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 1

Distorted portal triad and excessive biliary differentiation in Timp1−⁄−Timp3−⁄− liver.

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Distorted portal triad and excessive biliary differentiation in Timp1−⁄−...
(A) Timp1−⁄−Timp3−⁄− (T1−⁄−T3−⁄−) liver exhibiting abnormal superficial blood vessels (arrowheads). (B) Masson’s trichrome staining of liver sections; magnified area shows periportal collagen deposition in blue (black arrowheads) and sinusoid dilatation (yellow arrowheads). Portal collagen quantification; percentage of blue area on total tissue area. WT n = 3, T1−⁄−T3−⁄− n = 8 livers. (C) CK19+ immunofluorescence shows cholangiocytes forming bile ducts (BD; asterisks); αSMA+ smooth muscle cells form hepatic arteries (HA) and portal veins (PV). Arrows show abnormal periportal HA formation. Quantification of hepatic arteries per portal vein. WT n = 3, T1−⁄−T3−⁄− n = 5 livers. Mann-Whitney test, *P < 0.05. (D) Ki67 immunostaining highlights proliferating cholangiocytes (open arrowhead) in bile ducts (asterisks), interstitial cells (black arrows), and hepatocytes (black arrowhead) in T1−⁄−T3−⁄− portal area. (E) Immunofluorescence showing biliary duct formation. At E16.5, portal veins are surrounded by a single layer of Epcam+ cholangiocytes (yellow arrowheads) in WT, and a double layer of Epcam+ cells forming primitive ductal structures in T1−⁄−T3−⁄− embryos. Quantification of number of portal veins surrounded by Epcam+ cells in longitudinal sections of E16.5 livers (n = 6 livers per group) and number of Epcam+ cells per portal vein circumference. WT n = 6, T1−⁄−T3−⁄− n = 5 portal veins per genotype. Mann-Whitney test, *P < 0.05, **P < 0.01. (F) Immunofluorescence for SOX9, acetylated tubulin (AcT), and CK19 highlights mature duct cells. Data represent the average of the mean of mean fluorescence intensity of periportal SOX9+ nuclei. WT 11, T1−⁄−T3−⁄− 9 portal veins per genotype. Mann-Whitney test, ***P < 0.001. (G) Ten days after birth, Epcam+ cholangiocytes belong to bile ducts (yellow arrowheads) or are found in clusters scattered in the periportal area (white arrowheads). αSMA+ VSMCs highlight portal vein (PV) and arteries (HA). Bile duct/PV ratio at 10 days after birth (P10); n = 4 livers per genotype. Mann-Whitney test, *P < 0.05.