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 7

ADAM inhibitors prolong LPC expansion and organoid survival.

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ADAM inhibitors prolong LPC expansion and organoid survival. (A) T1−⁄−T3...
(A) T1−⁄−T3−⁄− biliary organoids were treated with ADAM inhibitors for 14 days. Time course of organoid growth using maximum diameter in the presence of the ADAM inhibitors GI254023X (GI; n = 4 livers) and TAPI-1 (n = 3 livers). Two-way ANOVA with Šidák’s multiple-comparison test. (B) Bright-field images of organoid cultures in the presence of TAPI-1 and GI254023X, showing collapsed organoids as black cell aggregates (arrows) and size of the largest organoid at day 14. (C) Relative number of T1−⁄−T3−⁄− live organoids in the presence of ADAM inhibitors compared with DMSO (100%). One-way ANOVA with Bonferroni’s multiple-comparison test. (D) WT organoids treated with ADAM and Notch inhibitors for 7 days. Maximum organoid size and relative number of WT live organoids obtained in the presence of TAPI-1, GI254023X, and DAPT compared with DMSO; n = 4 livers. One-way ANOVA with Bonferroni’s multiple-comparison test. (E) WT biliary organoids treated with TAPI-1 from day 4 to day 7. Bright-field images of the largest organoids and percentage of live organoids at day 7; n = 3 livers. Two-tailed paired t test. (F) TAPI-1–induced gene expression regulation of Alb (hepatocyte); Krt19, Hnf1b, and Epcam (cholangiocyte); Hes1 and Sox9 (biliary differentiation); and Mki67 and Ccnd1 (proliferation) in day 7 organoids, expressed as log2 of the fold change (TAPI-1/DMSO). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.