| Objective:1. To investigate the isolation, culture and identification of human amniotic mesenchymal stem cells, observe its biological characteristics and study its feasibility for neural regeneration therapies.2. To investigate the plasticity of human amniotic mesenchymal stem cells into retinal neuron-like cells through the experiment of induced differentiation in vitro.3. To study the development of sodium ion channel in human amniotic mesenchymal stem cells during its differentiation.Methods:1. Six placenta samples through cesarean delivery were collected aseptically with the informed consents of parturients. Human amnion was peeled from placenta by mechanical method. After being rinsed with PBS solution, the tissue was cut into pieces with microsurgical scissors and digested with 0.25% trypsin-EDTA at 37℃four times (10 minutes each). The cells were cultured and purified with complete medium consisting of DMEM/F12/10% FBS/10ng/mL EGF.2. The morphology of human amniotic mesenchymal stem cells was observed under phase contrast microscope every day, and the growth curve was drawn. The cell surface antigenic characteristics of the cultured amniotic mesenchymal stem cells at passage 1-3 were analyzed by flow cytometry. The expressions of neural stem/progenitor cell markers were evaluated by immunofluorescent staining and flow cytometry.3. The passage 3 of the expanded amniotic mesenchymal stem cells were induced with 2% DMSO/200μmol/L BHA/10ng/mL bFGF in vitro. The specific markers of neural stem/progenitor cells, neurons, glia and photoreceptors were detected by immunofluorescent staining and flow cytometry before and after induction. The levels of nestin, rhodopsin and SCN2A mRNA were assayed by real-time quantitative polymerase chain reaction at 0,3rd and 7th day’s induction. Whole cell patch clamp technique was performed to record the current of Na+Results:1. Human amniotic mesenchymal stem cells were adherent cells with a fibroblastoid, spindle-shaped morphology. It could be expanded in vitro. The flow cytometry analyses revealed that the expression of surface antigens, such as CD90(Thy-1) and CD49d were positive, but CD45 was negative. Nestin and vimentin positive cells made up 80～90% of total cells, and more than 90% of cells were found to be Musashi-1 positive, suggesting that they have neural differentiation potency.2. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. The number of nestin positively stained cells decreased remarkably after 7 day’s induction (from 81.3% to 24.9%, P<0.05), whereas the number of MAP2, rhodopsin and NSE positively stained cells increased significantly after induction (from 15.7% to 91.3%,10.3% to 97.1%,20.9% to 94.3% respectively, P<0.001). Meanwhile, GFAP positive cells increased from 1.4% to 18.0%(P<0.05). The expression of nestin mRNA decreased precipitately relative to the starting material. However, steady and time-dependent increases in the expression of rhodopsin and SCN2A mRNA were observed when cells were cultured in neural induction media. The inward current was absent (0/13) before induction, whereas some neuron-like cells (4/12) could be stimulated to show the current of sodium ion after induction (P=0.039). Conclusions:1. Human amniotic mesenchymal stem cells can be cultured and expanded in vitro with stable biological characteristics. These amniotic cell populations have phenotypes of neural stem/progenitor cells and might be of value in therapeutic applications that require human neural cells.2. Human amniotic mesenchymal stem cells can differentiate in vitro into excitable retinal neuron-like cells. However, further studies are necessary to determine whether such in vitro differentiated cells can function in vivo as neural cells. |
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