title: Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny.
abstract: Although Lgr5(+) intestinal stem cells have been expanded in vitro as organoids, homogeneous culture of these cells has not been possible thus far. Here we show that two small molecules, CHIR99021 and valproic acid, synergistically maintain self-renewal of mouse Lgr5(+) intestinal stem cells, resulting in nearly homogeneous cultures. The colony-forming efficiency of cells from these cultures is ~100-fold greater than that of cells cultured in the absence of CHIR99021 and valproic acid, and multilineage differentiation ability is preserved. We made use of these homogeneous cultures to identify conditions employing simultaneous modulation of Wnt and Notch signaling to direct lineage differentiation into mature enterocytes, goblet cells and Paneth cells. Expansion in these culture conditions may be feasible for Lgr5(+) cells from the mouse stomach and colon and from the human small intestine. These methods provide new tools for the study and application of multiple intestinal epithelial cell types.
authors.affiliation: 1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2] Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Cambridge, Massachusetts, USA. [3] Harvard Medical School, Boston, Massachusetts, USA. [4] Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA. [5] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.
authors.forename: Xiaolei
authors.initials: X
authors.lastname: Yin
authors.affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht, Utrecht, The Netherlands.
authors.forename: Henner F
authors.initials: HF
authors.lastname: Farin
authors.affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht, Utrecht, The Netherlands.
authors.forename: Johan H
authors.initials: JH
authors.lastname: van Es
authors.affiliation: Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht, Utrecht, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
authors.affiliation: 1] David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA. [2] Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA. [3] Department of Chemical Engineering, MIT, Cambridge, Massachusetts, USA.
authors.forename: Robert
authors.initials: R
authors.lastname: Langer
authors.affiliation: 1] Center for Biomedical Engineering, Department of Medicine, Brigham and Women's Hospital, Cambridge, Massachusetts, USA. [2] Harvard Medical School, Boston, Massachusetts, USA. [3] Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA. [4] Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Karp
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D002875:Chromosomes / Q000648:ultrastructure; D003106:Colon / Q000166:cytology*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006579:Heterozygote; D007421:Intestine, Small / Q000166:cytology*; D007621:Karyotyping; D051379:Mice; D018613:Microscopy, Confocal; D019879:Paneth Cells / Q000166:cytology; D011725:Pyridines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology*; D013270:Stomach / Q000166:cytology; D014635:Valproic Acid / Q000737:chemistry
chemical_list: C473711:Chir 99021; C493597:Lgr5 protein, mouse; D011725:Pyridines; D011743:Pyrimidines; D043562:Receptors, G-Protein-Coupled; D049452:Green Fluorescent Proteins; D014635:Valproic Acid