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

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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Figure 1: Establishment of intestinal crypt culture system.
Figure 2: Single Lgr5 + cells generate crypt-villus structures.
Figure 3: Colony-forming efficiency of single cells sorted in individual wells.
Figure 4: Composition of single stem cell-derived organoids.

References

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

    ADS  CAS  Article  Google Scholar 

  2. Bjerknes, M. & Cheng, H. Intestinal epithelial stem cells and progenitors. Methods Enzymol. 419, 337–383 (2006)

    CAS  Article  Google Scholar 

  3. Barker, N., van de Wetering, M. & Clevers, H. The intestinal stem cell. Genes Dev. 22, 1856–1864 (2008)

    CAS  Article  Google Scholar 

  4. Evans, G. S., Flint, N., Somers, A. S., Eyden, B. & Potten, C. S. The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J. Cell Sci. 101, 219–231 (1992)

    PubMed  Google Scholar 

  5. Whitehead, R. H., Demmler, K., Rockman, S. P. & Watson, N. K. Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. Gastroenterology 117, 858–865 (1999)

    CAS  Article  Google Scholar 

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

    PubMed  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  9. Pinto, D., Gregorieff, A., Begthel, H. & Clevers, H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium. Genes Dev. 17, 1709–1713 (2003)

    CAS  Article  Google Scholar 

  10. Kuhnert, F. 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)

    ADS  CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  15. Sasaki, T., Giltay, R., Talts, U., Timpl, R. & Talts, J. F. 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)

    CAS  Article  Google Scholar 

  16. Stingl, J., Eaves, C. J., Zandieh, I. & Emerman, J. T. Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res. Treat. 67, 93–109 (2001)

    CAS  Article  Google Scholar 

  17. St Clair, W. H. & Osborne, J. W. Crypt fission and crypt number in the small and large bowel of postnatal rats. Cell Tissue Kinet. 18, 255–262 (1985)

    CAS  PubMed  Google Scholar 

  18. Batlle, E. 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)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  24. Li, L. 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)

    CAS  Article  Google Scholar 

  25. Cheng, H. & Leblond, C. P. 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)

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  27. Yen, T. H. & Wright, N. A. The gastrointestinal tract stem cell niche. Stem Cell Rev. 2, 203–212 (2006)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  29. Spradling, A., Drummond-Barbosa, D. & Kai, T. Stem cells find their niche. Nature 414, 98–104 (2001)

    ADS  CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

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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.

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Correspondence to Hans Clevers.

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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.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1-9 with Legends. (PDF 1698 kb)

Supplementary Information

This file contains Supplementary Table 1 and Legends for Supplementary Table 1 and Movies 1-2. (PDF 367 kb)

Supplementary Movie 1

This movie shoes differential interference contrast microscopy movie of the first three days of culture of a single crypt. (MOV 4383 kb)

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). (MOV 3445 kb)

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Sato, T., Vries, R., Snippert, H. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009). https://doi.org/10.1038/nature07935

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