Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis
Ryan E. Feaver, … , Brian R. Wamhoff, Ajit Dash
Ryan E. Feaver, … , Brian R. Wamhoff, Ajit Dash
Published December 8, 2016
Citation Information: JCI Insight. 2016;1(20):e90954. https://doi.org/10.1172/jci.insight.90954.
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Resource and Technical Advance Hepatology Therapeutics

Development of an in vitro human liver system for interrogating nonalcoholic steatohepatitis

Abstract

A barrier to drug development for nonalcoholic steatohepatitis (NASH) is the absence of translational preclinical human-relevant systems. An in vitro liver model was engineered to incorporate hepatic sinusoidal flow, transport, and lipotoxic stress risk factors (glucose, insulin, free fatty acids) with cocultured primary human hepatocytes, hepatic stellate cells (HSCs), and macrophages. Transcriptomic, lipidomic, and functional endpoints were evaluated and compared with clinical data from NASH patient biopsies. The lipotoxic milieu promoted hepatocyte lipid accumulation (4-fold increase, P < 0.01) and a lipidomics signature similar to NASH biopsies. Hepatocyte glucose output increased with decreased insulin sensitivity. These changes were accompanied by increased inflammatory analyte secretion (e.g., IL-6, IL-8, alanine aminotransferase). Fibrogenic activation markers increased with lipotoxic conditions, including secreted TGF-β (>5-fold increase, P < 0.05), extracellular matrix gene expression, and HSC activation. Significant pathway correlation existed between this in vitro model and human biopsies. Consistent with clinical trial data, 0.5 μM obeticholic acid in this model promoted a healthy lipidomic signature, reduced inflammatory and fibrotic secreted factors, but also increased ApoB secretion, suggesting a potential adverse effect on lipoprotein metabolism. Lipotoxic stress activates similar biological signatures observed in NASH patients in this system, which may be relevant for interrogating novel therapeutic approaches to treat NASH.

Authors

Ryan E. Feaver, Banumathi K. Cole, Mark J. Lawson, Stephen A. Hoang, Svetlana Marukian, Brett R. Blackman, Robert A. Figler, Arun J. Sanyal, Brian R. Wamhoff, Ajit Dash

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Figure 1

Adaptation of the in vitro human liver system to mimic human nonalcoholic fatty liver under lipotoxic stress.

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Adaptation of the in vitro human liver system to mimic human nonalcoholi...
(A) Liver sinusoidal hemodynamics were applied to the human liver system using a cone-and-plate viscometer incorporated into a transwell multiculture model of nonparenchymal cells (NPCs) (top of transwell) and hepatocytes (bottom of the transwell). Rotation of the cone (orange triangle) imparts shear stress onto the transwell. Medium is continually perfused to recapitulate interstitial flow, as indicated by the inflow and outflow ports. (B) Representative photomicrographs (original magnification, ×20) of NPCs and hepatocytes are shown. NPCs include hepatic stellate cells (reelin+, green) and macrophages (CD68+, red). Hepatocytes are stained for E-cadherin (green). Nuclei stained with DAPI (blue). Scale bars: 100 μm and 50 μm for NPCs and hepatocytes, respectively. (C) Hepatocyte protein-protein interaction network was visualized and colored according to signaling community. (D) The relative directionality of many pathways identified in C were explored by calculating the FDR-scaled fold change for each, revealing key signaling pathways perturbed at the RNA level by lipotoxic stress. *Rotation gene set tests (ROAST), FDR < 5%.