Synergy between oxygenation and co-culture with liver sinusoidal endothelial cells drives expansion of functional liver organoids

Induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) hold great promise for drug discovery and cell therapy, but their immature phenotypes and limited proliferative capacity remain major challenges. We previously showed that direct oxygenation using an oxygen-permeable membrane enhances the differentiation of iPSC-derived liver progenitor cells (LPCs), although proliferation ceases during the terminal phase of maturation. Here, we demonstrate that combining direct oxygenation with co-culture of iPSC-derived liver sinusoidal endothelial cells (LSECs) overcomes this limitation. Under oxygenated conditions, co-cultured organoids exhibited robust increase in volume, whereas either oxygenation or co-culture alone was insufficient to support growth, indicating a synergistic effect. Importantly, oxygenated co-culture organoids maintained hepatic maturation comparable to oxygenated LPC monoculture organoids, based on albumin secretion and cytochrome P450 metabolic activities. Furthermore, oxygenation promoted spatial reorganization in co-cultured organoids, with LPCs occupying the center and LSECs forming a peripheral layer, suggesting improved architectural fidelity to the liver microenvironment. Together, this approach enables the scalable generation of highly mature liver organoids with enhanced structural and functional fidelity. • Liver progenitor cells (LPCs) and liver sinusoidal endothelial cells (LSECs) were generated from human iPS cells. • Direct oxygenation promoted hepatocytic maturation but not LPC proliferation. • LSEC co-culture alone failed to enhance LPC proliferation. • Combined oxygenation and LSEC co-culture enabled LPC expansion without loss of function. • This system provides a scalable platform for functional human hepatocyte production.