Modeling pathogenesis and progression of metabolic dysfunction-associated steatotic liver disease and therapeutic drug screening using hESC-derived mature polarized hepatocyte organoids.

backgroundMetabolic dysfunction-associated steatotic liver disease (MASLD), the most prevalent chronic liver disorder worldwide, exhibits complex pathogenesis and lacks effective targeted therapeutics. Existing animal models are limited by prolonged induction periods and interspecies discrepancies, while conventional monolayer hepatocyte cultures fail to recapitulate disease pathology due to inadequate polarization and functional immaturity.

methodsTo overcome these limitations, we established an in vitro MASLD model by treating human embryonic stem cell (hESC)-derived mature polarized hepatocyte organoids (P-hep-orgs) with free fatty acids (FFAs). Pathogenesis and progression of MASLD in this model were characterized using multiple assays, and its utility for drug screening was validated with three known antioxidant or lipid-lowering agents.

resultsP-hep-orgs derived from hESCs expressed mature hepatocyte markers (e.g., ALB), exhibited polarized architecture (e.g., MRP2) and demonstrated functionalities of mature hepatocytes (e.g., urea production). Moreover, we developed an in vitro MASLD model by treating P-hep-orgs with FFAs. This model recapitulated key pathological progression hallmarks, including disrupted glucose/lipid metabolism, oxidative stress, apoptosis, loss of polarization, impaired liver function, and ductular reaction. Furthermore, transcriptomic analysis revealed that P-hep-orgs treated with FFAs for 10 days shared similar molecular signatures with human MASH liver tissues (581 overlap DEGs). Finally, this model was used to assess the potential efficacy of established antioxidant or lipid-lowering agents (e.g., Vitamin E) in alleviating pathological phenotypes, including lipid accumulation and oxidative stress.

conclusionsIn summary, we established a novel in vitro MASLD model using hESC-derived P-hep-orgs. This model faithfully recapitulates key aspects of MASLD progression and serves as a valuable platform for investigating disease mechanisms and screening potential therapeutic compounds.