873 Opsin Expression in human amniotic mesenchymal stem cells in vitro

Abstract

Objective To determine the mRNA and protein expression of opsin in human amniotic mesenchymal stem cells. MethodsHuman amnion was harvested in normal cesarean section. The amnion pieces were subjected to 0.25% trypsin for digestion to remove epithelial cells. The tissue pieces were then placed in PBS containing collagenase IV (0.75 mg/mL) and DNase I (0.075 mg/mL) and incubated at 37°C for 60 minutes. The collected cells were cultured in DMEM/F12 medium containing 5% helios. Flow cytometry and cell-induced differentiation protocols were used to identify hAMSCs. The expression of opsins in hAMSCs were analyzed by the immunofluorescence technique under the fluorescence microscope and the confocal microscope in vitro. The mRNA leveland protein content of opsins in hAMSCs were detected by quantitative real-time PCR and western blotting analysis respectively. ResultsWe noticed fibroblast-like features in hAMSCs, which expressed representative mesenchymal cell surface markers CD105 and CD90 and also high levels of CD73, but no CD45, CD34, CD79a, or HLA-2expression was detected. Red mineralized sediments were observed after osteogenic induction by alizarin red staining. Immunofluorescence assays showed that the expression of Opsin1-SW, Opsin2, Opsin3, Opsin4 and Opsin5 was detected on hAMSCs membranes. The results of real-time fluorescence quantitative PCR demonstrated that the expression of Opsin1-SW, Opsin2, Opsin3 ,Opsin4 and Opsin5 mRNA were detected in hAMSCsand opsin3 mRNA were significantly more abundant than other opsins(p<0.05). The results were consistent with that of opsins protein by western blot analysis. ConclusionsOur study is the first report of the expression of opsins in hAMSCs and opsin3 mRNA were significantly more abundant than other opsins.