02233nas a2200277   4500000000100000000000100001008004100002260001500043653002600058653002800084653003000112100001800142700002200160700002000182700001700202700001900219700001900238700002100257700002000278700002000298245006600318856005500384300000900439520149300448022001401941       2025                            d  c2025-04-1610aRegenerative Medicine10aStem-cell biotechnology10aStem-cell differentiation1 aHasan Al Reza1 aConnie Santangelo1 aKentaro Iwasawa1 aAbid Al Reza1 aSachiko Sekiya1 aKathryn Glaser1 aAlexander Bondoc1 aJonathan Merola1 aTakanori Takebe00aMulti-zonal liver organoids from human pluripotent stem cells  uhttps://www.nature.com/articles/s41586-025-08850-1  a1-103 aDistinct hepatocyte subpopulations are spatially segregated along the portal–central axis and are critical to understanding metabolic homeostasis and injury in the liver1. Although several bioactive molecules, including ascorbate and bilirubin, have been described as having a role in directing zonal fates, zonal liver architecture has not yet been replicated in vitro2,3. Here, to evaluate hepatic zonal polarity, we developed a self-assembling zone-specific liver organoid by co-culturing ascorbate- and bilirubin-enriched hepatic progenitors derived from human induced pluripotent stem cells. We found that preconditioned hepatocyte-like cells exhibited zone-specific functions associated with the urea cycle, glutathione synthesis and glutamate synthesis. Single-nucleus RNA-sequencing analysis of these zonally patterned organoids identifies a hepatoblast differentiation trajectory that dictates periportal, interzonal and pericentral human hepatocytes. Epigenetic and transcriptomic analysis showed that zonal identity is orchestrated by ascorbate- or bilirubin-dependent binding of EP300 to TET1 or HIF1α. Transplantation of the self-assembled zonally patterned human organoids improved survival of immunodeficient rats who underwent bile duct ligation by ameliorating the hyperammonaemia and hyperbilirubinaemia. Overall, this multi-zonal organoid system serves as an in vitro human model to better recapitulate hepatic architecture relevant to liver development and disease.  a1476-4687