Generation of proliferative hESC-derived grape-clustered hepatocyte organoids with multipolar architecture as regenerative counterpart via synergy of YAP and IGF2 pathways.

Primary human hepatocyte (PHH)-derived organoids form grape-like clusters with proliferative capacity, hepatocyte functionality, and multipolar polarity, serving as valuable models for liver biology and therapeutics. However, deriving comparable organoids from human embryonic stem cells (hESCs) remains difficult. Here, we established a defined system to differentiate hESC-derived hepatoblast organoids into hepatocyte organoids (heporgs) with two morphologies: spheroid-like (S-heporgs) and grape-like (G-heporgs). S-heporgs predominated but displayed senescence and apoptosis, generating an inflammatory niche that facilitated G-heporg emergence. G-heporgs exhibited mature hepatocyte markers, binucleation, proliferative activity, and multipolar structures with branched bile canaliculi, closely resembling PHH-derived organoids. Transcriptomic and functional analyses identified IGF2-driven PI3K-AKT activation as essential for G-heporg formation, while YAP signaling supported their long-term expansion. IGF2 supplementation combined with YAP agonist treatment enabled stable G-heporg propagation for over 60 days. These expandable G-heporgs demonstrated regenerative competence and faithfully recapitulated hepatocyte polarity and functional bile canalicular networks, as evidenced by ATP7B copper-dependent translocation and drug-induced cholestasis assays. Our findings establish hESC-derived G-heporgs as expandable, functional counterparts to PHH-derived organoids, providing a robust platform for studying hepatocyte polarity, metabolite trafficking, and liver disease modeling.