Development and Characterization of a Cytochrome P450 CYP3A7 Fetal Hepatocyte-Like Cell Model Derived from Induced Pluripotent Stem Cells

Abstract ID 87829 Poster Board 426 Background CYP3A7 is the major cytochrome P450 (CYP) in the liver of the developing fetus and neonate, responsible for the metabolism of both endogenous and exogenous substrates. CYP3A7 metabolizes the adrenal steroid dehydroepiandrosterone sulfate (DHEA-S) to 16 αOH DHEA-S, a precursor for estriol synthesis in the placenta. Low estriol levels during pregnancy are associated with fetal growth restriction and risk for preterm labor. Therefore, any perturbation of this sensitive system, through xenobiotic inhibition or induction of CYP3A7, can have harmful outcomes for the fetus. Objective Due to the scarcity of human fetal hepatocytes, our goal was to develop and characterize a sustainably sourced fetal hepatocyte-like cell (HLC) model derived from induced pluripotent stem cells (iPSCs) in order to study CYP3A7 in a cell-based, in vitro system. Methods Our HLC differentiation protocol is based on that of Baxter et al. (J. Hepatol. 2015;62(3):581-589) with adjustments to increase the fetal characteristics. iPSCs were differentiated through three stages: definitive endoderm (DE), hepatoblast-like cells, and final HLCs. For differentiation to DE, several sub-protocols were tested to maximize cell survival and differentiation efficiency. At the end of the protocol, HLCs were characterized for fetal and liver markers via qPCR. Fold change in mRNA expression was calculated compared to undifferentiated iPSCs via the ΔΔCt method, with BACT as a reference. CYP3A7 enzymatic activity was assessed via incubations with 50-250 mM DHEA(S) at 37 °C for up to 4 hours, and formation of the 16 αOH metabolites was measured via LC-MS/MS. Results Inclusion of the supplement B27+ increased cell survival through DE differentiation, while also achieving high mRNA expression of the DE marker SOX17. After a 20-day protocol, fetal HLCs (fHLCs) displayed high levels of hepatic and fetal genes. Liver markers ALB and HNF4A displayed mRNA fold changes over 1800- and 410-compared to undifferentiated iPSCs, and fetal AFP was increased over 6 orders of magnitude. There were important differences in expression of the CYP3A isoforms: fetal CYP3A7 mRNA was increased 4321-fold over iPSCs, while adult CYP3A4 mRNA was only increased about 65-fold. CYP3A7 activity was highest with the substrate DHEA, showing increasing formation of the 16 αOH metabolite with time. Discussion and Conclusions We demonstrated that our current fHLC model displays high levels of both fetal and liver mRNA, accompanied by CYP3A7 enzymatic activity. The issue of cell death observed in preliminary differentiation protocols was addressed by optimizing the DE stage to increase cell survival while maintaining differentiation efficiency. The HLC maturation stage is currently being refined to maximize CYP3A7 activity. Because DHEA-S does not cross cell membranes as readily as its non-sulfonated form, DHEA was used as a probe substrate to determine CYP3A7 activity by monitoring formation of the major metabolite 16 αOH DHEA. In the future, metabolite ratios will be calculated to help determine contributions from both CYP3A7 and CYP3A4, due to their overlapping substrate specificity and varying regiospecificity. After a rigorous characterization, this work will yield a sustainable fetal hepatocyte model which can be used to gauge CYP3A7-mediated drug-hormone interactions while avoiding the use of fetal tissue.