The liver is the largest solid organ and the only one capable of using regenerative mechanisms to recover its mass fully. Although liver regeneration from acute injuries has been effective and extensively studied, chronic liver damage has adverse effects on hepatic histology, such as fibrosis or cirrhosis, suggesting that regenerative mechanisms have been disrupted. The most common form of chronic liver disorder, non-alcoholic fatty liver disease (NAFLD), is a growing cause of end-stage liver disease worldwide and is estimated to increase 2-3-fold by 2030. Despite the enormous amount of ongoing research on this topic, no treatment is available, leaving liver transplantation as the only current option. One approach to revert this situation focuses on facultative liver stem cells derived from the biliary epithelium. This recent alternative comes from the breakthrough that biliary epithelial cells (BEC) can be assembled into complex three-dimensional organoid structures in vitro from bile duct-derived stem cells. These BEC-organoids can be expanded as stem cells, with a similar phenotype to injury-induced BECs in vivo, and also can be differentiated into functional hepatocyte-like cells with engraftment ability upon transplantation. Considering the potential of BEC-organoids, I focused on dissecting the role and unveiling the potential of BECs in the context of NAFLD. BECs undergo rapid reprogramming and proliferation in chronic liver diseases, including the fibrotic stage of NAFLD, a process known as ductular reaction (DR). Thus, studying DR provides new insights into the BEC expansion mechanisms. For this purpose, I first investigated the effect of hepatic overload on BECs to study the initiation of DR during the early stages of NAFLD. I demonstrated that lipid overload induces the conversion of adult cholangiocytes into proliferating BECs and promotes their expansion via the activation of the E2F transcription factors, which drives cell cycle progression while promoting glycolytic metabolism. These observations, while correlative, reveal unexpected connections between lipid metabolism and stemness, and set the ground for future research in understanding the role and the therapeutic potential of lipid metabolism and E2Fs in controlling BEC activation. On the other hand, DR correlates closely with the severity of fibrosis in NAFLD, even though the function is largely unknown. Thus, I studied the effect of the mechanical properties of the fibrotic environment in BEC-organoid cultures by using the synthetic matrix we developed together with colleagues. The investigation of BECs cultured in a defined mechanical environment mimicking fibrotic stiffness revealed decreased stem cell capacities and increased inflammation, hepatic injury, and matrix metalloproteases. Finally, by using recently published data comparing BEC-organoid cultures from healthy and NASH patients, I revealed that hydrogels with fibrotic stiffness mimic the phenotype of organoids derived from NASH patients. As a result, investigating aberrant stiffness will enable the development of powerful DR models and future therapeutics for enhancing stem cell-mediated liver regeneration. Overall, the results from this thesis should spark future enthusiasm for the potential of BECs in studying DR regenerative mechanisms and developing therapies.