Metalloproteinase inhibitors regulate biliary progenitor cells through sDLK1 in organoid models of liver injury
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
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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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 4

Timp1 and Timp3 loss impairs liver organoid formation.

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Timp1 and Timp3 loss impairs liver organoid formation. (A) Schematic of...
(A) Schematic of biliary fragment–derived liver organoid cultures. (B) Bright-field images of organoids, maximum diameter, and number of organoids per Matrigel dome during 4 days of culture; n = 5 livers. Two-way ANOVA with Šidák’s multiple-comparison test. (C) Images depict aberrations observed in T1−⁄−T3−⁄− organoids. Top middle, cell aggregates and absence of lumen; bottom middle, thickening of outer cell layer; top and bottom right, asymmetric growth. (D) Gene expression for progenitor marker Prom1 and proliferation (Ccnd1, Mki67) during organoid growth; n = 3 livers per group. Two-tailed Student’s t test. (E) Immunofluorescence for markers of biliary cells (Epcam) and mature cholangiocytes (osteopontin, OPN) at day 4 culture of WT and T1−⁄−T3−⁄− organoids. Image inset in T1−⁄−T3−⁄− organoids panel shows OPN apical expression in bile duct (BD) from liver tissue; asterisk highlights lumen. (F) RT-PCR for cell population markers; n = 3 livers. Two-tailed Student’s t test. (G) Schematic of biliary fragment–derived primary organoids and their subsequent passages. Gene expression profiling of primary and passaged organoids; n ≥ 3 livers. Ratio paired t test, *P < 0.05, **P < 0.01, ***P < 0.001.