title: Adult Murine Pancreatic Progenitors Require Epidermal Growth Factor and Nicotinamide for Self-Renewal and Differentiation in a Serum- and Conditioned Medium-Free Culture.
abstract: Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and tri-lineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture components, such as serum and conditioned medium, has prevented a complete understanding of the signals required for progenitor cell growth. Here, we have established a serum-free, conditioned medium-free colony assay with the inclusion of seven defined factors: epidermal growth factor (EGF), R-Spondin 1 (RSPO1), Noggin, nicotinamide, exendin-4, activin B, and vascular endothelial growth factor (VEGF)-A. The requirements for colony growth were characterized and we found that EGF and nicotinamide were necessary and sufficient for the colony growth and long-term self-renewal of these progenitors. However, the seven factor (7F) culture medium better induced colony size and self-renewal in long-term culture than EGF plus nicotinamide alone. Individual 3-week-old colonies grown in the 7F culture medium expressed ductal, acinar, and endocrine lineage markers, suggesting that tri-lineage differentiation of the tri-potent progenitors was occurring without genetic manipulation. A delayed inhibition of Notch signaling using small molecules in 2-week-old cultures enhanced endocrine gene expression in 3-week-old colonies. This better-defined colony assay system will enable our and other laboratories for in-depth mechanistic studies on the biology of these progenitor cells.
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Lena
authors.initials: L
authors.lastname: Wedeken
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Angela
authors.initials: A
authors.lastname: Luo
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.|2 Irell & Manella Graduate School of Biological Sciences , Duarte, California.
authors.forename: Jacob R
authors.initials: JR
authors.lastname: Tremblay
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Rawson
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Liang
authors.initials: L
authors.lastname: Jin
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Dan
authors.initials: D
authors.lastname: Gao
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.
authors.forename: Janine
authors.initials: J
authors.lastname: Quijano
authors.affiliation: 1 Department of Translational Research and Cellular Therapeutics, Diabetes and Metabolism Research Institute , Beckman Research Institute of City of Hope, Duarte, California.|2 Irell & Manella Graduate School of Biological Sciences , Duarte, California.
authors.forename: Hsun Teresa
authors.initials: HT
authors.lastname: Ku
mesh_terms: D061354:Acinar Cells / Q000166:cytology / Q000378:metabolism; D028341:Activins / Q000494:pharmacology; D053687:Adult Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D000077270:Exenatide; D016326:Extracellular Matrix Proteins / Q000494:pharmacology; D005260:Female; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism; D008297:Male; D051379:Mice; D009536:Niacinamide / Q000494:pharmacology*; D010179:Pancreas / Q000166:cytology*; D010455:Peptides / Q000494:pharmacology; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D014688:Venoms / Q000494:pharmacology
chemical_list: D017077:Culture Media, Conditioned; D016326:Extracellular Matrix Proteins; D010455:Peptides; D051880:Receptors, Notch; D042461:Vascular Endothelial Growth Factor A; D014688:Venoms; D028341:Activins; D009536:Niacinamide; D004815:Epidermal Growth Factor; D000077270:Exenatide
keywords: Notch signaling pathway; differentiation; epidermal growth factor; nicotinamide; pancreatic colony-forming units; self-renewal