Gastroenterology

Gastroenterology

Volume 141, Issue 5, November 2011, Pages 1762-1772
Gastroenterology

Original Research
Basic and Translational—Alimentary Tract
Long-term Expansion of Epithelial Organoids From Human Colon, Adenoma, Adenocarcinoma, and Barrett's Epithelium

https://doi.org/10.1053/j.gastro.2011.07.050Get rights and content

Background & Aims

We previously established long-term culture conditions under which single crypts or stem cells derived from mouse small intestine expand over long periods. The expanding crypts undergo multiple crypt fission events, simultaneously generating villus-like epithelial domains that contain all differentiated types of cells. We have adapted the culture conditions to grow similar epithelial organoids from mouse colon and human small intestine and colon.

Methods

Based on the mouse small intestinal culture system, we optimized the mouse and human colon culture systems.

Results

Addition of Wnt3A to the combination of growth factors applied to mouse colon crypts allowed them to expand indefinitely. Addition of nicotinamide, along with a small molecule inhibitor of Alk and an inhibitor of p38, were required for long-term culture of human small intestine and colon tissues. The culture system also allowed growth of mouse Apc-deficient adenomas, human colorectal cancer cells, and human metaplastic epithelia from regions of Barrett's esophagus.

Conclusions

We developed a technology that can be used to study infected, inflammatory, or neoplastic tissues from the human gastrointestinal tract. These tools might have applications in regenerative biology through ex vivo expansion of the intestinal epithelia. Studies of these cultures indicate that there is no inherent restriction in the replicative potential of adult stem cells (or a Hayflick limit) ex vivo.

Section snippets

Reagents

Reagents used in the culture experiments are shown in Supplementary Table 1.

Mice

Lgr5-EGFP-ires-creERT2 mice,11 APCfl/fl mice,25 Axin2-lacZ mice,26 and C57B/6 wild-type mice (6–12 weeks old) were used for experiments. Lgr5-EGFP-ires-creERT2 mice were crossed with APCfl/fl mice. Cre enzyme activity was induced by intraperitoneal injections of tamoxifen (2 mg/mouse). Murine small intestines and colons were opened longitudinally, cut in small pieces, and washed with cold phosphate-buffered saline

Establishment of a Mouse Colon Culture System

In an attempt to establish a mouse colon culture system, we explored our small intestinal culture condition (here termed ENR). In our experience, initial growth of colon epithelium is often observed under the ENR culture condition but is invariably abortive. Organoid formation was studied using epithelium isolated from the distal part of the mouse colon. The plating efficiency of single distal colonic crypts was much lower than that of small intestine (1%–3% vs >90%), and these organoids could

Discussion

The protocols developed here allow robust and long-term culture of primary human epithelial cells isolated from small intestine, colon, adeno(carcino)mas, and Barrett's esophagus. In contrast to murine small intestine, murine colonic epithelial cells require Wnt ligand in the culture medium. We have previously reported that CD24hi Paneth cells produce Wnt-3/11, which are essential for stem cell maintenance in the small intestine.29 Wnt-6 and -9b messenger RNA are expresses at the bottom of

Acknowledgments

The authors thank M. van den Born, J. Korving, and H. Begthel for technical assistance.

Dr Sato's current affiliation is: Department of Gastroenterology, School of Medicine, Keio University, 35 Shinanomachi, Shinnjukuku, Tokyo, 160-8582, Japan.

References (41)

  • J.M. Denu

    Vitamin B3 and sirtuin function

    Trends Biochem Sci

    (2005)
  • L. Hayflick

    The cell biology of aging

    J Invest Dermatol

    (1979)
  • G.S. Evans et al.

    The development of a method for the preparation of rat intestinal epithelial cell primary cultures

    J Cell Sci

    (1992)
  • H. Fukamachi

    Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture

    J Cell Sci

    (1992)
  • A. Ootani et al.

    Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche

    Nat Med

    (2009)
  • T. Sato et al.

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

    Nature

    (2009)
  • V. Korinek et al.

    Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4

    Nat Genet

    (1998)
  • D. Pinto et al.

    Canonical Wnt signals are essential for homeostasis of the intestinal epithelium

    Genes Dev

    (2003)
  • F. Kuhnert 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 U S A

    (2004)
  • K.A. Kim et al.

    Mitogenic influence of human R-spondin1 on the intestinal epithelium

    Science

    (2005)
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    Conflicts of interest The authors disclose the following: T.S. and H.C. are inventors on several patents involving the culture system. The remaining authors disclose no conflicts.

    Funding T.S. is supported by the EU ERC/232814, S.V. is supported by the Dutch Cancer Foundation (KWF/PF-Hubr2007-3956), D.E.S. and R.V. are supported by the CBG, J. van Es is supported by TiParma/T3-106, and M.F. is supported by the FWO (Funds for Scientific Research, Belgium).

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