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- Matthias Zilbauer (United Kingdom)
- Walter A. Mihatsch (Germany)
EPIGENETIC SIGNATURES, SINGLE CELL RNA SEQUENCING AND ORGANOIDS - ON THE ROAD TO IDENTIFY THE PATHOGENESIS OF IBD
- Matthias Zilbauer (United Kingdom)
Abstract
Abstract Body
The intestinal epithelium as the most inner layer of the human intestine plays a critical role in maintaining homeostasis. Indeed, altered function of this single cell layer has been linked to the development of related pathologies including the inflammatory bowel diseases (IBD) Crohn’s Disease (CD) and Ulcerative Colitis. Constant exposure to a wide range of luminal antigens requires the epithelium to balance barrier function against harmful contents with tolerance towards essential nutrients and beneficial microbes. Complex cross talk between host epithelium and adjacent gut microbiota as well as exposure to other antigens implicates the potential role of epigenetic mechanisms that operate at the interface between environmental factors and the cellular genome. In recent years we have been investigating the role of DNA methylation as one of the main epigenetic mechanisms in regulating intestinal epithelial cell function. We have identified distinct, epithelial cell specific DNA methylation changes in the intestinal epithelium of children diagnosed with IBD. Using patient derived intestinal epithelial organoids (IEOs) allowed us to investigate functional implications of epigenetic changes and correlate them to clinical phenotype. Furthermore, applying single cell RNS sequencing to both primary epithelium and patient derived organoids provides unique opportunities to enhance our understanding of IBD pathogenesis.
GENETIC ENGINEERING IN GUT ORGANOIDS FOR CYSTIC FIBROSIS AND STEM CELL RESEARCH
- Bon-Kyoung Koo (Austria)
Abstract
Abstract Body
The identification of LGR5+ intestinal stem cells helped us to understand various aspects of adult stem cells and led to the establishment of primary 3D intestinal organoid culture system from mouse and human tissues. This novel culture system faithfully recapitulates various aspects of the intestinal epithelium in vitro with remarkable long-term expansion capacity and genetic stability. Thus, the model is recognised as a suitable in vitro model system for genetic studies. To exploit all the potential of this culture, protocols have been fully optimised for primary establishment, maintenance, cryopreservation, plasmid transfection and viral transduction. A number of examples will be shown to introduce how to apply CRISPR technology and organoid models for genetic studies, including simultaneous paralogue knockout, functional genetic screening and precise gene correction.