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In vitro production of human hepatocytes is of primary importance in basic research, pharmacotoxicology and biotherapy of liver diseases. We have developed a protocol of differentiation of human embryonic stem cells (ES) towards hepatocyte-like cells (ES-Hep). Using a set of human adult markers including CAAT/enhancer binding protein (C/EBPalpha), hepatocyte nuclear factor 4/7 ratio (HNF4alpha1/HNF4alpha7), cytochrome P450 7A1 (CYP7A1), CYP3A4 and constitutive androstane receptor (CAR), and fetal markers including alpha-fetoprotein, CYP3A7 and glutathione S-transferase P1, we analyzed the expression of a panel of 41 genes in ES-Hep comparatively with human adult primary hepatocytes, adult and fetal liver. The data revealed that after 21 days of differentiation, ES-Hep are representative of fetal hepatocytes at less than 20 weeks of gestation. The glucocorticoid receptor pathway was functional in ES-Hep. Extending protocols of differentiation to 4 weeks did not improve cell maturation. When compared with hepatocyte-like cells derived from adult liver non parenchymal epithelial (NPE) cells (NPE-Hep), ES-Hep expressed several adult and fetal liver makers at much greater levels (at least one order of magnitude), consistent with greater expression of liver-enriched transcription factors Forkhead box A2, C/EBPalpha, HNF4alpha and HNF6. It therefore seems that ES-Hep reach a better level of differentiation than NPE-Hep and that these cells use different lineage pathways towards the hepatic phenotype. Finally we showed that lentivirus-mediated expression of xenoreceptor CAR in ES-Hep induced the expression of several detoxification genes including CYP2B6, CYP2C9, CYP3A4, UDP-glycosyltransferase 1A1, solute carriers 21A6, as well as biotransformation of midazolam, a CYP3A4-specific substrate. Electronic supplementary material The online version of this article (doi:10.1007/s12015-010-9225-3) contains supplementary material, which is available to authorized users.

Cytochromes P450 (CYP) from families CYP1, CYP2 and CYP3 are involved in xenobiotic detoxication and are regulated by numerous agents including xenobiotics, bile salts and cytokines. Nuclear receptors PXR (Pregnane X Receptor, NR1I3) and CAR (Constitutive Androstane Receptor, NR1I2) control the expression of distinct but overlapping arrays of genes including the CYP2 and CYP3 families. These receptors are involved in a tangle of regulatory networks including those pathways controlling vitamin D and cholesterol/bile salt homeostasis, are expressed under the control of the glucocorticoid receptor and are inhibited by the short heterodimer partner (SHP). Such functional interferences provide new views for the understanding of xenobiotic adverse effects.