Expression and co-expression of surface markers of pluripotency on human amniotic cells cultured in different growth media.

OBJECTIVES: Despite constant advances in the field of biology and medical application of human embryonic stem cells, the molecular mechanism of pluripotency remains largely unknown. So far, definitions of pluripotent stem cells (SC) have been based on a limited number of antigenic markers and have not allowed for unambiguous determination of the homogeneity of each subpopulation. Moreover, the use of some crucial pluripotency markers such as SSEA-3 and SSEA-4 has recently been questioned due to the possibility that the pattern of surface glycans may be changed depending on the content of the cell culture medium. AIM: Quantitative analysis of amniotic SC subpopulations cultured in different media, based on the following pluripotency surface markers: SSEA-3, SSEA-4, TRA- 1-60 and TRA- 1-81 expression and co-expression. MATERIAL AND METHODS: Immunofluorescence and fluorescence microscopy were used to identify and localize SC within a normal human placenta at term. The number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells with co-expression of the above mentioned markers, cultured in media containing different protein supplements of animal origin, was counted by flow cytometry RESULTS AND CONCLUSIONS: Cells with characteristics of embryonic SC were identified in the amniotic epithelium and the chorion, but not in the decidua basalis. Amniotic epithelium contained various types of SC, with SSEA-4+ as the most numerous. Disproportion in the number of SSEA-4+, SSEA-3+, TRA-1-60+ and TRA-1-81+ cells and cells characterized by co-expression of these antigens, as well as lack of quantitative differences between SC subpopulations cultured in different media, was observed. In conclusion, the amniotic epithelium is composed of SC at different stages of the development but human amnion might become an alternative source of SSEA-4+ embryonic-like SC. The composition of the evaluated media, characterized by different content of animal-derived proteins, does not influence the number of cells identified within the SC subpopulations.

Expression of cytochrome P450 2C and 3A in female rat liver after long-term administration of gonadoliberin analogs.

OBJECTIVES: Gonadoliberin (GnRH) analogs may be expected to indirectly modify growth hormone (GH) total concentration and its 24-h secretion profile. As a consequence, changes in the levels of GH may modify the mechanism of sex-dependent cytochromes P450 (CYP450) synthesis, including the expression of transcriptional factors. The aim of the study has been to evaluate the effect of long-term administration of a low dose of GnRH analogs on hepatic expression of CYP2C and CYP3A isoforms, and the transcription factors: pregnane X receptor (PXR), hepatocyte nuclear factor 4α (HNF4α), HNF6 and signal transducers and activators of transcription 5b (STAT5b). MATERIAL AND METHODS: The study was carried out on adult female Sprague-Dawley rats during a 3-month treatment with dalarelin (GnRH agonist) and cetrorelix (GnRH antagonist), at a daily intraperitoneal injection (i.p.) dose of 6 µg/kg body weight/day, and 1, 2, and 4 weeks after treatment discontinuation. The concentrations of ovarian hormones and GH in the blood serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) method, respectively. Then, the expression of hepatic CYP450s (reverse transcription polymerase chain reaction - RT-PCR, Western blot and immunohistochemistry) and transcription factors (RT-PCR) was evaluated. RESULTS: We have found that cetrorelix induces changes in the circadian pattern of GH secretion and enhances GH blood concentrations. These changes may cause increased expression of both, female-specific CYP450s (especially CYP3A9), and HNF4α/HNF6 transcription factors. Decrease in GH blood concentrations, resulting from the effect of dalarelin, may promote inhibition of female-specific CYP2C12 and CYP3A9 isoforms as well as STAT5b transcription factor. Slight changes in sex-independent CYP3A1 protein expression caused by GnRH analogs were also observed. CONCLUSIONS: In adult female rats, HNF4α/HNF6 and STAT5b seem to be crucial for the regulation of GnRH antagonist/GH- and GnRH agonist/GH-dependent pattern of CYP450 expression, respectively.

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine.

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague-Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, ß-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b(5) reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.

Cytochrome P450 mRNA expressions along with in vitro differentiation of hepatocyte precursor cells from fetal, young and old rats.

Non-differentiated cells are attractive targets for cell therapy. During liver regeneration oval cells intensively proliferate and differentiate extending their metabolic activity. Hepatic cytochromes P450 (CYPs) can be linked either with metabolic activation of toxic compounds or drug metabolism. We investigated the differentiation and biotransformative potential of non-differentiated cells in primary cell cultures isolated from livers of fetuses (16-days-old), young (4-months-old) and old (20-months-old) rats. Under the conditions of experimental hepatocarcinogenesis, adult rats were fed for three weeks with CDE diet. Liver cells were cultured and precursor cells were differentiated to hepatocytes following induction with sodium butyrate (SB) or dimethyl sulphoxide (DMSO) in culture on MesenCult medium. We identified a number of cells expressing Thy-1, CD34, alpha-fetoprotein, cytokeratines--CK18 or CK19 and glutathione transferases--GSTpi or GSTalpha. In vitro differentiation of these cells, isolated from CDE-treated rats begun earlier as compared to non-treated ones. Age-dependent changes in the cell differentiation sequence, as well as CYPmRNA expression sequence accompanying precursor cells differentiation, were also observed. mRNA expression of CYP1A2, CYP2B1/2 and CYP3A1 was higher in the cells of young rats, but in the case of CYP2E1--in the cells of old rats. It was concluded that both proliferation and differentiation potential of oval cells, decreased with age.