title: Biphasic induction of Pdx1 in mouse and human embryonic stem cells can mimic development of pancreatic beta-cells.
abstract: Embryonic stem (ES) cells represent a possible source of islet tissue for the treatment of diabetes. Achieving this goal will require a detailed understanding of how the transcription factor cascade initiated by the homeodomain transcription factor Pdx1 culminates in pancreatic beta-cell development. Here we describe a genetic approach that enables fine control of Pdx1 transcriptional activity during endoderm differentiation of mouse and human ES cell. By activating an exogenous Pdx1VP16 protein in populations of cells enriched in definitive endoderm we show a distinct lineage-dependent requirement for this transcription factor's activity. Mimicking the natural biphasic pattern of Pdx1 expression was necessary to induce an endocrine pancreas-like cell phenotype, in which 30% of the cells were beta-cell-like. Cell markers consistent with the different beta-cell differentiation stages appeared in a sequential order following the natural pattern of pancreatic development. Furthermore, in mouse ES-derived cultures the differentiated beta-like cells secreted C-peptide (insulin) in response to KCl and 3-isobutyl-1-methylxanthine, suggesting that following a natural path of development in vitro represents the best approach to generate functional pancreatic cells. Together these results reveal for the first time a significant effect of the timed expression of Pdx1 on the non-beta-cells in the developing endocrine pancreas. Collectively, we show that this method of in vitro differentiation provides a template for inducing and studying ES cell differentiation into insulin-secreting cells.
authors.affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom.
authors.forename: Andreia S
authors.initials: AS
authors.lastname: Bernardo
authors.forename: Candy H-H
authors.initials: CH
authors.lastname: Cho
authors.forename: Sharon
authors.initials: S
authors.lastname: Mason
authors.forename: Hilary M
authors.initials: HM
authors.lastname: Docherty
authors.forename: Roger A
authors.initials: RA
authors.lastname: Pedersen
authors.forename: Ludovic
authors.initials: L
authors.lastname: Vallier
authors.forename: Kevin
authors.initials: K
authors.lastname: Docherty
mesh_terms: D028341:Activins / Q000494:pharmacology; D000682:Amyloid / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D055415:Bone Morphogenetic Protein 4 / Q000494:pharmacology; D002096:C-Peptide / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000235:genetics; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D058228:Islet Amyloid Polypeptide; D060850:LIM-Homeodomain Proteins; D051379:Mice; D016133:Polymerase Chain Reaction; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014157:Transcription Factors
chemical_list: D000682:Amyloid; C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D002096:C-Peptide; D018398:Homeodomain Proteins; D007328:Insulin; D058228:Islet Amyloid Polypeptide; D060850:LIM-Homeodomain Proteins; C491328:Nkx6-1 protein, mouse; D015534:Trans-Activators; D014157:Transcription Factors; C422405:activin A; C063843:insulin gene enhancer binding protein Isl-1; C084010:pancreatic and duodenal homeobox 1 protein; D013629:Tamoxifen; D028341:Activins; C016601:afimoxifene