In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture

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.

[1]  S. Nishikawa,et al.  A ROCK inhibitor permits survival of dissociated human embryonic stem cells , 2007, Nature Biotechnology.

[2]  Hans Clevers,et al.  Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. , 2011, Gastroenterology.

[3]  Terry P Yamaguchi,et al.  Wnt5a Potentiates TGF-β Signaling to Promote Colonic Crypt Regeneration After Tissue Injury , 2012, Science.

[4]  Hans Clevers,et al.  Intestinal Crypt Homeostasis Results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells , 2010, Cell.

[5]  Hans Clevers,et al.  Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. , 2010, Cell stem cell.

[6]  H. Clevers,et al.  Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche , 2009, Nature.

[7]  Takeshi Oshima,et al.  Mitogenic Influence of Human R-Spondin1 on the Intestinal Epithelium , 2005, Science.

[8]  H. Clevers,et al.  Controlled gene expression in primary Lgr5 organoid cultures , 2011, Nature Methods.

[9]  R. Nusse,et al.  Wnt signaling and stem cell control , 2008, Cell Research.

[10]  R. Behringer,et al.  Manipulating the Mouse Embryo: A Laboratory Manual , 2002 .

[11]  Toshiro Sato.  Novel intestinal stem cell culture system , 2012 .

[12]  H. Clevers,et al.  Wnt signaling, lgr5, and stem cells in the intestine and skin. , 2009, The American journal of pathology.

[13]  I. Weissman,et al.  Wnt proteins are lipid-modified and can act as stem cell growth factors , 2003, Nature.

[14]  H. Clevers,et al.  Identification of stem cells in small intestine and colon by marker gene Lgr5 , 2007, Nature.