Ischemic preconditioning promotes hepatic differentiation in human liver organoids

Introduction: Liver regeneration is essential for successful outcomes after liver transplantation. However, ischemia-reperfusion injury (IRI) remains a major determinant of graft dysfunction that can profoundly affect hepatic regenerative responses. Although ischemic stress has been implicated in both tissue damage and regenerative signaling, its direct impact on progenitor differentiation and hepatocyte maturation remains poorly understood, partly due to the lack of controllable human experimental models. In this study, we investigated how controlled ischemic stress influences hepatocyte differentiation and regenerative signaling using a human liver organoid (HLiO) model. Methods: Organoids were expanded as undifferentiated cultures and subjected to a stepwise differentiation protocol toward hepatocyte-like cells. An in vitro IRI model was generated by exposing organoids to 16 h of cold ischemia (O2 0%) followed by reperfusion under normoxic conditions (O2 20%). Molecular, imaging, and functional analyses were performed to evaluate progenitor status, hepatocyte differentiation, and the release of inflammatory mediators. Results: Differentiation of HLiOs induced a shift from progenitor-associated gene expression toward hepatocyte-specific programs, accompanied by increased albumin secretion and expression of mature markers. Controlled ischemia caused a transient reduction in viability and triggered the release of High Mobility Group Box 1 (HMGB1), Interleukin 1 Beta (IL-1β), Interleukin 8 (IL-8), and Oxidized Low Density Lipoprotein Receptor 1 (LOX-1), followed by recovery during reperfusion. Notably, ischemic preconditioning enhanced hepatocyte maturation, characterized by stronger downregulation of progenitor markers, increased expression of Cytochrome P450 3A4 (CYP3A4), Hepatocyte Nuclear Factor 4 Alpha (HNF4A), Alpha-1 Antitrypsin (A1AT), and albumin, and improved functional output compared with standard differentiation. Discussion: These findings suggest that sub-lethal ischemic stress may act as a regenerative stimulus, potentially mediated by a progenitor-associated HMGB1-LOX-1-IL-8 signaling axis. Despite the absence of non-parenchymal liver cells, this organoid platform provides a controllable system to study intrinsic regenerative responses to ischemia, and indicates that appropriately modulated ischemic cues might promote hepatocyte differentiation, and improve graft recovery after liver transplantation.