Abstract
BackgroundNon-alcoholic fatty liver disease (NAFLD) begins as simple hepatic steatosis, but further progress to chronic liver diseases results in severe liver damage and hepatic failure. However, therapeutic options are scarce due to the lack of reliable human in vitro liver models for understanding disease progression mechanisms and developing therapies.ResultsWe describe here a novel method for generating 3D hepatic spheroids using HepaRG cells, vascular endothelial cells, and mesenchymal stem cells cultured on a thick layer of soft matrix in a narrow conical tube; this method improved self-organization efficiency and functional competence. We further developed a 3D hepatic steatosis model with excess glucose and palmitate, accurately recapitulating steatosis phenotypes such as neutral lipid accumulation, enhanced expression of lipogenesis and gluconeogenesis markers, increased intracellular triglyceride content, and reduced glucose uptake. The expression and activity of cytochrome P450 4A (CYP4A), a hepatic glucose and lipid homeostasis enzyme, that is highly expressed in liver tissues from NAFLD patients, was induced in our in vitro steatosis model, and inhibiting CYP4A with the selective inhibitor HET0016 or a specific siRNA ameliorated steatosis-related pathology through reduced ER stress and improved insulin signaling.ConclusionsWe provide here a novel 3D human cell-based hepatic model that can be easily generated and reliably simulate hepatic steatosis pathology. We have experimentally validated its potential for target validation and drug evaluation by focusing on CYP4A, which may serve as a translational platform for drug development.
Highlights
Non-alcoholic fatty liver disease (NAFLD) begins as simple hepatic steatosis, but further progress to chronic liver diseases results in severe liver damage and hepatic failure
We previously identified 54 cytochrome P450 (CYP450) proteins that were upregulated in db/db and high-fat diet (HFD) diabetic mouse livers compared with normal mice and found that the CYP4a family, which includes important enzymes in lipid homeostasis that catalyze omega-hydroxylation of endogenous fatty acids and prostaglandin [20], was the most enriched isoform [21]
A novel method for spontaneous self-organization of 3D hepatic spheroids A self-organization protocol had been previously described in which hepatocytes derived from primary human tissue [27] or from pluripotent stem cells [28] were co-assembled with supporting cells on a thick layer of a soft matrix, such as Matrigel, in a 24-well plate
Summary
Non-alcoholic fatty liver disease (NAFLD) begins as simple hepatic steatosis, but further progress to chronic liver diseases results in severe liver damage and hepatic failure. Therapeutic options are scarce due to the lack of reliable human in vitro liver models for understanding disease progression mechanisms and developing therapies. Non-alcoholic fatty liver disease (NAFLD) starts with hepatic steatosis and can progress to chronic liver diseases, including non-alcoholic. Various mouse models of NAFLD, including dietary, chemical, and genetic models, are used in preclinical drug development [9], there is a need for human cell-based liver models that would provide additional and more refined alternatives to animal experiments. A hepatic model predicting human responses for drug evaluation is urgently needed, and patient-derived primary human hepatocytes (PHHs) are the ideal model for testing drug targets. HepaRG cells, a liver progenitor cell line that is widely used as an alternative and sustainable cell source for hepatocytes, proliferate and exhibit long-term phenotypical and functional stability after differentiation [11,12,13]. We provide a novel method to efficiently generate a multicellular 3D organotypic human hepatic steatosis model and further validate the feasibility of this platform for mechanism studies and for the validation of drugs targeting hepatic steatosis
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