[ effects of FGF8 and Shh on expression of dopaminergic markers from human amniotic epithelial cells]

Recently, interest has increased in the potential employment of embryonic stem cells for the treatment of Parkinson's disease, which has been considered as an alternative therapeutic strategy. Due to their pluripotent differentiation potential, the finding that they do not induce carcinoma and the fact that they do not raise the ethical concerns connected with human embryonic stem cells, human amniotic epithelial cells are considered to be a very promising cell source. The aim of this study was to investigate the effects of FGF8 and Shh on the expression of dopaminergic markers from human amniotic epithelial cells. In this study, we examined the differentiation of dopaminergic neurons in vitro from AECs using the expression of several markers including TH, DAT and D beta H. For dopaminergic differentiation, sonic hedgehog [Shh] and FGF8 were added to cultures and the cultures were allowed to differentiate for 21 days. Analysis of AECs derived dopaminergic neurons was performed at the TH, DAT, beta-tubulin III and D beta H expression levels by immunocitochemistry. The significance of the data was tested by Student's t-test [between two groups] and one-way analysis of variance [ANOVA] followed by Tukey post-test. [p<0/01, p<0/05]. Combination of Shh and FGF8 showed the higher level of TH in comparison to control group or these factors alone. Moreover, Shh is more effective than FGF8 on DAT expression in comparison to expression of D beta H. These results show the capability of AECs to express dopaminergic neural markers and this ability is affected by Shh and FGF8

[Induced in vitro differentiation of beta -tubulin III expressed cells from human amniotic epithelial cells]

The differentiation of neural cells from embryonic stem cells is influenced by several factors. Amniotic epithelial cells [AECs] share many of the same characteristics as embryonic stem cells, and therefore, those factors may similarly affect the derivation of neural cells from AECs. In this study, we examined the differentiation of neural cells in vitro from AECs using the expression of Beta-tubulin III marker, after AECs treatment with retinoic acid [RA], and the impact of basic fibroblast growth factor [bFGF]. We also studied whether blocking bone morphogenetic protein [BMP] signaling the use of its antagonist, noggin, affects the derivation of neural cells from AECs. AECs were isolated from fresh human amniotic membrane and aggregated for 5 days in bacteriological dishes. The dissociated cells were transferred to adherent culture dishes and treated with noggin and bFGF for 7 days. In some cultures, bFGF was removed and RA was added and the cultures were allowed to differentiate for 21 days. Analysis of AECs derived neural cells was performed at the Beta-tubulin III expression levels by immunocitochemistry. All cultures treated with noggin showed the higher levels of Beta-tubulin III expression than noggin free cultures. Combined treatment with bFGF and RA showed the highest level of Beta-tubulin III in all treatment groups with or without noggin. bFGF withdrawal did not promote expression of Beta-tubulin III, while its maintenance increased the expression of Beta-tubulin III. These results show the capability of AECs to express neural cell marker and this potential is affected by some factors including noggin, bFGF, RA

[Effect of gonadal steroid hormones on GIRK2 gene transcription in rat's spinal cord]

Considerable evidences have indicated the differences between different sexes in their pain responses and opioid antinociception in which Opioids have been more potent in males than females. GIRK[2], the primary post-synaptic effectors of opioids in the CNS, possibly contributes in these sex-related differences. In the present study, we examined the role and effect of gonadal steroid hormones on GIRK[2] gene transcription in rats. Male and female Wistar rats were divided into four groups of intact infant or adult, GDX, sham and GDX+T groups. "semiquantitave RT-PCR" was used to determine the GIRK[2] gene expression in spinal cord tissue of the rats. Our results showed that in the spinal cord of male rats, gene transcription of infant group did not fluctuate along with expression level of this gene in GDX or adult intact groups. Elimination of gonadal hormones in male rats significantly decreased the expression level of GIRK2 gene. These changes were not restored by testosterone replacement. However, in females, a greater expression of GIRK[2] gene was found in intact adult or GDX rats than those of infants. Ovariectomy of animals failed to alter the GIRK2 mRNA level. No significant preferential differences were observed in GIRK[2] gene transcription between both sexes of intact, sham and infant groups. These findings show that regulation of GIRK[2] gene transcription by gonadal hormones is male-dominant. Whereas, other sex-dependent developmental factors prominently affect on GIRK[2] gene transcription in female animals. Further investigations are needed to clarify the exact mechanism