title: The generation of definitive endoderm from human embryonic stem cells is initially independent from activin A but requires canonical Wnt-signaling.
abstract: The activation of the TGF-beta pathway by activin A directs ES cells into the definitive endoderm germ layer. However, there is evidence that activin A/TGF-beta is not solely responsible for differentiation into definitive endoderm. GSK3beta inhibition has recently been shown to generate definitive endoderm-like cells from human ES cells via activation of the canonical Wnt-pathway. The GSK3beta inhibitor CHIR-99021 has been reported to generate mesoderm from human iPS cells. Thus, the specific role of the GSK3beta inhibitor CHIR-99021 was analyzed during the differentiation of human ES cells and compared against a classic endoderm differentiation protocol. At high concentrations of CHIR-99021, the cells were directed towards mesodermal cell fates, while low concentrations permitted mesodermal and endodermal differentiation. Finally, the analyses revealed that GSK3beta inhibition rapidly directed human ES cells into a primitive streak-like cell type independently from the TGF-beta pathway with mesoderm and endoderm differentiation potential. Addition of low activin A concentrations effectively differentiated these primitive streak-like cells into definitive endoderm. Thus, the in vitro differentiation of human ES cells into definitive endoderm is initially independent from the activin A/TGF-beta pathway but requires high canonical Wnt-signaling activity.
authors.affiliation: Institute of Clinical Biochemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Lower Saxony, Germany, naujok.ortwin@mh-hannover.de.
authors.forename: Ortwin
authors.initials: O
authors.lastname: Naujok
authors.forename: Ulf
authors.initials: U
authors.lastname: Diekmann
authors.forename: Sigurd
authors.initials: S
authors.lastname: Lenzen
mesh_terms: D028341:Activins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004707:Endoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors; D006801:Humans; D007124:Immunoenzyme Techniques; D008648:Mesoderm / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D010179:Pancreas / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C473711:Chir 99021; D011725:Pyridines; D011743:Pyrimidines; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D051153:Wnt Proteins; C422405:activin A; D028341:Activins; D038362:Glycogen Synthase Kinase 3