br Experimental Procedures br Author Contributions br

Experimental Procedures
Author Contributions
Acknowledgments The work was supported by the Max Planck Society. M.D.V. is a Helmholtz Association young investigator. We thank M. Saitou (Kyoto University) and M. Boiani (MPI Muenster) for ESC lines, G. Salinas (Goettingen University) for sequencing, and H. Jäckle for his hospitality.
Introduction A specific adult liver stem cell population acting as second line of defense in liver regeneration has been described in several species, including rodents, humans, canines, and felines (Wang et al., 2003; Roskams et al., 2003; Kruitwagen et al., 2014; Ijzer et al., 2009). Recently, a three-dimensional and highly proliferative organoid culture system was developed for mouse, human, and dog liver stem proton pump inhibitors list (Huch et al., 2013, 2015; Nantasanti et al., 2015). Liver organoids have been proposed as in vitro disease-modeling tool for several genetic liver diseases, such as α1-antitrypsin deficiency, Alagille syndrome, and canine copper storage disease (Huch et al., 2013, 2015; Nantasanti et al., 2015). However, many liver diseases do not have monogenetic etiology and have a more complex pathophysiology. We aimed to explore the potential of liver organoids to model non-genetic metabolic liver disease. One of the most common metabolic liver diseases in humans is liver steatosis, also known as non-alcoholic fatty liver disease (NAFLD) (Younossi et al., 2016). Interestingly, a severe type of hepatic steatosis also occurs in cats (feline hepatic lipidosis, FHL) (Center et al., 1993). Both in human and feline steatosis, hepatocyte lipid overload arises from an increased amount of free fatty acids (FFA) that are offered to the liver, and obesity and insulin resistance are known risk factors for its development (Center, 2005; Cohen et al., 2011). NAFLD can result in hepatocyte degeneration and inflammation (non-alcoholic steatohepatitis), and ultimately in excessive liver fibrosis and hepatocellular carcinoma (Cohen et al., 2011).
Results
Discussion Feline liver organoids retain characteristics similar to liver organoids of other species, including massive proliferation capacity, an epithelial nature, and their gene expression pattern (Huch et al., 2013, 2015; Nantasanti et al., 2015). Feline liver organoids were positive for progenitor/biliary markers as well as early hepatocyte specification markers, indicative of a hepatic progenitor cell phenotype (Roskams et al., 2003). In addition, in some parts or cell clusters within single organoids albumin or ZO1 was expressed, whereas the rest of the structure was negative, indicating that there are different maturation levels within an organoid. This has also been described for mouse small intestinal organoids, which harbor a crypt and villus domain representative of a stem cell pool and a more mature progeny, respectively (Sato et al., 2009). Feline liver organoid differentiation was associated with an abrupt cease in proliferation, a phenomenon also observed in cultures of primary hepatocytes, which cannot be expanded and rapidly dedifferentiate in vitro (Fraczek et al., 2013). Although feline liver organoids showed a higher expression of liver-specific genes and had increased albumin secretion and aspartate aminotransferase and CYP3A activity upon differentiation, they did not reach full maturation (e.g., they remained negative for HepPar-1). Until now it has not been possible to accomplish full hepatocyte maturation in vitro from an immature cell type, such as embryonic stem cells or induced pluripotent stem cells (Ochiya et al., 2010; Zhang et al., 2013), nor to maintain maturation status of primary hepatocytes in culture (Fraczek et al., 2013). Future research is needed to elucidate pathways that are important for terminal differentiation of hepatocytes. We compared several liver organoid species (mouse, human, dog, and cat), and in all observed lipid accumulation when liver organoids were provided with FFA. Oleate and palmitate represent the most abundant fatty acid species in healthy and steatotic human and cat liver (Araya et al., 2004; Fujiwara et al., 2015). Hence, oleate and palmitate are widely used in lipidomics research and are considered physiologically relevant for modeling hepatic lipid accumulation (Gómez-Lechón et al., 2007). Hepatocytes have three major routes to handle FFA. They can (1) enter β-oxidation to provide energy or a substrate for ketogenesis, or they can be re-esterified to triglycerides, and either (2) become excreted in very-low-density lipoproteins (VLDL) or (3) be stored as intracellular lipid droplets. There were marked species differences in the extent of lipid accumulation on a cellular level: feline liver organoids accumulated more lipids than did human liver organoids. This exaggerated phenotype of lipid overload in feline liver cells complies with the fact that steatosis in cats often leads to liver failure and severe disease (Center, 2005). We can speculate that the other metabolic pathways handling excess FFA (β-oxidation, VLDL secretion) are quickly saturated in feline hepatocytes, leading to extensive lipid-droplet formation. Both human and feline organoids upregulated PLIN2 expression after FFA treatment, an essential machinery protein in lipid accumulation in lipid droplets. However, we also observed differences in transcriptional activation between human and feline organoids after FFA treatment, which could be explained by differences in activation of essential lipid regulatory pathways (mainly via PPARA and PPARG). Future research might focus on species differences in PPARA and PPARG signaling in response to excess FFA and their effects on hepatocellular lipid catabolism and storage.