Abstract
It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell–enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell–enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–266 (2009).
Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010).
Miyoshi, H., Ajima, R., Luo, C.T., Yamaguchi, T.P. & Stappenbeck, T.S. Wnt5a potentiates TGF-β signaling to promote colonic crypt regeneration after tissue injury. Science 338, 108–113 (2012).
Nusse, R. et al. Wnt signaling and stem cell control. Cold Spring Harb. Symp. Quant. Biol. 73, 59–66 (2008).
Haegebarth, A. & Clevers, H. Wnt signaling, Lgr5, and stem cells in the intestine and skin. Am. J. Pathol. 174, 715–721 (2009).
Kim, K.A. et al. Mitogenic influence of human R-spondin1 on the intestinal epithelium. Science 309, 1256–1259 (2005).
Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003).
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
Koo, B.K. et al. Controlled gene expression in primary Lgr5 organoid cultures. Nat. Methods 9, 81–83 (2011).
Nagy, A. Manipulating the Mouse Embryo: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 2003).
Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762–1772 (2011).
Sato, T. Novel stem cell culture system. Inflamm. Regen. 32, 43–47 (2012).
Snippert, H.J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).
Acknowledgements
We thank K.L. VanDussen and K.K. Patel for comments on the manuscript. This work was funded by the US National Institutes of Health (DK071619), The Crohn's and Colitis Foundation of America (CCFA) Genetics Initiative, the Pew Scholars Foundation and the Washington University Digestive Disease Research Core (NIH P30-DK52574).
Author information
Authors and Affiliations
Contributions
H.M. performed the experiments. H.M. and T.S.S. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Schematic representation of vectors for transfection into L-Wnt3a cells.
(a) A dual expression vector for R-spondin 3 and noggin. (b) An expression vector for R-spondin 3. Both vectors were constructed based on pVITRO2-hygro-mcs (Invivogen). PacI: enzyme digestion sites for linearization. More information about the original vector is available from manufacturer's website (http://www.invivogen.com).
Supplementary Figure 2 The effect of harvest timing on activity of conditioned media.
Primary culture media was incubated with L-WRN cells for the indicated number of days. Colonic spheroids were incubated with each conditioned media and relative expression levels of Axin2 (a target of canonical Wnt signaling) and Lgr5 (a stem cell marker) were determined by quantitative RT-PCR. Plots of mean (+SD) relative mRNA expression levels were determined by quantitative RT-PCR analysis (n=3 samples/group). Data were analyzed using one-way ANOVA followed by Tukey's post-test. P=0.0005 (Axin2), P=0.0006 (Lgr5). There was no significant difference between the activities of 1-, 2-, and 3-day media.
Supplementary Figure 3 Application of the culture protocol to non-gastrointestinal stem cells and tumor cells.
(a) Typical morphology of pancreatic, tracheal, lung, and thymic spheroids. Bars=1 mm. (b) Typical morphology of colonic tumor spheroids and colonic spheroids derived from an ApcMin mouse. (c) Typical morphology of colonic tumor spheroids derived from an AOM/DSS-treated mouse. Spheroids derived from normal tissues were cultured in 50% L-WRN conditioned media containing 10 μM Y27632 and 10 μM SB431542, whereas tumor spheroids were cultured in the basal media (0% conditioned media) containing 10 μM Y27632 and 10 μM SB431542. Bars=1 mm.
Supplementary Figure 4 Preparation of the mouse small intestine.
A piece of dissected small intestine (ileum) before (a) and after (b) scraping villi was shown. Individual crypts are visible after scraping (inset). Bars=0.5 mm.
Supplementary Figure 5 Efficient gene transduction of intestinal spheroids with concentrated lentiviruses
(a, b). Small intestinal (a) and colonic (b) spheroids were infected with unconcentrated (1 ml) or concentrated (100 μl) lentiviruses. Representative fluorescence (upper) and bright field images (lower) are shown. Bars=0.5 mm.
Supplementary information
Supplementary Figure 1
Schematic representation of vectors for transfection into L-Wnt3a cells. (PDF 661 kb)
Supplementary Figure 2
The effect of harvest timing on activity of conditioned media (PDF 242 kb)
Supplementary Figure 3
Application of the culture protocol to non-gastrointestinal stem cells and tumor cells. (PDF 1253 kb)
Supplementary Figure 4
Preparation of the mouse small intestine. (PDF 907 kb)
Supplementary Figure 5
Efficient gene transduction of intestinal spheroids with concentrated lentiviruses. (PDF 727 kb)
Generation of L-WRN and L-WR cells
Supplementary Methods (PDF 121 kb)
Rights and permissions
About this article
Cite this article
Miyoshi, H., Stappenbeck, T. In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc 8, 2471–2482 (2013). https://doi.org/10.1038/nprot.2013.153
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2013.153
This article is cited by
-
Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1
Stem Cell Reviews and Reports (2024)
-
Effect of aging on the formation and growth of colonic epithelial organoids by changes in cell cycle arrest through TGF-β-Smad3 signaling
Inflammation and Regeneration (2023)
-
Mutational landscape of intestinal crypt cells after long-term in vivo exposure to high fat diet
Scientific Reports (2023)
-
Uropathogenic Escherichia coli infection-induced epithelial trained immunity impacts urinary tract disease outcome
Nature Microbiology (2023)
-
An epithelial cell-derived metabolite tunes immunoglobulin A secretion by gut-resident plasma cells
Nature Immunology (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.