Abstract
The epithelium of the gastrointestinal tract represents the interface between the luminal contents of the gut and that of the host tissues and plays a central role not only in regulating absorption of dietary nutrients but also in providing a barrier to prevent the entry of bacteria and other pathogens. Repair and replacement of damaged aging cells within the epithelium is modulated by stem cells, which are located in the intestinal crypts of the small intestine.
Two distinct populations of intestinal stem cells have been described in the literature, one population at the very base of the crypt and a second population of long-lived stem cells located just above the Paneth cell zone. Herein, we describe a method to label this population of long-lived GFP label retaining cells. This method is free from confounding factors of previous methodologies based on radioactive tracers and also enables functional studies not previously possible using the radioactive tracer techniques described in the literature.
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References
Peterson LW, Artis D (2014) Intestinal epithelial cells: regulators of barrier function and immune homeostasis. Nat Rev Immunol 14(3):141–153. doi:10.1038/nri3608
Thaiss CA, Zmora N, Levy M, Elinav E (2016) The microbiome and innate immunity. Nature 535(7610):65–74. doi:10.1038/nature18847
van der Flier LG, Clevers H (2009) Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol 71:241–260. doi:10.1146/annurev.physiol.010908.163145
Watson AJ, Hughes KR (2012) TNF-alpha-induced intestinal epithelial cell shedding: implications for intestinal barrier function. Ann N Y Acad Sci 1258:1–8. doi:10.1111/j.1749-6632.2012.06523.x
Hughes KR, Gandara RM, Javkar T, Sablitzky F, Hock H, Potten CS, Mahida YR (2012) Heterogeneity in histone 2B-green fluorescent protein-retaining putative small intestinal stem cells at cell position 4 and their absence in the colon. Am J Physiol Gastrointest Liver Physiol 303(11):G1188–G1201. doi:10.1152/ajpgi.00080.2012
Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003–1007. doi:10.1038/nature06196
Potten CS, Gandara R, Mahida YR, Loeffler M, Wright NA (2009) The stem cells of small intestinal crypts: where are they? Cell Prolif 42(6):731–750. doi:10.1111/j.1365-2184.2009.00642.x
Henning SJ, von Furstenberg RJ (2016) GI stem cells - new insights into roles in physiology and pathophysiology. J Physiol 594(17):4769–4779. doi:10.1113/JP271663
Potten CS, Owen G, Booth D (2002) Intestinal stem cells protect their genome by selective segregation of template DNA strands. J Cell Sci 115(Pt 11):2381–2388
Potten CS (1977) Extreme sensitivity of some intestinal crypt cells to X and gamma irradiation. Nature 269(5628):518–521
Cheng H, Merzel J, Leblond CP (1969) Renewal of Paneth cells in the small intestine of the mouse. Am J Anat 126(4):507–525. doi:10.1002/aja.1001260409
Foudi A, Hochedlinger K, Van Buren D, Schindler JW, Jaenisch R, Carey V, Hock H (2009) Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 27(1):84–90. doi:10.1038/nbt.1517
Williams JM, Duckworth CA, Vowell K, Burkitt MD, Pritchard DM (2016) Intestinal preparation techniques for histological analysis in the mouse. Curr Protoc Mouse Biol 6(2):148–168. doi:10.1002/cpmo.2
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Hughes, K.R., Mahida, Y.R. (2018). Determination of Histone 2B–Green Fluorescent Protein (GFP) Retention in Intestinal Stem Cells. In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7371-2_6
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DOI: https://doi.org/10.1007/978-1-4939-7371-2_6
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