Spatially segregated defects and <scp>IGF1</scp>‐responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome

Iqbal, Afshan; Van Hul, Noemi; Belicova, Lenka; Corbat, Agustin A.; Hankeova, Simona; Andersson, Emma R.

doi:10.1111/liv.15789

Cited by 3 publications

(1 citation statement)

References 69 publications

(155 reference statements)

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“…This is one of key bottlenecks in organoid-based compound screening. Recently, Sun Bioscience developed Gri3D ® microwell array plate 56 for high-throughput organoid culture and imaging for compound testing. Although it is a significant advancement in the field, this platform only supports static culture of organoids, which don't require ECM encapsulation.…”

Section: Discussionmentioning

confidence: 99%

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Shrestha,

Acharya,

Kang

et al. 2024

Preprint

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Human liver organoids (HLOs) differentiated from embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) can recapitulate structure and function of human fetal liver tissues, thus, considered as a promising tissue model for liver diseases and predictive compound screening. Nonetheless, there are still several technical challenges to adopt HLOs in the drug discovery process, which include relatively long-term cell differentiation with multiple culture media (3 - 4 weeks) leading to batch-to-batch variation, short-term hepatic function after maturation (3 - 5 days), low assay throughput due to Matrigel dissociation and HLO transfer to a microtiter well plate, and insufficient maturity as compared to primary hepatocytes. To address these issues, expandable HLOs (Exp-HLOs) derived from human iPSCs were generated by optimizing differentiation protocols, which were rapidly printed on a 144-pillar plate with sidewalls and slits (144PillarPlate) and dynamically cultured for up to 20 days into differentiated HLOs (Diff-HLOs) in a 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for in situ organoid culture and analysis. Dynamically cultured Diff-HLOs were generated robustly and reproducibly in the pillar/perfusion plate with higher maturity as compared to those in statically cultured HLOs by differentiating Exp-HLOs for 10 days. In addition, Diff-HLOs in the pillar/perfusion plate were tested with acetaminophen and troglitazone for 3 days to assess drug-induced liver injury (DILI) and then incubated in an expansion medium for 10 days to evaluate the recovery of the liver from DILI. The assessment of liver regeneration post injury is critical to understand the mechanism of recovery and determine the threshold drug concentration beyond which there will be a sharp decrease in the liver's regenerative capacity. We envision that bioprinted Diff-HLOs in the pillar/perfusion plate could be used for high-throughput screening (HTS) of hepatotoxic compounds due to short-term differentiation of passage-able Exp-HLOs necessary, stable hepatic function after maturation, high reproducibility, and high throughput with capability of in situ organoid culture, testing, staining, imaging, and analysis.

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Section: Discussionmentioning

confidence: 99%

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Shrestha,

Acharya,

Kang

et al. 2024

Preprint

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Jag1 insufficiency alters liver fibrosis via T cell and hepatocyte differentiation defects

Mašek,

Filipovic,

Van Hul

et al. 2024

EMBO Mol Med

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Fibrosis contributes to tissue repair, but excessive fibrosis disrupts organ function. Alagille syndrome (ALGS, caused by mutations in JAGGED1) results in liver disease and characteristic fibrosis. Here, we show that Jag1Ndr/Ndr mice, a model for ALGS, recapitulate ALGS-like fibrosis. Single-cell RNA-seq and multi-color flow cytometry of the liver revealed immature hepatocytes and paradoxically low intrahepatic T cell infiltration despite cholestasis in Jag1Ndr/Ndr mice. Thymic and splenic regulatory T cells (Tregs) were enriched and Jag1Ndr/Ndr lymphocyte immune and fibrotic capacity was tested with adoptive transfer into Rag1−/− mice, challenged with dextran sulfate sodium (DSS) or bile duct ligation (BDL). Transplanted Jag1Ndr/Ndr lymphocytes were less inflammatory with fewer activated T cells than Jag1+/+ lymphocytes in response to DSS. Cholestasis induced by BDL in Rag1−/− mice with Jag1Ndr/Ndr lymphocytes resulted in periportal Treg accumulation and three-fold less periportal fibrosis than in Rag1−/− mice with Jag1+/+ lymphocytes. Finally, the Jag1Ndr/Ndr hepatocyte expression profile and Treg overrepresentation were corroborated in patients’ liver samples. Jag1-dependent hepatic and immune defects thus interact to determine the fibrotic process in ALGS.

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Jag1 Insufficiency Disrupts Neonatal T Cell Differentiation and Impairs Hepatocyte Maturation, Leading to Altered Liver Fibrosis

Mašek

Filipovic

Hankeová³

et al. 2022

Preprint

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Background and Aims: Alagille syndrome (ALGS) is a pediatric genetic disorder, caused by mutations in the Notch ligand JAGGED1, presenting with cholestasis due to intrahepatic bile duct paucity. Despite chronic liver disease, few patients develop severe fibrosis or liver cancer, compared to other chronic liver diseases. In contrast, about 1/3 of ALGS patients suffer from reoccurring infections. Notch signaling regulates both liver and immune system development, but how immune system defects interact with liver pathology in ALGS to influence disease course is not known. Here, we aimed to determine whether a mouse model of ALGS mimics fibrosis and liver cancer, and whether ALGS immune system compromise could positively modulate inflammation or liver disease course. Methods: We used single cell RNA sequencing and 25-color flow cytometry to characterize cell populations in embryonic and postnatal liver in an ALGS mouse model (Jag1Ndr/Ndr mice). We analyzed liver cancer prevalence in Jag1Ndr/Ndr mice, in a cancer-prone genetic background, and thymic and spleen cell composition using flow cytometry. Finally, to test the functionality of the Jag1Ndr/Ndr immune system we performed adaptive immune cell transfer experiments into Rag1-/- mice and assessed inflammatory capacity in an ulcerative colitis model and in a DDC model of hepatocellular damage. Results: Adult Jag1Ndr/Ndr mice do not develop liver tumors (0%) while 45% of control mice do. Interestingly, Jag1Ndr/Ndr mice display ALGS-like chicken-wire hepatocellular fibrosis concomitant with mild inflammation shortly after the onset of liver cholestasis. Comprehensive single cell analysis revealed that liver CD4+ and CD8+ T cells of Jag1Ndr/Ndr mice are dysregulated in favor of CD4+, and Regulatory T-cells (Tregs) manifest reduced activation. Trans-plantation experiments show no difference between Jag1Ndr/Ndr and Jag1+/+ lymphocytes in DDC-induced liver damage. In contrast, Jag1Ndr/Ndr lymphocytes do not mount an inflammatory response to bacterial infection. Conclusion: Here we report immune dysregulation and cancer protection in the hypomorphic Jag1 mouse model of ALGS. Disrupted thymocyte differentiation coincides with limited activation of hepatocytes, blunting the inflammatory response to cholestasis. This may contribute to the pericellular fibrosis and prevent carcinogenesis. These results prompt the question of the relative contribution of Jag1 to liver cell pathology vs immune system in ALGS and high-light the need of future studies focused on dissecting apart how Jag1 modulates susceptibility to infection versus cancer risk.

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Spatially segregated defects and IGF1‐responsiveness of hilar and peripheral biliary organoids from a model of Alagille syndrome

Cited by 3 publications

References 69 publications

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Regenerative human liver organoids (HLOs) in a pillar/perfusion plate for hepatotoxicity assays

Jag1 insufficiency alters liver fibrosis via T cell and hepatocyte differentiation defects

Jag1 Insufficiency Disrupts Neonatal T Cell Differentiation and Impairs Hepatocyte Maturation, Leading to Altered Liver Fibrosis

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