Stem Cell Characteristics Of Human Amniotic Epithelial Cells And Their Differentiation Into Neuron - Like Cells Induced By Different Concentrations Of Retinoic Acid

OBJECTIVE:1. Establish a system of isolation and culture of human amniotic epithelial cells (HAECs),to isolate,culture,identify,purify and amplify the cells (hAECs) in vivo.2. Study the biological characteristics and investigate the transdifferentiation ability into neural-like cells of HACEs.3. Explore the optimal concentration of retinotic acid when inducing human amniotic epithelial cells into neural-like cells.METHODS:1. After cleaned and cut up, the amniotic membrane was digested by 0.25% type Ⅱ collagenase for 2 hours,then 0.25% trypsin/0.02% EDTA for 15 min. The tissue was digested into cell suspension which contains thousands of separated cells. The separated cells were cultivated in the culture flask with the density of 1 × 107/ml,observed by microscope sooner.Using immunocytochemistry and immunofluorescence technology to identify the characteristics of the adhered cells.2. Use immunohistochemical technology to identify the expression of SSEA-4,Oct 4,nestin and musashi in human amniotic epithelial cells(HAECs).Study the stem cell characteristics of human amniotic epithelial cells(HAECs).3. HAECs were induced in different concentrations(1 × 10-8M,1×10-7M、1 × 10-6M、 1 × 10-5M) of retinoic acid for seven days, then investigated the expression of Oct-4,nestin,MAP-2 and GFAP by immunohistochemical technology,western-blotting and Real-time PCR technology.RESULTS:1. The separated cells adhered after 2-3 days,observing by microscope. The cells had a bigger size,reduced refraction and dimmed contour. The separated cells had varied shapes, such as polygon, eye-like, fusiform and so on. The separated cells had a clear shape.The cells had abundant cytoplasm and a round/ovoid nuclear centered.The amniotic epithelial cells growed fast in exponential phase, and the cells are irregular polygon. After 5-7 days, the cells became confluent, which look like radially arranged and spirally shaped. As the cell immunohistochemistry and immunofluorescence showed, the specific marker of epithelial cells-CK19, was strongly positive expressed in all the adherent cells. On the contrary, specific marker of mesenchymal cells-vimentin, was weakly positive expressed in some cells.2. As immunocytochemistry showed:the specific markers of embryonic stem cells-- SSEA -4,Oct 4 and the specific markers of neural stem cells-nestin and musashi, were positive expressed in the human amniotic epithelial cells in vivo.3. As immunocytochemistry showed:the expression of Oct-4 in the experimental group is reduced compared with the control group.However, the expression of nestin、MAP-2 and GFAP were increased. With the increased concentration of the retinoic acid, the expression of Oct-4 was gradually reduced and the expression of nestin MAP-2 and GFAP were gradually raised. The cells began to die when added 1 × 10-5 M retinoic acid. Western-blotting showed: compared with the control group, the expression of nestin、MAP-2 and GFAP was significantly increased in the experimental group, which has a significant difference observed in statistics(p<0.05). The expression of nestim MAP-2 and GFAP in the cells under the concentration of 1 × 10-7M increased compared with the cells under the concentration of 1×10-8M, which has a significant difference observed in statistics(p<0.05). The expression of nestin nestin、MAP-2 and GFAP in the cells under the concentration of 1 × 10-6M increased compared with the cells under the concentration of 1 × 10-7M, which has a significant difference observed in statistics(p<0.05). The expression of nestin、MAP-2 and GFAP in the cells under the concentration of 1 × 10-5M increased compared with the cells under the concentration of 1×10-6M, which has no difference observed in statistics(p>0.05). Compared with the control group, the expression of Oct-4 was significantly increased in the experiment group, which has a significant difference observed in statistics(p<0.05). The expression of Oct-4 in the cells under the concentration of 1 ×10-7M increased compared with the cells under the concentration of 1 ×10-8M, which has a significant difference observed in statistics(p<0.05). The expression of Oct-4 in the cells under the concentration of 1×10-6M increased compared with the cells under the concentration of 1×10-7M, which has a significant difference observed in statistics(p<0.05). The expression of Oct-4 in the cells under the concentration of 1×10-5M increased compared with the cells under the concentration of 1 × 10-6M, which has no difference observed in statistics(p>0.05).Real-time PCR:compared with the control, the expression of nestin、MAP-2 and GFAP was significantly increased in the experiment group, which has a significant difference observed in statistics(p<0.05). The expression of nestin、MAP-2 and GFAP in the cells under the concentration of 1×10-7M increased compared with the cells under the concentration of 1 ×10-8M, which has a significant difference observed in statistics(p<0.05). The expression of nestin、MAP-2 and GFAP in the cells under the concentration of 1×10-6M increased compared with the cells under the concentration of 1 ×10-7 M, which has a significant difference observed in statistics(p<0.05). The expression of nestin、MAP-2 and GFAP in the cells under the concentration of 1×10-5 M increased compared with the cells under the concentration of 1 ×10-6M, which has no difference observed in statistics(p>0.05). Compared with the control, the expression of Oct-4 was significantly increased in the experiment group, which has a significant difference observed in statistics(p<0.05). The expression of Oct-4 in the cells under the concentration of 1×10-7M increased compared with the cells under the concentration of 1 ×10-8M, which has a significant difference observed in statistics(p<0.05). The expression of Oct-4 in the cells under the concentration of 1 × 10-6 M increased compared with the cells under the concentration of 1 ×10-7M, which has a significant difference observed in statistics(p<0.05). The expression of Oct-4 in the cells under the concentration of 1 ×10-5M increased compared with the cells under the concentration of 1 ×10-6M, which has no difference observed in statistics(p>0.05).CONCLUSIONS:1. Isolate, culture, identify, purify and amplify the cells (hAECs) in vivo.2. Human amniotic epithelial cells can express Oct-4,SSEA-4,nestinand musashi, so they had the characteristic of embryonic stem cells and neural stem cells. Maybe they have the potential to differentiate into neural-like cells.3. As immunocytochemistry, western blot and real-time PCR showed:Human amniotic epithelial cells can be induced into neural-like cells by retinoic acid. 1 ×10-6 M retinoic acid is the optimal concentration to induce human amniotic epithelial cells into neural-like cells.