Application of Multilineage Human Liver Organoids in Disease Modelling, Drug Testing and Genetic Intervention

BACKGROUND AND AIMS: Animal models have limitations in predicting human clinical outcomes, highlighting the need for advanced human-relevant models. We aimed to establish multilineage human liver organoids (hLOs) for modelling fibrosis and metabolic dysfunction-associated steatotic liver disease (MASLD), and for evaluating pharmacological and genetic interventions METHODS: hLOs were generated from human induced pluripotent stem cells by stepwise co-differentiation. Fibrosis was induced by transforming growth factor-β (TGF-β), and steatotic injury by free fatty acids. Hydronidone and resmetirom were used for pharmacological studies. Lentiviral knockdown of YB1 in hepatocytes and STAT3 in hepatic stellate cells was used for lineage-restricted genetic perturbation. RESULTS: TGF-β induced a fibrotic phenotype in hLOs, with stellate cell activation, increased extracellular matrix secretion, and Sirius Red-positive matrix deposition. Free fatty acid treatment induced an acute lipotoxic MASLD-like phenotype characterised by steatosis, hepatocyte apoptosis, inflammatory activation, and fibrotic remodelling. Hydronidone attenuated fibrotic responses, whereas resmetirom reduced steatosis, inflammation, hepatocyte injury, and fibrosis-associated changes. In parallel, YB1 knockdown in hepatocytes and STAT3 knockdown in stellate cells ameliorated steatotic and fibrogenic phenotypes, respectively. CONCLUSIONS: Multilineage hLOs provide a human-relevant platform for modelling fibrosis and MASLD and for evaluating therapeutic and genetic interventions in a multicellular liver context.