Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis.

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research

Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog

Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research

Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research

Aberrant hepatic lipid storage and metabolism in canine portosystemic shunts

<div><p>Non-alcoholic fatty liver disease (NAFLD) is a poorly understood multifactorial pandemic disorder. One of the hallmarks of NAFLD, hepatic steatosis, is a common feature in canine congenital portosystemic shunts. The aim of this study was to gain detailed insight into the pathogenesis of steatosis in this large animal model. Hepatic lipid accumulation, gene-expression analysis and HPLC-MS of neutral lipids and phospholipids in extrahepatic (EHPSS) and intrahepatic portosystemic shunts (IHPSS) was compared to healthy control dogs. Liver organoids of diseased dogs and healthy control dogs were incubated with palmitic- and oleic-acid, and lipid accumulation was quantified using LD540. In histological slides of shunt livers, a 12-fold increase of lipid content was detected compared to the control dogs (EHPSS <i>P</i><0.01; IHPSS <i>P</i> = 0.042). Involvement of lipid-related genes to steatosis in portosystemic shunting was corroborated using gene-expression profiling. Lipid analysis demonstrated different triglyceride composition and a shift towards short chain and omega-3 fatty acids in shunt versus healthy dogs, with no difference in lipid species composition between shunt types. All organoids showed a similar increase in triacylglycerols after free fatty acids enrichment. This study demonstrates that steatosis is probably secondary to canine portosystemic shunts. Unravelling the pathogenesis of this hepatic steatosis might contribute to a better understanding of steatosis in NAFLD.</p></div

Organoid LD540 fluorescent whole-mount staining.

<p>Representative images of LD540 fluorescent whole-mount staining of control (<i>n</i> = 4), extrahepatic (EHPSS, <i>n</i> = 4) and intrahepatic (IHPSS, <i>n</i> = 4) canine organoids cultured in a bovine serum albumin (BSA) medium (control) or the enriched free fatty-acid (FFA) medium for 24 hours (A). Blue represents DAPI–nuclei, green represents LD540 labelled TAGs. The accumulation of LD540 in lipid droplets is more pronounced in the FFA cultured organoids as displayed in a section (A). Quantitative measurement of the total intracellular lipid accumulation was ascertained by flow cytometry analysis (B). Data is expressed as percentage increase in median fluorescent intensity of cells in FFA containing media compared to BSA control (<i>n</i> = 4).</p

Principal component analysis of phospholipid species.

<p>PCA of phospholipid species in hepatic biopsies of healthy (red, <i>n</i> = 4), EHPSS (green, <i>n</i> = 7), and IHPSS (blue, (<i>n</i> = 5) dogs. Resulting scores of the samples (A) using the calculated loadings (B). Lipids are colored according to their lipid class and dot sizes correspond to relative abundance. Degree of unsaturation found in the acyl chains of PL (C). Note the higher levels of acyl chains with four unsaturations in control dogs, at the expense of acyl chains with six unsaturations.</p

Average lipid intensity using an Oil-red-O staining in hepatic tissue of control, EHPSS and IHPSS dogs.

<p>Representative pictures of the hepatic samples from healthy dogs (<i>n</i> = 4), dogs with extrahepatic portosystemic shunts (EHPSS, <i>n</i> = 7), and intrahepatic portosystemic shunts (IHPSS, <i>n</i> = 5) are displayed left (A). The average Oil-red-O intensity is displayed in Log₁₀ per sample group, representing neutral lipid staining in the observed liver samples calculated with a Students T-test and <i>P</i> < 0.05 was considered significant (B).</p