Zonation Hallmarks in Human PSC‐Derived Hepatic Organoids Integrated into an Immune‐Compatible Organ‐on‐Chip for Liver Injury Modeling

Liver metabolic zonation enables hepatocytes to carry out diverse, spatially organized functions essential for liver homeostasis and metabolism. However, in vitro recapitulation of zone‐specific diversity, particularly in human pluripotent stem cell (PSC)‐derived hepatocyte‐like cells (HLCs), remains challenging, as these cells typically exhibit an immature phenotype. Here, we report a scalable differentiation protocol for generating 3D organoids from PSCs with defined zone 1 (z1‐HLCs) or zone 3 (z3‐HLCs) identities. When benchmarked against primary human hepatocytes (PHHs), these HLCs exhibit zone‐specific enhanced postnatal metabolic functions. z1‐HLCs reveal high albumin and urea production, whereas z3‐HLCs show elevated glutamine secretion and cytochrome P450 (CYP) enzyme activity, including CYP3A4, CYP1A2, and CYP2C9. Hallmarks of functional zonation were further confirmed via proteomics, transcriptomics, metabolic assays, and oxygen consumption. z3‐HLCs show increased sensitivity to acetaminophen‐induced cytotoxicity and distinct immune interactions with primary monocytes. Collectively, the zonated liver organoids provide a promising platform for studying zone‐specific liver metabolism, drug metabolism, toxicology, and liver disease, including liver injury.