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Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche

Nature volume 459, pages 262265 (14 May 2009) | Download Citation

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Abstract

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5+ stem cells at the bottoms of small-intestinal crypts1. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5+ stem cells can also initiate these crypt–villus organoids. Tracing experiments indicate that the Lgr5+ stem-cell hierarchy is maintained in organoids. We conclude that intestinal crypt–villus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.

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References

  1. 1.

    et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007)

  2. 2.

    & Intestinal epithelial stem cells and progenitors. Methods Enzymol. 419, 337–383 (2006)

  3. 3.

    , & The intestinal stem cell. Genes Dev. 22, 1856–1864 (2008)

  4. 4.

    , , , & The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J. Cell Sci. 101, 219–231 (1992)

  5. 5.

    , , & Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. Gastroenterology 117, 858–865 (1999)

  6. 6.

    Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture. J. Cell Sci. 103, 511–519 (1992)

  7. 7.

    & Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures. Exp. Cell Res. 224, 354–364 (1996)

  8. 8.

    et al. Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nature Genet. 19, 379–383 (1998)

  9. 9.

    , , & Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 17, 1709–1713 (2003)

  10. 10.

    et al. Essential requirement for Wnt signaling in proliferation of adult small intestine and colon revealed by adenoviral expression of Dickkopf-1. Proc. Natl Acad. Sci. USA 101, 266–271 (2004)

  11. 11.

    et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005)

  12. 12.

    & Peptide growth factors in the intestine. Eur. J. Gastroenterol. Hepatol. 13, 763–770 (2001)

  13. 13.

    et al. De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine. Science 303, 1684–1686 (2004)

  14. 14.

    et al. Cell–cell contacts prevent anoikis in primary human colonic epithelial cells. Gastroenterology 132, 587–600 (2007)

  15. 15.

    , , , & Expression and distribution of laminin α1 and α2 chains in embryonic and adult mouse tissues: an immunochemical approach. Exp. Cell Res. 275, 185–199 (2002)

  16. 16.

    , , & Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res. Treat. 67, 93–109 (2001)

  17. 17.

    & Crypt fission and crypt number in the small and large bowel of postnatal rats. Cell Tissue Kinet. 18, 255–262 (1985)

  18. 18.

    et al. β-Catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111, 251–263 (2002)

  19. 19.

    et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001)

  20. 20.

    Generalized lacZ expression with the ROSA26 Cre reporter strain. Nature Genet. 21, 70–71 (1999)

  21. 21.

    et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006)

  22. 22.

    et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnol. 25, 681–686 (2007)

  23. 23.

    et al. Notch/γ-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435, 959–963 (2005)

  24. 24.

    et al. The human homolog of rat Jagged1 expressed by marrow stroma inhibits differentiation of 32D cells through interaction with Notch1. Immunity 8, 43–55 (1998)

  25. 25.

    & Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. I. Columnar cell. Am. J. Anat. 141, 461–479 (1974)

  26. 26.

    et al. Myofibroblasts. II. Intestinal subepithelial myofibroblasts. Am. J. Physiol. 277, C183–C201 (1999)

  27. 27.

    & The gastrointestinal tract stem cell niche. Stem Cell Rev. 2, 203–212 (2006)

  28. 28.

    et al. Intestinal epithelial–mesenchymal cell interactions. Ann. NY Acad. Sci. 859, 1–17 (1998)

  29. 29.

    , & Stem cells find their niche. Nature 414, 98–104 (2001)

  30. 30.

    & Stem cell niche: structure and function. Annu. Rev. Cell Dev. Biol. 21, 605–631 (2005)

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Acknowledgements

We thank M. van den Born, J. Korving, H. Begthel and S. van den Brink for technical assistance, and N. Ong and M. van den Bergh Weerman for technical assistance with electron microscopy.

Author information

Affiliations

  1. Hubrecht Institute and University Medical Center Utrecht, Uppsalalaan 8, 3584CT Utrecht, The Netherlands

    • Toshiro Sato
    • , Robert G. Vries
    • , Hugo J. Snippert
    • , Marc van de Wetering
    • , Nick Barker
    • , Daniel E. Stange
    • , Johan H. van Es
    •  & Hans Clevers
  2. Nuvelo, Inc., 201 Industrial Road, Suite 310, San Carlos, California 94070-6211, USA

    • Arie Abo
  3. The Netherlands Cancer Institute, Antoni van Leeuwenhoek Hospital, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

    • Pekka Kujala
    •  & Peter J. Peters

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Competing interests

H.C. is an inventor on several patents involving the culture system in this paper, as is T.S.

Corresponding author

Correspondence to Hans Clevers.

Supplementary information

PDF files

  1. 1.

    Supplementary Figures

    This file contains Supplementary Figures 1-9 with Legends.

  2. 2.

    Supplementary Information

    This file contains Supplementary Table 1 and Legends for Supplementary Table 1 and Movies 1-2.

Videos

  1. 1.

    Supplementary Movie 1

    This movie shoes differential interference contrast microscopy movie of the first three days of culture of a single crypt.

  2. 2.

    Supplementary Movie 2

    This Movie shows a 7-day-old organoid derived from an Lgr5-EGFP-ires CreERT2/Rosa26-YFP crypt (see file s2 for full Legend).

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DOI

https://doi.org/10.1038/nature07935

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