Periodic Reporting for period 4 - SysOrganoid (A systems biology approach to investigate cell fate switches in intestinal organoids) Reporting period: 2023-04-01 to 2023-09-30 Summary of the context and overall objectives of the project Every day, billions of new cells are produced in the intestine of a mouse. The generation of these cells is driven by adult stem cells, which residue in the intestinal epithelium. These stem cells divide every day, but they also differentiate towards distinct cell types that are involves in processes such as nutrient uptake and secretion of mucus and hormones. Intestinal organoid cultures recently emerged as a paradigm to study adult stem cell maintenance and differentiation. These ‘miniguts’ can be cultured in vitro and contain all the different cell types that present in the mouse small intestinal epithelium. Recently it was shown that small-molecule driven perturbations can be used to obtain organoids, which are strongly enriched for specific intestinal cell types. This system thus provides a perfect opportunity to study, for the first time, adult stem cell maintenance and (de)differentiation in a controlled manner. Using small molecule-driven perturbations and a unique combination of ‘omics’ technologies this project has provided a systems-wide view of the molecular (epigenetic) mechanisms that orchestrate cell fate changes in intestinal organoids. Our integrative approach has identified the major regulatory networks that define the remarkable cellular plasticity and homeostasis of the mouse small intestinal epithelium. The technologies and computational infrastructure that we developed in the project have also been and will be further applied to other organoid models, including cancer organoids. Insights from these studies may eventually result in new treatments for colon cancer. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far During the project we have uncovered differentiation trajectories and associated gene expression and epigenetics dynamics for various cell types in the mouse intestinal epithelium, including enterocytes, Paneth cells and Microfold cells. We have also developed various technologies in the context of the project. One of these, a new proximity labelling enzyme called ProtA-Turbo that facilitates interactions proteomics experiments in primary cells, including organoids. This enzyme has been licensed to Millipore and will soon become commercially available. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Several innovative technologies have been developed in the project, including a miniaturised interaction proteomics platform on a microfluidics chip, a new proximity labelling enzyme for interaction proteomics and a method to profile transcription factor binding affinities for native chromatin. Our multi-omics analyses of cellular differentiation in the mouse small intestinal epithelium has uncovered new regulators of cell fate in the intestine, which will have profound implications from a fundamental and applied scientific perspective. sysorganoid.png
