Exhibition at the EASL Congress 2024

Background and aims: Alcoholic fatty liver (AFL) and metabolic dysfunction-associated fatty liver (MAFL), representing the initial stages of steatotic liver disease, serve as prerequisites for disease progression to advanced stages. ALF and MAFL are distinguished solely by clinical context as these conditions share histological and radiographic features, aswell as genetic risk factors. Hence, it remains elusive whether they represent distinctive or shared pathophysiology. This study aims to characterize the hepatocytic response to the metabolic stress of AFL by contrasting it with that of MAFL, with the goal of advancing our understanding of how AFL primes the development of advanced stages of alcoholic liver disease (ALD). Method: Human hepatocytes (HH), either primary or derived from humanized liver chimeric mice (HLCM), were seeded onto collagen-coated plates and cultured with DMSO-containing medium for 7 days, promoting the reinstatement of genuine characteristics. Subsequently, HH were cultured with either ethanol (EtOH) or a free fatty acid-sugar cocktail for an additional 7 days, during which time DMSO was replaced with DMSO2 due to its potent inhibitory effect on a multitude of hepatic metabolic enzymes. were characterized via mRNA-seq, ribosome profiling, and quantitative proteomics to decipher the impact of metabolic stresses on the transcriptome, translatome, and proteome. The results obtained from in vitro studies were validated through in vivo experiments with HLCM. Results: In vitro cultured HH exhibits the efficient EtOH metabolism to acetate, the final metabolite, at a rate comparable that of the human liver. In parallel, HH treated with EtOH developed macrovesicular steatosis at a comparable level to AFL liver tissue. HH treated with the free fatty acid-sugar cocktail also developed macrovesicular steatosis to an extent indistinguishable from AFL-HH. Despite the identical appearance, mRNA-seq analysis revealed substantial differences in the number and type of differentially expressed genes, with only a small proportion exhibiting overlap. Furthermore, polysome profiling of AFL-HH but not MAFL-HH revealed substantial dissociation of mRNAs from ribosomes, indicating that metabolic stress in AFL results in potent suppression of translatome. Subsequent in vitro and in vivo biochemical and protein studies indicate that the translatome perturbation in AFL-HH is largely attributed to an impairment of elongation processes rather than initiation machineries. We also found that the translatome deficiency results in the sublethal activation of cell death pathways in AFL-HH but not in MAFL-HH upon exposure to inflammatory cytokines. Conclusion: Our work revealed a marked translatome perturbation at the elongation stage and consequential cell toxicity in HH of AFL, uncovering a previously unrecognized pathophysiology of ALD.

Background and aims: In vitro culture of human hepatocytes (HH) results in rapid quality deterioration, attributed to both the injury inflicted during cell procurement processes and the absence of a tissue-specific microenvironment. Our refined 2D culture approach, however, demonstrates that freshly isolated single-cell suspension HH undergo dynamic processes of in vitro wound healing, resulting in the reinstatement of genuine characteristics. These processes include the restoration of tight junctions, proper cellular polarity, morphological architecture, and the expression of hepatocyte marker genes at levels comparable to those observed in liver tissue. Our work also shows that the degree of fate restoration and maintenance is inversely correlated with the frequency of culture medium replacement, indicating the involvement of hepatocyte-derived humoral factors in this process. This study aims to decipher the autocrine role of hepatocyte-intrinsic humoral factors in determining the cell fate of HH. Method: HH, either primary or derived from humanized liver chimeric mice (HLCM), were seeded onto collagen-coated plates and cultured for 14 days in medium containing hepatocyte-specific supplements. Subsequently, the culture medium was pooled as the conditioned medium (CM), and its impact on the fate restoration and maintenance of freshly isolated HH was compared with that of fresh medium (FM) via transcriptome analysis to identify responsible pathways, and loss of function approaches were employed for validation. Immunofluorescent and electron microscopic analyses were also conducted to characterize the impact of the conditioned medium (CM) on cellular and extracellular architectures. Lastly, HH cultured with the CM or FM were transplanted into uPA/SCID mice to determine their fate status by assessing the capacity to establish HLCM. Results: The transcriptome of HH cultured with the CM exhibits a pattern highly resembling that of freshly isolated HH, normal human liver tissue, aswell as the liver of HLCM. In contrast, HH cultured with FM showa significant upregulation of the TGFβ-YAP/TAZ pathway, as well as signature genes indicative of dedifferentiation or transdifferentiation to cholangiocytes. Microscopic analyses reveal that CM treatment promotes the accumulation of extracellular matrix (ECM) composed of fibrin, fibronectin, laminin, and vitronectin, preventing HH from developing morphological deformities. Studies employing a loss-of-function approach indicate that ECM formation is the key fate-protective effect of the CM. Lastly, the transplantation of HH cultured with the CM, but not with FM into HLCM host mouse showed efficient engraftment. Conclusion: This study demonstrates the critical role of hepatocytederived humoral factors in shaping the fate of in vitro cultured HH by establishing ECM that mimics the liver tissue microenvironment.