Metalloproteinase inhibitors regulate biliary progenitor cells through sDLK1 in organoid models of liver injury
Virginie Defamie, … , Paul D. Waterhouse, Rama Khokha
Virginie Defamie, … , Paul D. Waterhouse, Rama Khokha
Published December 19, 2024
Citation Information: J Clin Invest. 2025;135(3):e164997. https://doi.org/10.1172/JCI164997.
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Research Article Hepatology

Metalloproteinase inhibitors regulate biliary progenitor cells through sDLK1 in organoid models of liver injury

Abstract

Understanding cell fate regulation in the liver is necessary to advance cell therapies for hepatic disease. Liver progenitor cells (LPCs) contribute to tissue regeneration after severe hepatic injury, yet signals instructing progenitor cell dynamics and fate are largely unknown. Tissue inhibitor of metalloproteinases 1 (TIMP1) and TIMP3 control the sheddases ADAM10 and ADAM17, key for NOTCH activation. Here we uncover the role of the TIMP/ADAM/NOTCH/DLK1 axis in LPC maintenance and cholangiocyte specification. Combined TIMP1/TIMP3 loss in vivo caused abnormal portal triad stoichiometry accompanied by collagen deposits, dysregulated Notch signaling, and increased soluble DLK1. The MIC1-1C3+CD133+CD26– biliary progenitor population was reduced following acute CCl4 or chronic DDC liver injury and in aged TIMP-deficient livers. Single-cell RNA sequencing data interrogation and RNAscope identified portal mesenchymal cells coexpressing ADAM17/DLK1 as enzymatically equipped to process DLK1 and direct LPC differentiation. Specifically, TIMP-deficient biliary fragment–derived organoids displayed increased propensity for cholangiocyte differentiation. ADAM17 inhibition reduced Sox9-mediated cholangiocyte differentiation, prolonging organoid growth and survival, whereas WT organoids treated with soluble DLK1 triggered Sox9 expression and cholangiocyte specification in mouse and patient-derived liver organoids. Thus, metalloproteinase inhibitors regulate instructive signals for biliary cell differentiation and LPC preservation within the portal niche, providing a new basis for cell therapy strategies.

Authors

Virginie Defamie, Kazeera Aliar, Soumili Sarkar, Foram Vyas, Ronak Shetty, Swami Reddy Narala, Hui Fang, Sanjay Saw, Pirashaanthy Tharmapalan, Otto Sanchez, Jennifer J. Knox, Paul D. Waterhouse, Rama Khokha

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Figure 3

Impaired LPC expansion after liver injury.

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Impaired LPC expansion after liver injury. (A) Schematic of tissue analy...
(A) Schematic of tissue analyses following 0.1% DDC– and CCl4-induced liver injury. Left hepatic lobe was harvested (yellow dashed line) for biochemistry and tissue staining, and the remaining organ was digested to obtain a single non-parenchymal cell (NPC) suspension for flow cytometry analysis. For CCl4-treated livers, NPCs were flow-profiled or cultured in CFC assay. (B) Timp expression in WT hepatic tissue following DDC or CCl4 administration relative to uninjured liver; n ≥ 3 livers per time point. One-way ANOVA with Šidák’s multiple-comparison test. (C) Liver tissue characterization after 4 weeks of DDC treatment. Fibrosis assessed with Masson’s trichrome staining; collagen deposition appears in blue. Ductular reaction (yellow arrowheads) seen by immunofluorescence for CK19 and laminin. Ki67 marks proliferative cholangiocytes (open arrowheads) and hepatocytes (black arrowheads). Immunofluorescence for specific markers (HNF4α, hepatocytes; SOX9, cholangiocytes; αSMA, VSMCs) within the ductular reaction. White arrowheads point to double-positive SOX9+HNF4α+ cells in the vicinity of bile ducts (asterisks). (D and E) Flow cytometry analysis of MIC1-1C3+CD133+CD26– population (D) and whole-tissue expression of Trop2 (E) after DDC treatment; n ≥ 3 livers. Two-way ANOVA with Tukey’s multiple-comparison test. (F) Characterization of WT and T1−⁄−T3−⁄− livers 3 days after CCl4 insult. H&E staining showing pericentral necrotic area (yellow dashed lines). Immunofluorescence for Ki67 shows proliferative hepatocytes in zone 1/2 (white arrowheads) and desmin-positive mesenchymal cell migration within the pericentral necrotic area. (G) Flow cytometry analysis of MIC1-1C3+CD133+CD26– population at 6 days after CCl4 injury; n ≥ 3 livers per genotype per time point. Two-way ANOVA with Tukey’s multiple-comparison test. (H) Representative plates of colony-forming assay from untreated and post-CCl4 livers. Colony quantification graphed as absolute number of colonies and percentage of colony type (n ≥ 3). Two-way ANOVA with Tukey’s multiple-comparison test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.