Abstract
Intestinal stem cell research has greatly aided our understanding of the biology of intestinal self-renewal but has also shed light on the role of cancer stem cells (CSCs) in carcinogenesis, cancer growth, and dissemination. With new possibilities for CSC targeting, there is a need to have established techniques for quantifying (cancer) stem cell clonogenicity, particularly in organoid cultures. Here, we describe a detailed methodology for the isolation and expansion of mouse intestinal crypts from three different locations—the colon, proximal, and distal small intestine. In addition, we describe techniques that allow the measurement of stem cell clonogenicity and its manipulation using two approaches—organoid counting and immunohistochemistry.
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References
Nassar D, Blanpain C (2016) Cancer stem cells: basic concepts and therapeutic implications. Annu Rev Pathol 11:47–76
Medema JP (2013) Cancer stem cells: the challenges ahead. Nat Cell Biol 15:338–344
Vermeulen L, Todaro M, de Sousa MF, Sprick MR, Kemper K et al (2008) Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity. Proc Natl Acad Sci U S A 105:13427–13432
Oskarsson T, Batlle E, Massagué J (2014) Metastatic stem cells: sources, niches, and vital pathways. Cell Stem Cell 14(3):306–321
Colak S, Zimberlin CD, Fessler E, Hogdal L, Prasetyanti PR et al (2014) Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells. Cell Death Differ 21(7):1170–1177
Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459:262–265
Yui S, Nakamura T, Sato T, Nemoto Y, Mizutani T et al (2012) Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5+ stem cell. Nat Med 18:618–623
Van Der Heijden M, Zimberlin CD, Nicholson AM, Colak S, Kemp R et al (2016) Bcl-2 is a critical mediator of intestinal transformation. Nat Commun 7:10916
Acknowledgments
This work was supported by Oncode Institute, Transcan-2 grant Tactic and Dutch Cancer Society (KWF) Grants UvA2015-7587 and 10150. D.J. Huels was supported by an EMBO long-term fellowship ALTF 1102-2017.
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Ramesh, P., Kirov, A.B., Huels, D.J., Medema, J.P. (2018). Isolation, Propagation, and Clonogenicity of Intestinal Stem Cells. In: Turksen, K. (eds) Stem Cell Niche. Methods in Molecular Biology, vol 2002. Humana, New York, NY. https://doi.org/10.1007/7651_2018_179
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DOI: https://doi.org/10.1007/7651_2018_179
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