Abstract: Objective To investigate the time-dependent transcriptomic alterations during liver organoid formation from HepaRG cells under three-dimensional culture from 0 to 120 h, and to characterize molecular features associated with the programmatic transition from proliferation-related processes to hepatic functional differentiation. Methods HepaRG cells were mixed with Matrigel (1∶1, v/v) and seeded into ultra-low-attachment 96-well plates. Samples collected at 0 h, 14 h, 24 h, 72 h, and 120 h were subjected to RNA sequencing (RNA-seq), followed by principal component analysis (PCA)/correlation assessment, DESeq2-based differential expression, time-course clustering with enrichment analysis, and protein-protein interaction (PPI) network analysis. Results HepaRG cells self-assembled into spheroid organoids under three-dimensional culture, accompanied by increased albumin (ALB) expression. The number of differentially expressed genes (DEGs) peaked at 72 h and 120 h. Six temporal clusters revealed an early-to-late transition from proliferation/cell-cycle programs to hepatic functional programs (lipid metabolism, lipoprotein remodeling, and the complement pathway). Conclusions HepaRG organoid formation involves dynamic transcriptomic remodeling. The 72-120 h interval constitutes a critical stage with enhanced hepatic function-related programs, offering transcriptomic support for maturation evaluation and mechanistic studies.

Key words: HepaRG cells, liver organoids, transcriptome, time-series analysis, protein-protein interaction network