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.
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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).
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H.M. performed the experiments. H.M. and T.S.S. wrote the manuscript.
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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)
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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
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DOI: https://doi.org/10.1038/nprot.2013.153
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