Six-compound cocktail for maturation of human pluripotent stem cell-derived liver spheroids for toxicity prediction.

Drug-induced liver injury remains a leading cause of drug development failure and a major clinical safety concern. Human induced pluripotent stem cell-derived hepatocytes offer a promising approach for modeling patient-specific drug responses, but their immature phenotype limits predictive performance. We report a chemically defined six-compound maturation strategy for human stem cell-derived liver spheroids. Matured spheroids acquire adult-like hepatic features, including increased cytochrome P450 activity, enhanced metabolic capacity, and a metabolic shift toward oxidative phosphorylation, supported by genome-wide pathway analysis and functional assays across multiple donors. When evaluated with twenty reference compounds, matured spheroids improve hepatotoxicity prediction and outperform primary human hepatocyte spheroids. Free fatty acids treatment further  induces features of metabolic dysfunction-associated steatotic liver disease and increases sensitivity to selected hepatotoxic drugs. In this work, we show that a defined six-compound cocktail enhances stem cell-derived liver spheroids maturation, improves drug-induced hepatotoxicity prediction, and enables modeling of steatotic liver disease.