Three-dimensional organoid culture enhances functional maturation of human pluripotent stem cell–derived hepatocytes

Background Human pluripotent stem cells (hPSCs) offer a promising source of hepatocytes for disease modeling and drug screening. However, hepatocytes derived in conventional two-dimensional (2D) cultures often exhibit incomplete maturation, limiting their physiological relevance. The molecular mechanisms underlying the improved differentiation observed in three-dimensional (3D) culture systems remain poorly understood. Results Induced PSCs (iPSCs) were differentiated into hepatic progenitors under 2D monolayer culture. At the hepatic progenitor stage, cells were either continued in 2D or re-aggregated into 3D organoids for hepatocyte maturation. Both conditions were cultured under identical experimental conditions throughout terminal differentiation for controlled comparison. At the end of differentiation, cultures were evaluated by immunostaining, gene and protein expression, functional assays, and RNA sequencing. Compared with 2D cultures, 3D hepatic organoids showed higher expression of mature hepatocyte markers (ALB, CPS1, AAT, and CYP3A4) and improved function, including increased albumin secretion, glycogen storage, and urea production. RNA sequencing identified 1,266 differentially expressed genes with upregulated genes enriched in hepatic metabolic pathways and downregulated genes associated with focal adhesion and extracellular (ECM)-receptor interaction. Conclusions These findings demonstrate that 3D organoid culture enhances iPSC-derived hepatocyte maturation by activating liver-specific transcriptional programs, while suppressing progenitor-associated gene signatures, supporting its use for liver disease modelling and drug metabolism studies.