Critical differences in toxicity mechanisms in induced pluripotent stem cell-derived hepatocytes, hepatic cell lines and primary hepatocytes
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.
KeywordsHepatotoxicity Drug-induced liver injury iPS cells HepaRG Acetaminophen Apoptosis
Human-induced pluripotent stem cells
Human-induced pluripotent stem cell-derived hepatocytes
Cryopreserved human hepatocytes
Principal component analysis
Periodic acid schiff
Liquid chromatography/mass spectrometry
Quantitative real-time PCR
Cycle of threshold
LC-/MS-radiochemical activity monitoring
Hepatocyte nuclear factor 4 alpha
Pregnane X receptor
This study was in part supported by SCR&Tox, which is funded by the European Commission within its seventh framework Programme and Cosmetics Europe, the European Cosmetics Association, as part of the SEURAT (Toward the replacement of in vivo repeated dose system toxicity) Cluster [Contract HEALTH-F5–2010-266573]. We thank Isabelle Jansson, Anette Persson-Kry and Anna Svensson (Discovery Sciences, Reagents and Assay Development, AstraZeneca R&D, Mölndal, Sweden) for contributing with cell culture work and Frank Seeliger (Drug Safety and Metabolism, Pathology Sciences, AstraZeneca R&D, Mölndal, Sweden) for histological evaluations of teratoma.
Conflict of interest
The authors declare no conflict of interest.
The manuscript does not contain clinical studies or patient data.
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