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UBC Theses and Dissertations

Inferring regulators of liver development using single cell sequencing and organoids Drissler, Sibyl


The liver regulates most chemical levels in the blood, breaking down and processing nutrients, metabolizing drugs, and excreting waste products through a product called bile. These critical tasks are mainly fulfilled by cells called hepatocytes, which are supported by endothelial, mesenchymal, and immune cells. Herein, we analyze the earliest stages of mouse embryonic liver development using single cell transcriptomics of cells from embryonic day (E) 7.5 to E10.5 embryos. Single cell transcriptomics shows that by E10.5 the endothelial and mesenchymal cells already express many liver-specific markers, such as Lyve1 and Gdf2, respectively. In the first part of this thesis, I expanded the CellPhoneDB database to provide a list of possible ligand-receptor interactions in the early liver bud at E9.5 and E10.5. This analysis yields many known interactions, in addition to many interactions which do not yet have known roles in liver development. The novel interactions include signaling from the hepatoblast (hepatocyte precursor) ligand LECT2 to the liver sinusoidal endothelial cell receptor TIE1, and from the stellate cell ligands RSPO3 and DKK1 to hepatoblast receptors. Next, I developed a multilineage organoid model of liver development. Our goal is to use the organoids for high throughput screening of these ligand-receptor interactions using shRNA-based knockdown of genes or small molecules to activate or inhibit the interactions or their downstream pathways. This model starts from human pluripotent stem cells and uses a differentiation protocol that allows for concomitant differentiation of the mesenchymal, endothelial, and hepatocyte lineages. Preliminary experiments show evidence of these lineages, but further characterization is required to determine how closely they resemble their in vivo counterparts. The second part of this thesis focuses on transcription factor and gene regulatory analysis of the differentiating hepatoblasts, which will eventually give rise to hepatocytes. This analysis identified NR5A2 as a putative regulator of hepatoblast development and/or differentiation in E9.5 livers. The critical role of NR5A2 in Zebrafish development, but currently unvalidated role in human or mouse liver development suggests that further research into NR5A2 is warranted.

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