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 5

ADAMs and DLK1 are coexpressed in periportal mesenchymal cells.

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ADAMs and DLK1 are coexpressed in periportal mesenchymal cells. (A) UMAP...
(A) UMAP visualization of isolated hepatic mesenchymal cells, based on 23,689 PDGFRβ+ single-cell transcriptomes pooled from control mice (12,587 cells) and after 6 weeks CCl4 treatment (11,102 cells) from the Dobie et al. 2019 mouse dataset (52) (HSC, hepatic stellate cell; VSMC, vascular smooth muscle cell). (B) UMAP visualization of control and CCl4-treated cells. (C) Density plots showing expression of Dlk1, Adam10, and Adam17 in liver mesenchymal cell populations described in A. (D) Correlation plots representing fibroblasts expressing Dlk1, Adam17, and Adam10. The gray area contains cells coexpressing Dlk1/Adam17 and Dlk1/Adam10. (E) Scatterplots showing expression level of Dlk1, Adam10, and Adam17 in fibroblasts, HSCs, and VSMCs. Percentages indicate the number of cells expressing the gene of interest among the total cell population. (F) Representative RNAscope and immunofluorescence images of biliary ducts from WT and T1−⁄−T3−⁄− untreated adult livers (n = 3). CK19+ biliary cells forming ducts are delineated with yellow dashed lines. Yellow arrows point to Dlk1, Adam10, or Adam17 RNAscope positive staining in vimentin-positive (Vim) mesenchymal cells. (G) Immunofluorescence image of WT and T1−⁄−T3−⁄− portal triad showing CK19+ cholangiocyte, CD34+ fibroblast, and αSMA+ VSMC (PV, portal vein; HA, hepatic artery; BD, bile duct). (H) Western blot for cleaved DLK1 (sDLK1, 50 kDa) and SOX9 in postnatal and adult livers; each lane represents 1 animal. (I) Expression of Dlk1 in liver tissue following CCl4 or DDC injury (n ≥ 3). One-way ANOVA with Šidák’s multiple-comparison test, *P < 0.05.