title: Aggregate culture of human embryonic stem cell-derived hepatocytes in suspension are an improved in vitro model for drug metabolism and toxicity testing.
abstract: Early phase drug development relies on primary human hepatocytes for studies of drug metabolism, cytotoxicity, and drug-drug interactions. However, primary human hepatocytes rapidly lose metabolic functions ex vivo and are refractory to expansion in culture and thus are limited in quantity. Hepatocytes derived from human pluripotent stem cells (either embryonic stem (ES) or induced pluripotent stem (iPS) cells), have the potential to overcome many of the limitations of primary human hepatocytes, but to date the use of human pluripotent stem cell-derived hepatocytes has been limited by poor enzyme inducibility and immature metabolic function. Here, we present a simple suspension culture of aggregates of ES cell-derived hepatocytes that compared to conventional monolayer adherent culture significantly increases induction of CYP 1A2 by omeprazole and 3A4 by rifampicin. Using liquid chromatography-tandem mass spectrometry, we further show that ES cell-derived hepatocytes in aggregate culture convert omeprazole and rifampicin to their human-specific metabolites. We also show that these cells convert acetaminophen (APAP) to its cytotoxic metabolite (N-acetyl-p-benzoquinone imine (NAPQI)), although they fail to perform APAP glucuronidation. In summary, we show that human pluripotent stem cell-derived hepatocytes in aggregate culture display improved enzymatic inducibility and metabolic function and is a promising step toward a simple, scalable system, but nonetheless will require further improvements to completely replace primary human hepatocytes in drug development.
authors.affiliation: Morgridge Institute for Research, Madison, Wisconsin 53715 ssengupta@morgridgeinstitute.org.
authors.forename: Srikumar
authors.initials: S
authors.lastname: Sengupta
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Brian Patrick
authors.initials: BP
authors.lastname: Johnson
authors.affiliation: Morgridge Institute for Research, Madison, Wisconsin 53715.
authors.forename: Scott Allen
authors.initials: SA
authors.lastname: Swanson
authors.affiliation: Morgridge Institute for Research, Madison, Wisconsin 53715.
authors.forename: Ron
authors.initials: R
authors.lastname: Stewart
authors.affiliation: McArdle Laboratory for Cancer Research, Department of Oncology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin 53706.
authors.forename: Christopher Alan
authors.initials: CA
authors.lastname: Bradfield
authors.affiliation: Morgridge Institute for Research, Madison, Wisconsin 53715 Department of Cell & Regenerative Biology, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706 Department of Molecular, Cellular, & Developmental Biology, University of California Santa Barbara, Santa Barbara, California 93106.
authors.forename: James Alexander
authors.initials: JA
authors.lastname: Thomson
mesh_terms: D000082:Acetaminophen / Q000378:metabolism / Q000633:toxicity; D002449:Cell Aggregation; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D002853:Chromatography, Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis; D065694:Cytochrome P-450 CYP1A2 Inducers / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis; D065701:Cytochrome P-450 CYP3A Inducers / Q000494:pharmacology; D055808:Drug Discovery / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism; D004790:Enzyme Induction; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D009853:Omeprazole / Q000378:metabolism / Q000494:pharmacology; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D012293:Rifampin / Q000378:metabolism / Q000494:pharmacology; D053719:Tandem Mass Spectrometry; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D065694:Cytochrome P-450 CYP1A2 Inducers; D065701:Cytochrome P-450 CYP3A Inducers; D000082:Acetaminophen; C504573:CYP1A2 protein, human; D019388:Cytochrome P-450 CYP1A2; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D009853:Omeprazole; D012293:Rifampin
keywords: Drug testing; ES cells; Hepatocytes; Stem cells; Toxicity testing; iPS cells
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