title: Sonic hedgehog and other soluble factors from differentiating embryoid bodies inhibit pancreas development.
abstract: Success of cell-replacement therapy for diabetes will largely depend on the establishment of alternative sources of pancreatic islet grafts. Embryonic stem (ES) cell differentiation toward pancreatic insulin-producing cells offers such perspectives, but there are still many challenges to overcome. Our previous studies suggested that the limited amount of insulin-positive cells derived from ES cells is related to the activation of pancreas inhibitory signals. To confirm this hypothesis, we report here that exposure of mouse embryonic pancreas explants to soluble factors from embryoid bodies (EBs) inhibits growth, morphogenesis, and endocrine and exocrine differentiation as evaluated by explant size and mRNA and protein expression. Sonic Hedgehog (Shh), an established pancreas repressor both at early and late developmental stages, was produced and secreted by EBs, and participated in the inhibitory effect by inducing its target Gli1 in the explants. Inhibition of Hedgehog pathway rescued the differentiation of Insulin-positive cells in the explants. In contrast to pancreatic cells, hepatic progenitors exposed to EB-conditioned medium showed improved differentiation of albumin-positive cells. In a model system of ES cell differentiation in vitro, we found that definitive endoderm induction by serum removal or activin A treatment further increased Hedgehog production and activity in EBs. Concomitantly, downregulation of the pancreas marker Pdx1 was recorded in activin-treated EBs, a phenomenon that was prevented by antagonizing Hedgehog signaling with Hedgehog interacting protein. These data strongly suggest that Hedgehog production in EBs limits pancreatic fate acquisition and forms a major obstacle in the specification of pancreatic cells from ES-derived definitive endoderm. Disclosure of potential conflicts of interest is found at the end of this article.
authors.affiliation: Cell Differentiation Unit, Diabetes Research Centre, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
authors.forename: Josué K
authors.initials: JK
authors.lastname: Mfopou
authors.forename: Véronique
authors.initials: V
authors.lastname: De Groote
authors.forename: Xiabo
authors.initials: X
authors.lastname: Xu
authors.forename: Harry
authors.initials: H
authors.lastname: Heimberg
authors.forename: Luc
authors.initials: L
authors.lastname: Bouwens
mesh_terms: D028341:Activins / Q000494:pharmacology; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D017077:Culture Media, Conditioned; D004622:Embryo, Mammalian / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects; D004702:Endocrine Glands / Q000187:drug effects / Q000378:metabolism; D004707:Endoderm / Q000187:drug effects / Q000378:metabolism; D005088:Exocrine Glands / Q000187:drug effects / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D010179:Pancreas / Q000187:drug effects / Q000196:embryology*; D015398:Signal Transduction / Q000187:drug effects; D012995:Solubility / Q000187:drug effects; D015534:Trans-Activators / Q000378:metabolism
chemical_list: D017077:Culture Media, Conditioned; D053823:Hedgehog Proteins; D018398:Homeodomain Proteins; C465570:Shh protein, mouse; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D028341:Activins