Introduction: Cholangiocytes, the epithelial cells lining the bile ducts, are highly susceptible to ischemia-reperfusion injury (IRI), a major contributor to post-transplant biliary complications. However, the cellular heterogeneity and specific stress responses of human cholangiocytes under hypoxia remain poorly defined. We established a human extrahepatic cholangiocyte organoid (ECO) model to investigate hypoxia-induced injury and identify vulnerable subpopulations and regulatory targets.

Methods: Human intrahepatic biliary tissues were used to generate ECOs embedded in Matrigel and cultured in expansion medium. After 10 passages, organoids were subjected to hypoxia (1% O₂, 5% CO₂) for up to 72 h, followed by reoxygenation to simulate IRI. Apoptosis and proliferation were assessed by TUNEL, western blotting (Bax, Bcl-2, Ki67, PCNA), and immunofluorescence. Single-cell RNA sequencing (scRNA-seq) was performed on organoids under normoxic and hypoxic conditions to assess transcriptomic responses. The role of immediate early response gene 3 (IER3) was validated using siRNA-mediated knockdown followed by bulk RNA sequencing and functional assays.

Results: Human ECOs expressed canonical biliary markers (CK18, CK19, EpCAM, CFTR) and responded to hypoxia with progressive reductions in organoid diameter, increased TUNEL positivity, elevated Bax, and reduced Ki67 and PCNA levels, consistent with apoptosis and suppressed proliferation. scRNA-seq identified five cholangiocyte clusters, with one cluster—designated as the apoptosis-prone cholangiocyte cluster (APCC)—showing marked depletion post-H/R. While most stress-responsive genes were upregulated across clusters, IER3 was insufficiently induced specifically in APCC. GSEA implicated IER3 in regulating apoptosis and oxidative stress pathways. Functional experiments confirmed that IER3 knockdown exacerbated hypoxia-induced apoptosis, reduced organoid size, and altered global transcriptomic profiles, with enrichment of cell death and proliferation-related pathways.

Conclusion: Our study establishes a human ECO-based IRI model that reveals cholangiocyte heterogeneity and identifies an apoptosis-prone subpopulation with insufficient IER3 induction under hypoxic stress. These findings highlight IER3 as a potential regulatory target for modulating cholangiocyte injury and improving biliary outcomes post-transplant. This model offers a translational platform for investigating human biliary pathophysiology and therapeutic strategies in ischemic injury.