title: Induced Quiescence of Lgr5+ Stem Cells in Intestinal Organoids Enables Differentiation of Hormone-Producing Enteroendocrine Cells.
abstract: Lgr5+ adult intestinal stem cells are highly proliferative throughout life. Single Lgr5+ stem cells can be cultured into three-dimensional organoids containing all intestinal epithelial cell types at near-normal ratios. Conditions to generate the main cell types (enterocyte, goblet cells, Paneth cells, and M cells) are well established, but signals to induce the spectrum of hormone-producing enteroendocrine cells (EECs) have remained elusive. Here, we induce Lgr5+ stem cell quiescence in vitro by blocking epidermal growth factor receptor (EGFR) or mitogen-associated protein kinase (MAPK) signaling pathways in organoids and show that their quiescent state is readily reverted. Quiescent Lgr5+ stem cells acquire a distinct molecular signature biased toward EEC differentiation. Indeed, combined inhibition of Wnt, Notch, and MAPK pathways efficiently generates a diversity of EEC hormone-expressing subtypes in vitro. Our observations uncouple Wnt-dependent stem cell maintenance from EGF-dependent proliferation and provide an approach for the study of the elusive EECs in a defined environment.
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT, Utrecht the Netherlands; Cancer Genomics Netherlands, UMC Utrecht, 3584 GC, Utrecht, the Netherlands.
authors.forename: Onur
authors.initials: O
authors.lastname: Basak
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT, Utrecht the Netherlands; Cancer Genomics Netherlands, UMC Utrecht, 3584 GC, Utrecht, the Netherlands.
authors.forename: Joep
authors.initials: J
authors.lastname: Beumer
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT, Utrecht the Netherlands; Cancer Genomics Netherlands, UMC Utrecht, 3584 GC, Utrecht, the Netherlands.
authors.forename: Kay
authors.initials: K
authors.lastname: Wiebrands
authors.affiliation: Department of Gastroenterology and Hepatology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Seno
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT, Utrecht the Netherlands; Cancer Genomics Netherlands, UMC Utrecht, 3584 GC, Utrecht, the Netherlands.
authors.forename: Alexander
authors.initials: A
authors.lastname: van Oudenaarden
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584 CT, Utrecht the Netherlands; Cancer Genomics Netherlands, UMC Utrecht, 3584 GC, Utrecht, the Netherlands; Princess Máxima Centre, 3584 CT, Utrecht, the Netherlands. Electronic address: h.clevers@hubrecht.eu.
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D019858:Enteroendocrine Cells / Q000166:cytology*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D020869:Gene Expression Profiling; D006728:Hormones / Q000096:biosynthesis*; D007422:Intestines / Q000166:cytology*; D020935:MAP Kinase Signaling System; D051379:Mice; D009940:Organoids / Q000166:cytology*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D051880:Receptors, Notch / Q000378:metabolism; D017423:Sequence Analysis, RNA; D015398:Signal Transduction; D059010:Single-Cell Analysis; D013234:Stem Cells / Q000166:cytology*; D051153:Wnt Proteins / Q000378:metabolism
chemical_list: D006728:Hormones; C493597:Lgr5 protein, mouse; D043562:Receptors, G-Protein-Coupled; D051880:Receptors, Notch; D051153:Wnt Proteins; D066246:ErbB Receptors
keywords: EGFR signaling; Lgr5; enteroendocrine cell; intestinal stem cells; organoids; quiescence