Experimental Study On Mouse Retinal Müller Cells Differentiate Into Neural Stem Cells

Objective:To investigate whether the adult mouse retinal Müller cells could differentiate into neural stem cells under the cultural condition of neural stem cells,and the differentiation ability of Müller cell-derived neural stem cells.Methods:In this experiment,mouse retinal Müller cells were isolated and cultured through tissue block adherent culture method.Because of Müller cells have a strong adherent ability,we used the method of repeated trypsin digestion and multiple culture to purificate them.We collected P3 Müller cells to detect the expression of GS and Vimentin by immunocytochemistry.Then we digested these Müller cells into single cell suspension and adjust the cell density to 2×10~5/mL,and put them into the neural stem cells cultrue medium(20 ng/mL bFGF,20 ng/mL EGF,B27),the experssion of neural stem cell marker Nestin was detected by immunocytochemistry.Then these cells were cultured in serum-containing medium to observe the differentiate ability of the neuropheres.After that,the expressions of these differentiated cells were tested by immunocytochemisty,including Müller cell markers GS,Vimentin,neuron markers?-tubulin III,MAP2ab,astrocyte marker GFAP,oligodendrocyte marker RIP,amacrine cell marker Syntaxin,and bipolar cell marker PKC?.Results:1.Müller cells could be effectively obtained through the tissue culture method.Müller cells grow in clusters at an early stage with the shape of long fusiform.Immunocytochemistry showed that the expression of GS and Vimentin were positive in the P3 generation of Müller cells.2.The P3 generation of Müller cells were gathered together and formed"mulberry-like"spherical structure after cultured in neural stem cells medium for 3-5 days,the expression of Nestin was positive by immunocytochemistry.3.After Müller cells-derived neural stem cells were cultured in serum-containing medium for 7 days,the cells were adherent with various shapes.Immunocytochemistry demonstrated that the expression of GS,Vimentin,?-tubulin III,MAP2ab,GFAP,RIP,PKC?were positive.Conclusion:1.Müller cells could be effectively obtained through the tissue culture method.2.Mouse retinal Müller cells can be differentiate into neural stem cells by serum-free suspension culture in vitro.3.Müller cell-derived neural stem cells can be differentiate into glia-like cells and neuron-like cells in vitro.