cytology; D004707:Endoderm / Q000166:cytology* / Q000254:growth & development; D005260:Female; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D011247:Pregnancy; D055760:SOXF Transcription Factors / Q000235:genetics; D051153:Wnt Proteins / Q000378:metabolism; D060449:Wnt Signaling Pathway / Q000187:drug effects / Q000502:physiology*; D000509:alpha-Fetoproteins / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism; D000099274:Frizzled-Related Proteins
chemical_list: D028341:Activins; D006023:Glycoproteins; D047908:Intracellular Signaling Peptides and Proteins; D055760:SOXF Transcription Factors; D051153:Wnt Proteins; D000509:alpha-Fetoproteins; D051176:beta Catenin; D000099274:Frizzled-Related Proteins
keywords: cell differentiation; liver/hepatocytes; stem cells