Establishment of an in vitro culture system, based on small intestinal crypt organoids, for the investigation of putative small intestinal stem cells
Gulino, Maria E. (2016) Establishment of an in vitro culture system, based on small intestinal crypt organoids, for the investigation of putative small intestinal stem cells. PhD thesis, University of Nottingham.
Small intestinal (SI) stem cells differentiate into short-lived progeny, except lysozyme-expressing Paneth cells. In vivo, the Tet-Op histone 2 B (H2B) - green fluorescent protein (GFP) fusion protein transgenic mouse has been employed to analyse the slow-cycling putative small intestinal epithelial stem cells, at cell position +4 (cp4 cells), through doxycycline-inducible transient expression of H2B-GFP. The aim of the study was to employ the same genetic mouse model in order to develop a culture system in which was possible to detect and investigate H2B-GFP-retaining putative SI stem cells. SI crypts isolated from 6-12 weeks old Tet-Op-H2B-GFP transgenic mice were established in culture (designated organoids) using growth factors and Matrigel. For in vitro transgene expression, doxycycline was added to the complete culture medium for 24/48 hours (pulse). H2B-GFP and lysozyme expression was studied by confocal and fluorescence microscopy. Percentages of H2B-GFP-retaining putative SI stem cells and of H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring GFP-immunoreactive cells. Ulex europaeus-I lectin (UEA-I) cell labelling, combined with flow cytometry, was employed in a pilot study aimed at establishing a protocol for the separation of H2B-GFP-retaining putative SI stem cells from H2B-GFP-retaining Paneth cells persisting in the small intestine of chased mice.
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