MASH Background Confers Enhanced Disease Susceptibility and Acetaminophen Toxicity in iPSC‐Derived Liver Organoids
ABSTRACT Organoids derived from human induced pluripotent stem cells (iPSCs) serve as advanced multicellular models for studying human organ development and disease. Recent liver organoid platforms focus on achieving multicellular organization while minimizing reliance on xenogeneic extracellular matrices to support future clinical translation. Building on these advances, this study establishes a xenogeneic‐free strategy that develops the hepatic cellular repertoire with interdigitating vasculature using an air‐liquid interface approach, generating highly vascularized, multicellular, and functional liver organoids from de‐identified control and metabolic dysfunction‐associated steatohepatitis (MASH) donor‐derived iPSCs. Phenotypic and functional characterization confirms the presence of hepatocytes, cholangiocytes, stellate cells, sinusoidal endothelial cells, and Kupffer‐like cells. These organoids demonstrate the capacity to model steatohepatitis following free‐fatty acid exposure and predict acetaminophen‐induced drug toxicity. Organoids derived from MASH‐donors exhibit increased susceptibility to steatosis, inflammation, fibrosis, and acetaminophen‐induced toxicity. Lipidomic profiling reveals that MASH phenotype in organoids induces global lipidomic shifts that closely resemble those observed in MASH liver biopsies. Further post‐transplantation into mice, the organoids retain hepatic cell repertoire, establish functional anastomoses with host vasculature, display intraluminal erythrocytes, and secrete human‐specific albumin, validating their translational potential. This approach provides a robust, xenogeneic‐free platform for disease modeling, evaluating drug responses, and exploring regenerative therapies.