title: Differentiation of Mouse Embryonic Stem Cells toward Functional Pancreatic β-Cell Surrogates through Epigenetic Regulation of Pdx1 by Nitric Oxide.
abstract: Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that regulates the embryonic development of the pancreas and the differentiation toward β cells. Previously, we have shown that exposure of mouse embryonic stem cells (mESCs) to high concentrations of diethylenetriamine nitric oxide adduct (DETA-NO) triggers differentiation events and promotes the expression of Pdx1. Here we report evidence that Pdx1 expression is associated with release of polycomb repressive complex 2 (PRC2) and P300 from its promoter region. These events are accompanied by epigenetic changes in bivalent markers of histones trimethylated histone H3 lysine 27 (H3K27me3) and H3K4me3, site-specific changes in DNA methylation, and no change in H3 acetylation. On the basis of these findings, we developed a protocol to differentiate mESCs toward insulin-producing cells consisting of sequential exposure to DETA-NO, valproic acid, and P300 inhibitor (C646) to enhance Pdx1 expression and a final maturation step of culture in suspension to form cell aggregates. This small molecule-based protocol succeeds in obtaining cells that express pancreatic β-cell markers such as PDX1, INS1, GCK, and GLUT2 and respond in vitro to high glucose and KCl.
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Center for Network Research in Diabetes and Metabolic Diseases (CIBERDEM) Instituto de Salud Carlos III, Madrid, Spain.|Cell Therapy Network, Madrid (RED-TERCEL), Instituto de Salud Carlos III, Madrid, Spain.|Fundación Progreso y Salud, Seville, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Salguero-Aranda
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Pablo de Olavide University, Seville, Spain.
authors.forename: Rafael
authors.initials: R
authors.lastname: Tapia-Limonchi
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Pablo de Olavide University, Seville, Spain.
authors.forename: Gladys Margot
authors.initials: GM
authors.lastname: Cahuana
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Center for Network Research in Diabetes and Metabolic Diseases (CIBERDEM) Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Ana Belen
authors.initials: AB
authors.lastname: Hitos
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Center for Network Research in Diabetes and Metabolic Diseases (CIBERDEM) Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: Irene
authors.initials: I
authors.lastname: Diaz
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Cell Therapy Network, Madrid (RED-TERCEL), Instituto de Salud Carlos III, Madrid, Spain.|Fundación Progreso y Salud, Seville, Spain.
authors.forename: Abdelkrim
authors.initials: A
authors.lastname: Hmadcha
authors.affiliation: Department of Epigenetics, Oncologic Institute of Principado of Asturias, Oviedo, Spain.
authors.forename: Mario
authors.initials: M
authors.lastname: Fraga
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Center for Network Research in Diabetes and Metabolic Diseases (CIBERDEM) Instituto de Salud Carlos III, Madrid, Spain.|Pablo de Olavide University, Seville, Spain.
authors.forename: Franz
authors.initials: F
authors.lastname: Martín
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Center for Network Research in Diabetes and Metabolic Diseases (CIBERDEM) Instituto de Salud Carlos III, Madrid, Spain.|Cell Therapy Network, Madrid (RED-TERCEL), Instituto de Salud Carlos III, Madrid, Spain.|Fundación Progreso y Salud, Seville, Spain.
authors.forename: Bernat
authors.initials: B
authors.lastname: Soria
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Center for Network Research in Diabetes and Metabolic Diseases (CIBERDEM) Instituto de Salud Carlos III, Madrid, Spain.|Cell Therapy Network, Madrid (RED-TERCEL), Instituto de Salud Carlos III, Madrid, Spain.|Pablo de Olavide University, Seville, Spain.
authors.forename: Juan Rigoberto
authors.initials: JR
authors.lastname: Tejedo
authors.affiliation: Andalusian Center for Molecular Biology and Regenerative Medicine, Seville, Spain.|Center for Network Research in Diabetes and Metabolic Diseases (CIBERDEM) Instituto de Salud Carlos III, Madrid, Spain.|Cell Therapy Network, Madrid (RED-TERCEL), Instituto de Salud Carlos III, Madrid, Spain.|Pablo de Olavide University, Seville, Spain.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Bedoya
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D019175:DNA Methylation / Q000187:drug effects; D050881:E1A-Associated p300 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D005941:Glucokinase / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D051273:Glucose Transporter Type 2 / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D063151:Polycomb Repressive Complex 2 / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014635:Valproic Acid / Q000494:pharmacology
chemical_list: D051273:Glucose Transporter Type 2; D006657:Histones; D018398:Homeodomain Proteins; D007328:Insulin; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D009569:Nitric Oxide; D014635:Valproic Acid; D063151:Polycomb Repressive Complex 2; D050881:E1A-Associated p300 Protein; C494361:Ep300 protein, mouse; D005941:Glucokinase; D005947:Glucose
keywords: Cell differentiation; Diabetes; Embryonic stem cells (ESCs); Insulin-producing cells; Nitric oxide (NO)