Microbial-Derived Lithocholic Acid and Vitamin K2 Drive the Metabolic Maturation of Pluripotent Stem Cells-Derived and Fetal Hepatocytes

Yishai Avior, Gahl Levy, Michal Zimerman, Daniel Kitsberg, Robert Schwartz, Ronen Sadeh, Arieh Moussaieff, Merav Cohen, Joseph Itskovitz-Eldor, Yaakov Nahmias*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

70 Scopus citations

Abstract

The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4α) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R2 correlation of 0.94 between TC50 values obtained in stem cell-derived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins. Conclusion: Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology.

Original languageAmerican English
Pages (from-to)265-278
Number of pages14
JournalHepatology
Volume62
Issue number1
DOIs
StatePublished - 1 Jul 2015

Bibliographical note

Publisher Copyright:
© 2015 by the American Association for the Study of Liver Diseases.

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