Examination of drug metabolism using human hepatocytes is important in the early stages of drug development. However, primary human hepatocytes are short-lived and cannot be maintained in culture over the long term. Considerable donor-dependent variations are also problematic. Human embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts and are capable of differentiating into three embryonic germ layers and germ cells.1) The cells apparently differentiate into various types of mature cells, and are thereby an attractive source for routine access to large numbers of cells that can be used for the development of candidate drug-screening strategies replacing primary cells.2) However, ethical considerations have limited the availability of human ES cells. The phenotype of human ES cells is known to be similar to that of monkey ES cells but differs from that of mouse ES cells with regard to morphology, response to leukemia inhibitory factor, and gene expression patterns. 1,[3][4][5] Research using monkey ES cells is considered to be useful for investigation of differentiation mechanisms in primate ES cells and models of human ES cells. Recently, it has been shown that monkey ES cells can be differentiated into various cell types-including neurons, hematopoietic cells, and pancreatic cells-using growth factors. [6][7][8] Hepatocytes derived from monkey ES cells may be useful for pharmacokinetic examinations such as induction of drug metabolism enzymes and interactions of candidate drugs. To date however, there have been few reported studies describing the differentiation of monkey ES cells into hepatocytes. 9,10) After the emergence of the liver bud from the developing gut tube, the level of hepatic maturation is characterized by the expression of liver-and stage-specific genes. For example, alfa-fetoprotein (AFP) is an early hepatic marker, expressed by hepatoblasts in the liver bud until birth.11,12) The synthesis of AFP decreases dramatically after birth and only trace amounts are expressed in the adult liver. In contrast, albumin (ALB), the most abundant protein synthesized by hepatocytes, is initially expressed at lower levels in early fetal hepatocytes but this increases as the hepatocytes mature, reaching a maximum in adult hepatocytes. 13,14) The mRNA expression of cytochrome P450 7A1 (CYP7A1) which is a rate-limiting enzyme in the conversion of cholesterol to bile acids in liver 15) is detected in fetal liver of third trimester of pregnancy.16) After birth, CYP7A1 increases several-fold with age both at the enzyme activity level and the mRNA level. 17,18) The in vitro approaches involve the formation of embryoid bodies (EBs) to mimic the inductive microenvironment required for liver organogenesis [19][20][21] and treatment with specific growth factors and cytokines critical for hepatic differentiation.22) At present, culture systems for ES cells have mainly used gelatin-or collagen-coated plates as the matrix for the maintenance of cells in an undifferentiated state an...

Cytochrome P450 represent a superfamily of heme proteins that play a critical role in the oxidative metabolism of xenobiotics and endogenous substances, and there are several isoforms. The human CYP3A subfamily contains mainly three isoforms, CYP3A4, CYP3A5, and CYP3A7.3)CYP3A4 is a major isoform that accounts for about 30% of the amount of total CYP in human adult livers and is responsible for approximately 50% of the metabolism by CYPs of known drugs in humans. 4,5) CYP3A5 is expressed polymorphically throughout development in the liver.6,7) CYP3A7 is a major CYP isoform in the human fetal and newborn liver. 8)It is known that the human placenta contains multiple CYP isoforms in the mitochondria and endoplasmic reticulum of trophoblastic cells.9) The type and amount of expressed CYPs varies depending on the period of gestation and maternal health status. 10,11) In general, it appears that the placenta expresses a wider variety of CYP isoforms in the first trimester than at term. 12,13) Thus it has been suggested that xenobiotic metabolism in the placenta may be feto-protective during the critical embryogenic and organogenesis stage prior to the second trimester, when it is most susceptible to the effects of teratogens, the expression of CYP mRNAs is maximal.14) However, the central nervous system develops throughout the pregnancy and may also be affected in the later stages of pregnancy. Even though, as described above, various studies of enzymes related to pharmacokinetics in human placenta have been reported, there is only limited research in fetal membranes. The fetal membranes, which extend from the placenta, are an important barrier between the fetus and mother. The membranous structure that surrounds the developing fetus and forms the amniotic cavity is derived from fetal tissue except for decidua and is composed of three layers: amnion (inner layer), chorion leave (middle layer) and decidua (outer layer). The amnion is a translucent structure adjacent to the amniotic fluid. The chorion leave is a more opaque membrane that is attached to the decidua (i.e., maternal tissue that lines the uterus during pregnancy). The amnion and chorion leave are separated by the exocelomic cavity until approximately three months gestation, when they become fused. These membranes regulate the access of substances including drugs and other harmful chemicals to and from the amniotic fluid and, hence, the fetus.15) In recent years, it has been reported that the multidrug resistance/P-glycoprotein gene (MDR1/P-gp) and breast cancer resistance protein (BCRP), belong to the ATP-binding cassette transporter family are expressed in human fetal membranes including decidua. 16,17) It is considered that the role of these transporters is to discharge drugs in amniotic fluid and to keep out transfer of drugs from the maternal side via the uterus. It is well established that P-glycoprotein transports many drugs that are metabolized by CYP3A4 and many modulators of P-glycoprotein also modulate the CYP3A gene family.18) To the best of o...

Pregnane X receptor (PXR) mRNA was detected in HepG2 cells by RT-PCR, but not in human fetal liver (HFL) cells. CYP3A4 was induced by rifampicin (RIF), mifepristone (RU486), clotrimazole (CTZ), and dexamethasone (DEX) in HepG2 cells, while these PXR ligands with the exception of DEX did not induce CYP3A4 mRNA expression in HFL cells. Ad-PXR infection increased mRNA levels of PXR and CYP3A4 in both cells despite the absence of PXR ligands. Similar results were observed in reporter gene assays. However, in HFL cells, RIF-mediated CYP3A4 induction was insufficient compared with HepG2 cells, despite PXR overexpression. The expression levels of five coactivators (HNF4α, PGC1α, SRC1, CBP, and P300) related to CYP3A4 expression in HepG2, HFL cells, and human adult liver were analyzed by RT-PCR. Expression levels of HNF4α and PGC1α in HFL cells were downregulated to 20% of those in the human adult liver. On the other hand, the expression level of HNF4α in HepG2 cells was higher than that in HFL cells, although PGC1α expression level was almost the same as that in HFL cells. HNF4α mRNA expression level in HepG2 cells was 57% of that in human adult liver, and the level in HFL cells was 30% of that in HepG2 cells. These results suggested that lower expression of HNF4α and PGC1α may impair RIF-mediated CYP3A4 induction under conditions of PXR overexpression in HFL cells.

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