Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes

Abstract

Human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Hep) hold great potential as an unlimited cell source for toxicity testing in drug discovery research. However, little is known about mechanisms of compound toxicity in hiPSC-Hep. In this study, modified mRNA was used to reprogram foreskin fibroblasts into hiPSC that were differentiated into hiPSC-Hep. The hiPSC-Hep expressed characteristic hepatic proteins and exhibited cytochrome P450 (CYP) enzyme activities. Next, the hiPSC-Hep, primary cryopreserved human hepatocytes (cryo-hHep) and the hepatic cell lines HepaRG and Huh7 were treated with staurosporine and acetaminophen, and the toxic responses were compared. In addition, the expression of genes regulating and executing apoptosis was analyzed in the different cell types. Staurosporine, an inducer of apoptosis, decreased ATP levels and activated caspases 3 and 7 in all cell types, but to less extent in Huh7. Furthermore, a hierarchical clustering and a principal component analysis (PCA) of the expression of apoptosis-associated genes separated cryo-hHep from the other cell types, while an enrichment analysis of apoptotic pathways identified hiPSC-Hep as more similar to cryo-hHep than the hepatic cell lines. Finally, acetaminophen induced apoptosis in hiPSC-Hep, HepaRG and Huh7, while the compound initiated a direct necrotic response in cryo-hHep. Our results indicate that for studying compounds initiating apoptosis directly hiPSC-Hep may be a good alternative to cryo-hHep. Furthermore, for compounds with more complex mechanisms of toxicity involving metabolic activation, such as acetaminophen, our data suggest that the cause of cell death depends on a balance between factors controlling death signals and the drug-metabolizing capacity.

Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused Three-Dimensional Multicompartment Bioreactor

“…6–9 These procedures mimic the embryonic development of the liver by adding different growth factors necessary for each developmental stage. The resulting hepatocyte-like cells (HLC) were successfully applied for in vitro studies on human drug exposure, 10,11 hepatitis B and C infection, 12,13 or malaria pathogenesis 14 among others, and they have been shown to repopulate the livers of chimeric mice and rescue the disease phenotype in these animals. 15 However, the HLC obtained with existing protocols still show an immature phenotype with reduced hepatic functionality when compared to PHH.…”

New markers for tracking endoderm induction and hepatocyte differentiation from human pluripotent stem cells

“…Human pluripotent stem cells (hPSCs), including both embryonic (hESCs) and induced pluripotent stem cells (hiPSCs), represent an unlimited source of differentiated cell types and tissues for modeling human development and disease in vitro, for developing new cell-based therapeutics and for establishing new drug discovery and predictive toxicology platforms (Cherry and Daley, 2012;Diecke et al, 2014;Fox et al, 2014;Holmgren et al, 2014;Leung et al, 2013;Medine et al, 2013;Roelandt et al, 2013;Sjogren et al, 2014;Szkolnicka et al, 2014;Trounson et al, 2012;Zhou et al, 2014). Translating this potential of stem cells into practice is, however, dependent on the availability of directed differentiation strategies that enable the efficient, reproducible and cost-effective generation of the lineage of interest.…”

Evaluation of CYP3A4 inhibition and hepatotoxicity using DMSO-treated human hepatoma HuH-7 cells

“…Therefore, robust, sensitive, and biologically relevant hepatoma cell lines are valuable as alternative models. Recently, various cell culture systems have been developed as in vitro models, such as human induced pluripotent stem cell-derived hepatocytes (Sjogren et al 2014), hepatic cell lines transfected with drug-metabolizing enzyme transgenes, terminally differentiated human bipotent progenitor cell line HepaRG (Donato et al 2013), and 3D cultured hepatoma cell lines (Malinen et al 2014). Although these systems are useful, they often require complicated development procedures and extensive maintenance, which are not ideal for higher-throughput screening.…”