title: Characterization of polyhormonal insulin-producing cells derived in vitro from human embryonic stem cells.
abstract: Human embryonic stem cells (hESCs) were used as a model system of human pancreas development to study characteristics of the polyhormonal cells that arise during fetal pancreas development. HESCs were differentiated into fetal-like pancreatic cells in vitro using a 33-day, 7-stage protocol. Cultures were ~90-95% PDX1-positive by day (d) 11 and 70-75% NKX6.1-positive by d17. Polyhormonal cells were scattered at d17, but developed into islet-like clusters that expressed key transcription factors by d33. Human C-peptide and glucagon secretion were first detected at d17 and increased thereafter in parallel with INS and GCG transcript levels. HESC-derived cells were responsive to KCl and arginine, but not glucose in perifusion studies. Compared to adult human islets, hESC-derived cells expressed ~10-fold higher levels of glucose transporter 1 (GLUT1) mRNA, but similar levels of glucokinase (GCK). In situ hybridization confirmed the presence of GLUT1 transcript within endocrine cells. However, GLUT1 protein was excluded from this population and was instead observed predominantly in non-endocrine cells, whereas GCK was co-expressed in insulin-positive cells. In rubidium efflux assays, hESC-derived cells displayed mild potassium channel activity, but no responsiveness to glucose, metabolic inhibitors or glibenclamide. Western blotting experiments revealed that the higher molecular weight SUR1 band was absent in hESC-derived cells, suggesting a lack of functional KATP channels at the cell surface. In addition, KATP channel subunit transcript levels were not at a 1:1 ratio, as would be expected (SUR1 levels were ~5-fold lower than KIR6.2). Various ratios of SUR1:KIR6.2 plasmids were transfected into COSM6 cells and rubidium efflux was found to be particularly sensitive to a reduction in SUR1. These data suggest that an impaired ratio of SUR1:KIR6.2 may contribute to the observed KATP channel defects in hESC-derived islet endocrine cells, and along with lack of GLUT1, may explain the absence of glucose-stimulated insulin secretion.
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Bruin
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Suheda
authors.initials: S
authors.lastname: Erener
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Javier
authors.initials: J
authors.lastname: Vela
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Xiaoke
authors.initials: X
authors.lastname: Hu
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
authors.forename: James D
authors.initials: JD
authors.lastname: Johnson
authors.affiliation: Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Harley T
authors.initials: HT
authors.lastname: Kurata
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Francis C
authors.initials: FC
authors.lastname: Lynn
authors.affiliation: Department of Chemical & Biological Engineering, Michael Smith Laboratories, University of British Columbia, Vancouver, BC, Canada.
authors.forename: James M
authors.initials: JM
authors.lastname: Piret
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Ali
authors.initials: A
authors.lastname: Asadi
authors.affiliation: BetaLogics Venture, Janssen R & D LLC, Raritan, NJ, USA.
authors.forename: Alireza
authors.initials: A
authors.lastname: Rezania
authors.affiliation: Laboratory of Molecular and Cellular Medicine, Department of Cellular & Physiological Sciences, Life Sciences Institute, University of British Columbia, Vancouver, BC, Canada; Department of Surgery, University of British Columbia, Vancouver, BC, Canada. Electronic address: tim.kieffer@ubc.ca.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Kieffer
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002096:C-Peptide / Q000378:metabolism; D002454:Cell Differentiation; D053595:Embryonic Stem Cells / Q000166:cytology*; D055098:Endocrine Cells / Q000166:cytology / Q000378:metabolism; D005941:Glucokinase / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D005905:Glyburide / Q000494:pharmacology; D006728:Hormones / Q000378:metabolism; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D054086:KATP Channels / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000378:metabolism; D064233:Sulfonylurea Receptors / Q000235:genetics / Q000378:metabolism; D000099161:Kcnj11 Channel
chemical_list: D000255:Adenosine Triphosphate; D002096:C-Peptide; D005941:Glucokinase; D051272:Glucose Transporter Type 1; D005905:Glyburide; D006728:Hormones; D007004:Hypoglycemic Agents; D054086:KATP Channels; D024661:Potassium Channels, Inwardly Rectifying; D064233:Sulfonylurea Receptors; D000099161:Kcnj11 Channel; C577507:ABCC8 protein, human