During the past few years several differentiation protocols to derive midbrain dopamine (DA) neurons from human embryonic stem (hES) cells have been developed, but the production of sufficient amounts of the ‘right’ therapeutic DA cells has not yet been accomplished. The aim of this study was to efficiently generate tyrosine hydroxylase (TH)-positive cells in vitro from our hES cells using a chemically defined culture system. At the end of differentiation, the vast majority of cells (>90%) were positive for both TH and β-tubulin isotype III (TuJ1). Other markers of dopaminergic cells, like dopamine transporter (DAT) and Nurr1 were also detected by immunofluorescence or RT–PCR. The functions of these cells were confirmed by measurements of DA release in vitro and by transplantation of derived cells into Parkinson's disease (PD) rats in vivo. We found these cells were able to release DA when depolarized by high K⁺. Moreover, 4 weeks after transplantation, the hES-derived cells could survive and reduce the apomorphine-induced rotation behaviour of the rats. In conclusion, the experimental system presented here provided a reliable protocol to produce a large number of hES-derived TH⁺ cells which may be used in cell therapy for PD in future.

Non-human primates are suitable models for preclinical research aimed at cell-replacement therapies. Recently, it has been reported that Rho-associated kinase inhibitor Y-27632 markedly reduced dissociation-induced apoptosis of human embryonic stem (hES) cells, and is expected as a novel supplement for hES cell maintenance or differentiation inductions; however, the effects of the chemical are still to be determined in model animals. Here, we demonstrated the effect of Y-27632 on cynomolgus monkey ES (cyES) cells. Also, in cyES cells, Y-27632 treatment dramatically improved the efficiency of colony formation from single cells without affecting the pluripotent state and karyotype. Y-27632 supplementation was also effective for feeder-free culture and differentiation induction. Neural stem cells directly induced from cyES cells could give rise to neurons, astrocytes and dopamine producing cells. The present result not only suggests that the chemical was effective for improving the culture system of primate ES cells, but also the similarity between cyES and hES cells regarding the reactions to the chemical, which might be further evidence that cyES cells are superior models for hES cells.

The presence of an isochromosome Xq in Klinefelter syndrome (KS) is an apparently rare condition. In all cases reported so far, patients showed the classic phenotype. We here describe a case of isochromosome Xq [47,X,i(Xq),Y] in a non-mosaic KS patient. The patient exhibited a normal androgenized phenotype, normal testes and normal cognitive abilities. Semen analysis revealed a medium oligozoospermia (5 x 10⁶ spermatozoa/ml). After the patient underwent intracytoplasmic sperm injection, he generated two cytogenetically healthy normal females. Fluorescence in situ hybridization analysis showed the presence of a dicentric Xq chromosome that did not show the presence of residual Xp arm up to the 57,820,478 bp position (Xp 1.1) of X chromosome sequence. Preferential inactivation of Xq isochromosome was demonstrated by bromodeoxyuridine replication analysis and transcriptional silencing by DNA methylation at the HUMARA locus. Furthermore, we demonstrated by quantitative RT–PCR an active XIST RNA expression in blood lymphocytes from Klinefelter patients, comparable to that observed in control females and over 30 000-fold greater than in control males. In conclusion, this qRT–PCR approach could be useful for screening of prepuberty males and for diagnosis or exclusion of cryptic Klinefelter mosaics.

The aims of the present study were to compare the levels of mRNA and protein expression of matrix metalloproteinase (MMP)-1, -3, -8 and -9 in human cervical tissue in preterm and term labor as well as not in labor and to determine if corticotropin-releasing hormone (CRH) has an effect on MMP-1, -3 and interleukin (IL)-8 secretion in both preterm and term cervical fibroblasts. Cervical biopsies were taken from 60 women: 18 at preterm labor, 7 at preterm not in labor, 18 at term labor and 17 at term not in labor. ELISA and Immulite were used for protein and real-time RT–PCR for mRNA analysis. Cervical fibroblast cultures were incubated for 18 h with different CRH concentrations (10^–13–10^–6 M). The mRNA expression of MMP-1, -3 and -9 was higher in laboring groups compared with term not in labor. Protein levels of MMP-8 and -9 were higher in term in labor group compared with non-laboring groups. There were no significant differences in mRNA and protein expression between the preterm and respective term control groups. CRH significantly increased secretion of IL-8 in preterm and term cervical fibroblasts compared with controls. The secretion of IL-8 and MMP-1 was significantly higher and MMP-3 secretion lower in preterm cervical fibroblasts. In conclusion, cervical ripening at preterm seems to be a similar inflammatory process as at term with CRH involved. However, preterm and term cervical fibroblasts might have different phenotypes based on different secretion patterns of IL-8, MMP-1 and MMP-3.

Human leukocyte antigen-G (HLA-G) is a non-classical class I HLA molecule that is expressed by extravillous cytotrophoblast cells. This protein may play a critical role in the protection of cytotrophoblasts from maternal immune response, allowing these semi-allogeneic cells to invade the uterus unimpeded. We have demonstrated that diminished placental HLA-G expression is associated with pre-eclampsia. In order to explore fundamental mechanisms underlying this reduced HLA-G expression in pre-eclampsia, we looked for, and found by sequences analysis, a single base-pair mutation in the HLA-G gene 3'-untranslated region (3'UTR) adjacent to an AUUUA motif. This mutation is significantly associated with pre-eclampsia, the severe form being more strongly associated with homozygosity for this mutation than the mild form. Since the null allele was discovered in the HLA-G mRNA 3'UTR adjacent to an AUUUA motif, we also examined the effect of this mutation on HLA-G mRNA stability, and found that half-lives of HLA-G mRNA with the mutation were significantly shorter than without the mutation. These data provide evidence that this mutation could be one of the fundamental mechanisms for lower levels of placental HLA-G protein expression in patients with pre-eclampsia.

Endometriosis is a debilitating disease in which apoptotic, genetic, immunological, angiogenic and environmental factors have been implicated. Endocrine-disrupting agents (e.g. dioxins) might be involved. Dioxins, via the arylhydrocarbon receptor (AhR), induce estrogen-metabolizing enzymes CYP1A1 and CYP1B1. Elevated expression of -SYNUCLEIN (-SYN) has been associated with hormone-related conditions. Tissue sets consisting of eutopic and ectopic (ovarian) endometrium from patients with stage 3 or 4 endometriosis were obtained. Following RNA extraction and reverse transcription, quantitative real-time reverse transcriptase–polymerase chain reaction was performed for anti-apoptotic B-cell leukaemia/lymphoma 2 (BCL-2), CYP1A1, CYP1B1, estrogen receptor (ER), ERβ and -SYN. Immunohistochemical analyses for -syn, ER, ERβ and CYP1A1 were also conducted. A 3–9-fold increase in intra-individual expression of CYP1A1 in ectopic (ovarian) endometrium compared with eutopic tissue was observed; immunohistochemical analyses pointed to CYP1A1 being localized to the glandular epithelium. This intra-individual expression profile was not observed for CYP1B1 or BCL-2. However, a 5–53-fold intra-individual increase in -SYN expression was also demonstrated in six of nine tissue sets (a further two showed an increase that was not considered significant) when comparing ectopic to eutopic endometrium; -syn positivity was associated with endothelial cells. An elevation in ERβ was also noted when comparing ectopic to eutopic endometrium; with regard to ER, this was inconsistent. These results suggest an up-regulation of dioxin-inducible CYP1A1 and -SYN occurs in endometriosis. Whether -syn may be a novel diagnostic marker for endometriosis remains to be ascertained.