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.

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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.

After both 24 hours and 48 hours exposure (pulse) to the optimal concentration of doxycycline, the nuclei of all cells in SI organoids expressed H2B-GFP. During subsequent culture, in absence of doxycycline (chase period), there was gradual loss (due to cell division) of H2B-GFP. After 6 day chase, small numbers of H2B-GFP-retaining putative slow-cycling stem cells and Paneth cells were seen. Flow cytometric analyses indicated that to label SI crypt cells with UEA-I lectin is a promising approach for isolating putative SI stem cells for functional characterization.

A method to induce ubiquitous transient expression of H2B-GFP in cultured SI organoids, compatible with regular organoid development, has been optimized. It allows detection, after a short chase period, of the slow-cycling H2B-GFP-retaining putative SI epithelial stem cells. The developed in vitro culture system will enable further characterisation of these cells and Paneth cells.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Mahida, Y.
Keywords: Small intestinal stem cells, Small intestinal organoids
Subjects: QS-QZ Preclinical sciences (NLM Classification) > QU Biochemistry
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Medicine
Item ID: 33730
Depositing User: Gulino, Maria
Date Deposited: 15 Aug 2016 12:36
Last Modified: 19 Dec 2017 11:21

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