Periodic Reporting for period 1 - BESIDE (Bile acids and epithelial stem cells in intestinal development)
Periodo di rendicontazione: 2018-04-01 al 2020-03-31
WP1. Effects of BSH activity on proliferation of intestinal epithelial stem cells (IESCs): During this WP the fellow was trained in state-of-the-art flow cytometry techniques and she developed optimized and reproducible protocols for the isolation of IESCs from the murine small intestine and colon. Once isolated, the IESCs were successfully grown as 3D in vitro models. To determine the effects of BSH, different concentrations of bile acids at different times were added to these cells and the toxic effect was evaluated with different viability tests. Once the optimal concentration and exposure time was established, a screening of over 50 genes involved in differentiation and proliferation was carried out. This led to the selection of target genes to be analysed in the rest of the WP.
WP2. Effect of BSH activity impact on IESCs differentiation and crypt development: Isolated IESCs were grown in different media in order to promote differentiation. However, the IESCs failed to differentiate in all the media tested. Thus, crypts isolated from the small intestine and the colon, were used instead of IESCs. This gave us a robust protocol to grow both types of crypts, which were differentiated into organoids. Protocols for determination of proliferation and differentiation rates using flow cytometry were established. The RT-qPCR analyses for small intestine organoids were completed using the gene panel selected in WP1. Results showed that BSH led to a modulation in the expression of the differentiation and proliferation markers. The protocol for the fixation of organoids and staining with differentiation markers was optimized for analysis by confocal microscopy in University College Dublin. The RT-qPCR analyses of colon organoids and the confocal microscopy had to take place in March 2020 but, due to the COVID-19 outbreak, Universities in the Republic of Ireland are closed so they had to be postponed.
WP3. In vivo impact of BSH activity on GIT development in a murine germ-free model: Two separate animal trials were performed, one using a bacteria expressing BSH and one using individual bile acids to evaluate the effect in the GIT development and maturation. A panel of 25 genes was evaluated in both experiments. Results showed that BSH activity is able to modulate key genes involved in maturation. Histological and immunohistochemistry assays showed that BSH expression stimulated an increase in a number of differentiation parameters as well as for molecular markers. In addition, an extra deliverable was included in this WP. The analysis of metabolites was performed at the Centre for Omic Sciences (Tarragona), which allowed to strength the results obtained from these experiments.
WP4. Communication and Dissemination: The dissemination plan was discussed on regular meetings. There is a draft review paper and journal article in preparation. Also, there is a second paper expected to be released by 2021, as some of the experiments had to be postponed. The work carried out has been also shown in different workshops and to non-scientific audiences as a result of the participation in different outreach activities.
WP5 and WP6 were related to Training and Career Development and Transfer of Knowledge and Management. All deliverables and milestones included in these WPs have been achieved.
Regular meetings with the group facilitated the evaluation of the project’s progress and to troubleshoot any issue related to all WP progression.