title: Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells-derived and fetal hepatocytes.
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 R(2) 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.
  
  
  CONCLUSIONS
  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.
authors: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem, Israel.|Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Yishai
authors: Y
authors: Avior
authors: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem, Israel.|Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Gahl
authors: G
authors: Levy
authors: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem, Israel.|Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Michal
authors: M
authors: Zimerman
authors: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem, Israel.|Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Daniel
authors: D
authors: Kitsberg
authors: Department of Medicine, Weill Cornell Medical College, New York, NY.
authors: Robert
authors: R
authors: Schwartz
authors: Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.|School of Computer Science and Engineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Ronen
authors: R
authors: Sadeh
authors: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem, Israel.|Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Arieh
authors: A
authors: Moussaieff
authors: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem, Israel.|Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Merav
authors: M
authors: Cohen
authors: Stem Cell Center, Technion, Haifa, Israel.
authors: Joseph
authors: J
authors: Itskovitz-Eldor
authors: Department of Cell and Developmental Biology, Hebrew University of Jerusalem, Jerusalem, Israel.|Grass Center for Bioengineering, Hebrew University of Jerusalem, Jerusalem, Israel.|School of Computer Science and Engineering, Hebrew University of Jerusalem, Jerusalem, Israel.
authors: Yaakov
authors: Y
authors: Nahmias
mesh_terms: D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D065729:Cytochrome P-450 CYP2C9 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D008095:Lithocholic Acid / Q000494:pharmacology*; D039904:Pluripotent Stem Cells / Q000187:drug effects*; D000077297:Pregnane X Receptor; D011987:Receptors, Steroid / Q000378:metabolism; D017423:Sequence Analysis, RNA; D023382:Toxicity Tests, Acute; D024482:Vitamin K 2 / Q000031:analogs & derivatives / Q000494:pharmacology*
chemical_list: D000077297:Pregnane X Receptor; D011987:Receptors, Steroid; D024482:Vitamin K 2; C030814:menatetrenone; D008095:Lithocholic Acid; D065729:Cytochrome P-450 CYP2C9; D051544:Cytochrome P-450 CYP3A