P.01: Insufficient IER3 induction confers apoptosis vulnerability to a cholangiocyte subpopulation in organoid models of ischemic-type biliary injury.

作者：Xuechun Zhao, Guobin Huang, Yuanyuan Zhao, Zipei Wang, Dong Chen, Wei Lai, Zhishui Chen, Bo Yang · 发表于：Transplantation · 年份：2025 · DOI：10.1097/01.tp.0001170680.18614.24 · 研究领域：Liver Diseases and Immunity、Liver physiology and pathology、Organ Transplantation Techniques and Outcomes

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,...