The lack of registered drugs for nonalcoholic fatty liver disease (NAFLD) is partly due to the paucity of human-relevant models for target discovery and compound screening. Here we use human fetal hepatocyte organoids to model the first stage of NAFLD, steatosis, representing three different triggers: free fatty acid loading, interindividual genetic variability (PNPLA3 I148M) and monogenic lipid disorders (APOB and MTTP mutations). Screening of drug candidates revealed compounds effective at resolving steatosis. Mechanistic evaluation of effective drugs uncovered repression of de novo lipogenesis as the convergent molecular pathway. We present FatTracer, a CRISPR screening platform to identify steatosis modulators and putative targets using APOB−/− and MTTP−/− organoids. From a screen targeting 35 genes implicated in lipid metabolism and/or NAFLD risk, FADS2 (fatty acid desaturase 2) emerged as an important determinant of hepatic steatosis. Enhancement of FADS2 expression increases polyunsaturated fatty acid abundancy which, in turn, reduces de novo lipogenesis. These organoid models facilitate study of steatosis etiology and drug targets.

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.