Recapitulation of Bile Acid Metabolism in Hepatobiliary Organoids Derived from hiPSC

BACKGROUND & AIMS: Bile acids are crucial mediators of cholesterol homeostasis, lipid digestion, and detoxification, and reliable in vitro synthesis systems are essential for liver disease research and precision medicine. Although hiPSC-derived hepatic organoids model key human liver functions, fully recapitulating bile acid biosynthesis remains challenging. Here, we generated bile acid-hepatobiliary organoids (BA-HBOs) that capture major aspects of bile acid metabolism, thereby providing a more physiologically and pathologically relevant platform for metabolic studies and drug screening. METHODS: Maturation protocols were optimized with saikosaponin A to enhance bile acid biosynthesis in BA-HBOs. Fibrotic BA-HBOs (FiBA-HBOs) were further established by TGF-β treatment. Cellular identity and functional markers were evaluated by immunofluorescence, flow cytometry, and qPCR. Organoid heterogeneity was characterized by single-cell RNA sequencing, and bile acid composition and diversity were quantified by LC-MS/MS metabolomics. RESULTS: BA-HBOs demonstrated hepatic and biliary functions, including organized lineage segregation and robust synthetic and metabolic capacities. Compared with controls, BA-HBOs showed increased bile acid synthesis, improved bile duct structure, and higher transport protein expression (n ≥ 3, p < 0.05). Targeted metabolomics identified a complex spectrum of 33 bile acid species, predominated by glycine-conjugated forms, consistent with human physiology. Single-cell RNA sequencing revealed that BA-HBOs recapitulated the transcriptional landscape of adult liver tissue, and revealed hepatocyte subpopulation restructuring associated with enhanced bile acid metabolism. Moreover, modeling fibrosis-associated dysregulation generated FiBA-HBOs, which exhibited cholestasis-like changes and disease-relevant metabolomic profiles (n ≥ 3, p < 0.05). CONCLUSION: BA-HBOs recapitulate key aspects of human liver bile acid metabolism, hepatocyte zonation, and core metabolic processes in vitro, providing a physiologically relevant platform for mechanistic studies of liver disease and screening therapeutic candidates. IMPACT AND IMPLICATIONS: Our hiPSC-derived BA-HBOs synthesize and secrete diverse bile acid species, capturing key transcriptional and metabolic profiles of the human liver. They enable modeling of liver fibrosis accompanied by disruptions in bile acid metabolism, offering a tool to dissect disease mechanisms. These robust organoids create new opportunities for basic liver research, therapeutic development, and precision medicine.