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Nat Methods. 2014 Jan;11(1):106-12. doi: 10.1038/nmeth.2737. Epub 2013 Dec 1.

Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny.

Author information

1
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.
2
Hubrecht Institute for Developmental Biology and Stem Cell Research and University Medical Centre Utrecht, Utrecht, The Netherlands.
3
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.
4
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.

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.

PMID:
24292484
PMCID:
PMC3951815
DOI:
10.1038/nmeth.2737
[Indexed for MEDLINE]
Free PMC Article
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