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

Nature volume 459, pages 262–265 (2009)Cite this article

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.

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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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Authors and 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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  1. Toshiro Sato
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Corresponding author

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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