Expression and function of the urea cycle in widely‐used hepatic cellular models

• Published in: Journal of inherited metabolic disease Vol. 47; no. 6; pp. 1228 - 1238
• Main Authors: Makris, Georgios, Veit, Lara, Rüfenacht, Véronique, Klassa, Sven, Zürcher, Nadia, Matsumoto, Shirou, Poms, Martin, Häberle, Johannes
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.11.2024; Blackwell Publishing Ltd
• Subjects: Animal models; Animal welfare; Arginase; Arginase - metabolism; Cell Differentiation; Cell lines; Cognition; Complications; enzyme expression; Hep G2 Cells; HepaRG cells; hepatic cellular models; Hepatocytes; Hepatocytes - metabolism; Hepatoma; Humans; Immunoblotting; Immunocytochemistry; Induced Pluripotent Stem Cells - metabolism; Liver - metabolism; Liver cancer; Liver diseases; Liver transplantation; Molecular modelling; Ornithine; Pluripotency; stable isotope quantification; Urea; Urea - metabolism; urea cycle; Urea Cycle Disorders, Inborn - metabolism; ureagenesis; enzyme expression; hepatic cellular models; stable isotope quantification; ureagenesis; urea cycle; HepaRG cells
• ISSN: 0141-8955; 1573-2665; 1573-2665
• DOI: 10.1002/jimd.12701

The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma‐derived cell lines, induced pluripotent stem cell hepatocytes (iPSC‐Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma‐derived, 2‐week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC‐Heps, which are known to exhibit batch‐to‐batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs.