Modeling the personalized variations in liver disease due to α1-antitrypsin deficiency using induced pluripotent stem cell-derived hepatocyte-like cells

In the classical form of α1-antitrypsin deficiency (ATD), aberrant intracellular accumulation of misfolded mutant α1-antitrypsin Z (ATZ) in hepatocytes causes hepatic damage by a gain-of-function, “proteotoxic” mechanism. Whereas some ATD patients develop severe liver disease that necessitates liver transplantation, others with the same genetic defect completely escape this clinical phenotype. We investigated whether induced pluripotent stem cells (iPSCs) from ATD individuals with or without severe liver disease could model these personalized variations in hepatic disease phenotypes. Patient-specific iPSCs were generated from ATD patients and a control, and differentiated into hepatocyte-like cells (iHeps) having many characteristics of hepatocytes. Pulse-chase and endoglycosidase H analysis demonstrate that the iHeps recapitulate the abnormal accumulation and processing of the ATZ molecule, compared to the wild-type AT molecule. Measurements of the fate of intracellular ATZ show a marked delay in the rate of ATZ degradation in iHeps from severe liver disease patients, compared to those from no liver disease patients. Transmission electron microscopy showed dilated rough endoplasmic reticulum in iHeps from all individuals with ATD, not in controls, but globular inclusions that are partially covered with ribosomes were observed only in iHeps from individuals with severe liver disease. These results provide definitive validation that iHeps model the individual disease phenotypes of ATD patients with more rapid degradation of misfolded ATZ and lack of globular inclusions in cells from patients who have escaped liver disease. The results support the concept that “proteostasis” mechanisms, such as intracellular degradation pathways, play a role in observed variations in clinical phenotype and show that iPSCs can potentially be used to facilitate predictions of disease susceptibility for more precise and timely application of therapeutic strategies

Cellular Location of HNF4α is Linked With Terminal Liver Failure in Humans

Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in hepatocyte function, and HNF4α-based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4α RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4α localization and the pathways involved in post-translational modification of HNF4α in human hepatocytes from patients with decompensated liver function. RNA-sequencing analysis revealed that AKT-related pathways, specifically phospho-AKT, is down-regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4α were significantly reduced, and cytoplasmic expression of HNF4α was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho-AKT to nuclear localization of HNF4α was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4α acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET-AKT pathway directly correlate with HNF4α localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4α and pathways involved in HNF4α posttranslational modification may restore hepatocyte function in patients with terminal liver failure.Fil: Florentino, Rodrigo M.. Univeristy of Pittsburgh. School of Medicine; Estados Unidos. Universidade Federal de Minas Gerais; BrasilFil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Morita, Kazutoyo. University of Pittsburgh at Johnstown; Estados UnidosFil: Takeishi, Kazuki. University of Pittsburgh at Johnstown; Estados UnidosFil: Ostrowska, Alina. University of Pittsburgh at Johnstown; Estados UnidosFil: Achreja, Abhinav. Michigan State University; Estados UnidosFil: Animasahun, Olamide. Michigan State University; Estados UnidosFil: Haep, Nils. University of Pittsburgh at Johnstown; Estados UnidosFil: Arazov, Shohrat. University of Pittsburgh at Johnstown; Estados UnidosFil: Agarwal, Nandini. University of Pittsburgh at Johnstown; Estados UnidosFil: Collin de lHortet, Alexandra. University of Pittsburgh at Johnstown; Estados UnidosFil: Guzman Lepe, Jorge. University of Pittsburgh at Johnstown; Estados UnidosFil: Tafaleng, Edgar N.. University of Pittsburgh at Johnstown; Estados UnidosFil: Mukherjee, Amitava. University of Pittsburgh at Johnstown; Estados UnidosFil: Troy, Kris. University of Pittsburgh at Johnstown; Estados UnidosFil: Banerjee, Swati. University of Pittsburgh at Johnstown; Estados UnidosFil: Paranjpe, Shirish. University of Pittsburgh at Johnstown; Estados UnidosFil: Michalopoulos, George K.. University of Pittsburgh at Johnstown; Estados UnidosFil: Bell, Aaron. University of Pittsburgh at Johnstown; Estados UnidosFil: Nagrath, Deepak. Michigan State University; Estados UnidosFil: Hainer, Sarah J.. University of Pittsburgh at Johnstown; Estados UnidosFil: Fox, Ira J.. University of Pittsburgh at Johnstown; Estados UnidosFil: Soto Gutierrez, Alejandro. University of Pittsburgh at Johnstown; Estados Unido

Human Hepatocellular response in Cholestatic Liver Diseases

ABSTRACTPrimary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), the most common types of cholestatic liver disease (CLD), result in enterohepatic obstruction, bile acid accumulation, and hepatotoxicity. The mechanisms by which hepatocytes respond to and cope with CLD remain largely unexplored. This study includes the characterization of hepatocytes isolated from explanted livers of patients with PBC and PSC. We examined the expression of hepatocyte-specific genes, intracellular bile acid (BA) levels, and oxidative stress in primary-human-hepatocytes (PHHs) isolated from explanted livers of patients with PBC and PSC and compared them with control normal human hepatocytes. Our findings provide valuable initial insights into the hepatocellular response to cholestasis in CLD and help support the use of PHHs as an experimental tool for these diseases

Polymorphisms Associated With Metabolic Dysfunction-Associated Steatotic Liver Disease Influence the Progression of End-Stage Liver Disease

Background and Aims: Chronic liver injury that results in cirrhosis and end-stage liver disease (ESLD) causes more than 1 million deaths annually worldwide. Although the impact of genetic factors on the severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and alcohol-related liver disease (ALD) has been previously studied, their contribution to the development of ESLD remains largely unexplored. Methods: We genotyped 6 MASLD-associated polymorphisms in healthy (n = 123), metabolic dysfunction-associated steatohepatitis (MASH) (n = 145), MASLD-associated ESLD (n = 72), and ALD-associated ESLD (n = 57) cohorts and performed multinomial logistic regression to determine the combined contribution of genetic, demographic, and clinical factors to the progression of ESLD. Results: Distinct sets of factors are associated with the progression to ESLD. The PNPLA3 rs738409:G and TM6SF2 rs58542926:T alleles, body mass index (BMI), age, and female sex were positively associated with progression from a healthy state to MASH. The PNPLA3 rs738409:G allele, age, male sex, and having type 2 diabetes mellitus were positively associated, while BMI was negatively associated with progression from MASH to MASLD-associated ESLD. The PNPLA3 rs738409:G and GCKR rs780094:T alleles, age, and male sex were positively associated, while BMI was negatively associated with progression from a healthy state to ALD-associated ESLD. The findings indicate that the PNPLA3 rs738409:G allele increases susceptibility to ESLD regardless of etiology, the TM6SF2 rs58542926:T allele increases susceptibility to MASH, and the GCKR rs780094:T allele increases susceptibility to ALD-associated ESLD. Conclusion: The PNPLA3, TM6SF2, and GCKR minor alleles influence the progression of MASLD-associated or ALD-associated ESLD. Genotyping for these variants in MASLD and ALD patients can enhance risk assessment, prompting early interventions to prevent ESLD