The European Association for the Study of the Liver (EASL) Congress 2024 presents a diverse scientific programme tailored to various specialties and levels of expertise in hepatology, including: metabolism, alcohol, toxicity; cirrhosis; liver tumors; viral hepatitis; general hepatology; and more! 

PhoenixBio is excited to be sponsoring a booth and attending the EASL Congress 2024 in person in Milan, Italy on June 5th - 8th, 2024. Meet with our team at Booth #O2 to learn more about PhoenixBio's capabilities and how we can help you advance and deliver tomorrow’s technologies. 

While you're here, be sure to attend humanized liver chimeric mouse model related abstracts featured below and grab a chance to speak with our team!

Humanized Liver Related Abstracts:

THU-319: Hepatocyte response to metabolic stress in alcoholic fatty liver leads to sublethal cell toxicity through the perturbation of protein translation-elongation machinery

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.

FRI-540: Role of hepatocyte-intrinsic humoral factors in establishing bioscaffold that regulates the fate determination of primary hepatocytes

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.

SAT-364: Mathematical modeling of hepatitis B and D viral kinetics during coinfection in humanized mice suggests differences in their infectivity

Background and aims: Understanding of hepatitis D virus (HDV) and hepatitis B virus (HBV) dynamics during acute co-infection in humans is lacking due to the uncertainty of the exact time of infection and sparse kinetic data. We aim to provide insights into HDV/HBV acute co-infection by studying frequently sampled kinetic data in uPA/SCID mice with humanized livers from inoculation to steady state using a mathematical modeling approach. Method: 4 micewere inoculated with co-infected serum of HDV RNA (6.0 log cp/ml) and HBV DNA (6.0 log cp/ml). HBV DNA and HDV RNA levels were frequently measured from blood samples up to 12 weeks post infection. Using the model selection theory, we identified a mathematical model that best reproduced the observed serum HBV and HDV kinetics. The model incorporated four different states of human hepatocytes (termed cells), (i) uninfected, (ii) HBV monoinfected, (iii) HDV mono-infected, and (iv) HBV/HDV co-infected. Cellswere allowed to transition into infectious states by one virus at a time. Furthermore, in HBV and HBV/HDV infected cells, intracellular processes leading to the production of HBV virions was described as in [Hepatology 2023, 78 (Suppl.1): S530–S531]. This intracellular model included saturation in pre-genomic RNA (pgRNA) production from covalently closed circular DNA (cccDNA), and linear production rates of relaxed circular DNA (rcDNA) from pgRNA and rcDNA secretion. In contrast, HDV virions were assumed to be produced by co-infected cells at a constant rate. Several models exploring the impact on intracellular HBV processes by HDV in co-infected cells as well as differences in infectivity for different cell-infection status were tested. We fit the models to the observed serum viral data using a population, nonlinear mixed-effects approach. Results: The serum kinetics of HBV and HDV in all 4 humanized mice was successfully recapitulated using the best model which assumed different infectivity based on virus and cell type. HDV infectivity of uninfected cells (2 × 10−11 day−1.copies−1) is estimated to be ∼40-fold lower compared to HBV infectivity of uninfected cells (8 × 10−10 day−1.copies−1). Moreover, HBV infectivity of HDV monoinfected cells (1.7 × 10−3 day−1.copies−1) is estimated to be manyfold higher compared to HDV infectivity of HBV mono-infected cells (2.6 × 10−9 day−1.copies−1). Analysis also suggests that due to the presence of HDV, cccDNA per infected cell accumulates to a lower level in co-infected cells (∼1.9 copies) compared to HBV monoinfected cells (∼5 copies). All parameterswere identifiable in the best model. Conclusion: Modeling suggests that different HBV and HDV infectivity of uninfected and mono-infected cells plays a pivotal role in reproducing the observed viral kinetics from infection to steady state in humanized mice.

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