INVESTIGATIONS ON EPITHELIAL BIOLOGY USING ORGANOID DIFFERENTIATION AND CO-CULTURES: Building representative models of the snake venom gland and complex tissue interactions in cancer and metabolic diseases

The work described in this thesis revolves around one question: How can we utilize and develop organoid technology to elucidate previously intractable areas of biology?
My approaches to this question in the various chapters share three common threads: First, differentiating organoids towards the cell types required to address research questions ranging from intestinal cell specialization to snake venom secretion. Second, exposing organoids to microenvironmental components such as microbiota and stromal growth factors in a controlled fashion to infer causality. Third, characterizing organoid cultures on multiple levels, including single cell transcriptomics and analysis of their secreted products such as hormones and toxins.
A growing body of evidence implicates a role for the gut microbiota – the community of microorganisms residing in the intestine – in health and disease. However, to date, most of these studies have only been able to show a correlative relationship between the microbiota and gut (patho-)physiology. In the first part of this thesis, I describe how human intestinal and colonic organoids can be used to recapitulate epithelium-microbe interactions. We identify a bacterially induced mutational signature using organoids, serving as a prime example of how co-cultures enable a detailed mechanistic understanding of microbial contributions to diseases such as colorectal cancer.
While this study adds to a substantial body of knowledge on carcinogenesis in the intestinal tract, even the function of the healthy gut epithelium remains incompletely understood. Major intestinal cell types such as enterocytes and goblet cells are commonly treated as a homogeneous population with broad functions such as nutrient uptake and mucus secretion, respectively. Recently, the hormone-producing enteroendocrine cells (EECs) have been shown to change their hormone expression in response to varying levels of bone morphogenetic protein (BMP) as they travel along the crypt-villus axis in the mouse intestine. In the second section of this thesis, we show that this division of labor along the crypt-villus axis applies to all major cell types in the human intestine, thus providing a more nuanced view of functional subpopulations of enterocytes, goblet cells, and enteroendocrine cells. We anticipate that these findings will not only advance our understanding of intestinal physiology, but also open therapeutic avenues for diseases such as type 2 diabetes and gut motility disorders by specifically targeting the relevant cellular subpopulations.
Human and murine organoids are rapidly gaining relevance as a screening platform for patient-specific drug response prediction. In the shadows of this progress in the treatment of Western diseases, the global snakebite problem with more than 100,000 deaths each year had been untouched by organoid research. Despite the major impact of snakebite on human health, our understanding of venom production in snakes and the procedures for generating antivenom lag far behind other areas of cellular and molecular biology. In the last section of this thesis we lay the groundwork for a better understanding of snake venom production by deriving and characterizing organoids from the snake venom gland. Ultimately, this might pave the way for a novel antivenom production approaches to ameliorate the devasting effects of snakebite.