title: 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.
authors.affiliation: Cardiovascular and Metabolic Diseases Innovative Medicines, DMPK, AstraZeneca R&D, 431 83, Mölndal, Sweden, anna-karin.sjogren@astrazeneca.com.
authors.forename: Anna-Karin M
authors.initials: AK
authors.lastname: Sjogren
authors.forename: Maria
authors.initials: M
authors.lastname: Liljevald
authors.forename: Björn
authors.initials: B
authors.lastname: Glinghammar
authors.forename: Johanna
authors.initials: J
authors.lastname: Sagemark
authors.forename: Xue-Qing
authors.initials: XQ
authors.lastname: Li
authors.forename: Anna
authors.initials: A
authors.lastname: Jonebring
authors.forename: Ian
authors.initials: I
authors.lastname: Cotgreave
authors.forename: Gabriella
authors.initials: G
authors.lastname: Brolén
authors.forename: Tommy B
authors.initials: TB
authors.lastname: Andersson
mesh_terms: D000082:Acetaminophen / Q000378:metabolism / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000175:diagnosis / Q000209:etiology; D015925:Cryopreservation / Q000379:methods; D005347:Fibroblasts / Q000166:cytology; D052816:Foreskin; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D008297:Male; D025341:Principal Component Analysis; D019311:Staurosporine / Q000633:toxicity*; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D000082:Acetaminophen; D019311:Staurosporine