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 2

Increased Notch activation in the absence of TIMP1 and TIMP3.

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Increased Notch activation in the absence of TIMP1 and TIMP3. (A) UMAP v...
(A) UMAP visualization of isolated hepatic cells, based on 47,752 single-cell transcriptomes pooled from control (10,636), CCl4-treated (18,185), and bile duct–ligated mice (18,931) from the Yang et al. 2021 mouse dataset (28). (B) UMAP obtained for each condition. (C) Violin plots showing the expression of Timp, Adam, Notch ligands, receptors, and target genes in the hepatic cell populations defined in A, in control, CCl4-treated, and after bile duct ligation (BDL). (D) Western blot for cleaved Notch1 (NICD1) and Notch2 (NICD2; band density quantification, 2-tailed Student’s t test) in postnatal (P10) livers, and in 12-week-old livers. Notch1 cleavage correlates with severity of portal triad alterations (size of the dot) observed by Masson’s trichrome staining depicted in Figure 1B (based on collagen deposition and portal triad distortion). Black lines delineate lanes that were run on the same gel but were non-contiguous. (E) Expression of Notch target genes (n ≥ 11 livers) and Notch receptors and ligands (n ≥ 3 livers) in 12-week-old mice. *P < 0.05, **P < 0.01, 2-tailed Student’s t test. For Western blots, each lane represents 1 animal.