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Title: Development of small intestinal organoid cultures for the study of enteric pathogens
Author: Luu, L.
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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The gastrointestinal tract is the major route of entry for enteric pathogens. Current models are not suited for the study of early invasion which limits the use of systems biology approaches to study host-pathogen interactions. Therefore, there is a demand to develop a suitable model to study early-stage host-pathogen interactions. Recent developments in stem cell research have led to the establishment of "mini-gut" organoids that encapsulate the cellular diversity and structural arrangement of the small intestinal epithelium in vivo. Due to these features, they have been proposed as a promising tool for infection studies. Several aspects of organoid optimization are required before exploiting this model for infection. Firstly, recent works have demonstrated the amenability of organoid cultures to be driven toward Paneth or goblet cell phenotype for the study of specialized host defence functions. However this model requires further validation. Secondly, luminal access is restricted, and infection can only be established via microinjection of pathogens. Lastly, mass spectrometry approaches have not yet been validated for use in these models. This doctoral thesis addresses these issues by firstly validating the use of drug-skewed organoid models using quantitative label free mass spectrometry, and then by modifying cultures to establish differentiated and polarized murine epithelial monolayers with an accessible apical surface. This collagen-supported monolayer is exploited to study hostproteome changes during early infection with Toxoplasma gondii via quantitative label-free mass spectrometry. Label-free mass spectrometry of Paneth and goblet-enriched organoids detected unique organoid proteome profiles that reflect the phenotype of these subpopulations as expected in vivo, and demonstrate the suitability of mass spectrometry to study changes in protein abundance in this model. Here, we also identified a protein, DNAJC3, enriched within Paneth cells which may play a role in specialized cell function or differentiation. Modification of 3D organoid culture on collagen scaffolds has established a novel stem cell derived collagen-supported epithelial monolayer culture. Confocal microscopy demonstrated retention of differentiated features, such as the presence of Paneth and goblet cell subpopulations and apical polarization. Label-free mass spectrometry was used to detect the presence of intestinal epithelial cell markers in both 3D and collagen-supported epithelial sheets. The findings here demonstrate that many of the differentiated epithelial cell markers are detectable in the collagen-supported epithelial monolayer, and that this model can mimic the in vivo epithelial composition. A series of pilot studies and time-course infection studies were performed to establish a working protocol to exploit the collagen-supported epithelial sheet for infection studies. Quantitative label-free mass spectrometry was exploited to examine host-proteome changes in response to infection with two strains of Toxoplasma gondii; Veg (type III, avirulent) and RH (type I, virulent). In agreement with current literature, we observed modulation of proteins associated with host cell cycling, apoptosis and lipid metabolism. Interestingly, we observed a lag in parasite replication compared to reported observations in cell line cultures and an aggregative behavior along epithelial sheet perimeters previously not reported. The findings in this chapter demonstrate the suitability of this model for infection studies, and highlights some parasite behaviours that are not observed in cell line models.
Supervisor: Coombes, Janine ; Wastling, Jonathan ; Wigley, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral