Atoh7Promotes The Mechanism Of Differentiation Of Miiller Cells Into Retinal Ganglion Cells

Chapter1purification culture and dedifferentiation of retinal Muller cellsObjective:To study purity culture and dedifferentiation of rat retinal Muller cells in vitro and identification.Methods:Retina of SD rat at postnatal10-21days were dissociated from the eye balls. The passage culture method of Muller cells was carried out by repeated incomplete pancreatic enzyme digestion method. Fhe third passage of retinal Muller cells was detected by fluorescence-activated cell sorter (FACS), immunohistochemistry technology and RT-PCR to determine the purity. The third passage of retinal Muller cells was induced in the serum-free dedifferentiation medium (containing1x N2,2x B27,20ng/ml EGF,10ng/ml bFGF). The cell proliferation state was observed under an inverted microscope. The expression of the specific markers Ki-67and Nestin of retinal stem cells was measured by RT-PCR and Wester blot. The cell proliferation of retinal stem cells was detected by Edu staining.Results:FACS demonstrated that the purity of retinal Muller cells was more98.01%. Under the fluorescence microscope, about98.50%±1.08%of the cells were GS positive. RT-PCR analysis indicated that the specific markers of Muller cells(GS、Vimentin) mRNA was at a higher level; and the specific marker of other retinal cells mRNA was almost no expression. The purified Muller cells were cultured in stem cell-conditioned medium for3-7days, the proliferation was clonal, and a few spherical or mulberry-shaped cell spheres appeared. Immunofluorescence staining showed that stem cells within the spheres were positive for retinal stem cell-specific markers Nestin (green fluorescence,92.94±6.48%) and Ki-67(red fluorescence,85.96±6.04%). Meanwhile, RT-PCR analysis showed that cell spheres in the culture expressed a battery of transcripts characteristic of stem cells such as Nestin and Ki-67, which were absent in the Muller cells. Western blot analysis further confirmed the expression of Nestin and Ki-67in the cell spheres but not in the Muller cells. Edu staining showed that most of the nuclei within the cell spheres were stained red (82.80±6.65%), suggesting that the new cell spheres have the capacity for effective proliferation.Conclusion:The repeated incomplete pancreatic enzyme digestion method is an efficient and practical method to purify retinal Muller cells. Retinal stem cells were successfully cloned in the dedifferentiation medium. Retinal Muller cells were an accessible source of retinal stem cells.Chapter2Atoh7promotes the differentiation of retinal stem cells derived from Muller cells into retinal ganglion cells in vitroObjective:To investigate the regulation of Atoh7on retinal stem cells derived from Muller cells dedifferentiating into ganglion cells in vitro.Methods:Purified retinal Muller cells were induced to differentiate into retinal stem cells in vitro. The multiplicity of infection (MOI) and infection efficiency were determined by preliminary experiments. Retinal stem cells were divided into three groups randomly:group A was transfected by PGC-FU-Atoh7-GFP lentivirus; group B was transfected by PGC-FU-GFP vector; group C was non-transfection as contrast. After replacing the differentiation medium, the intervention was preformed in according to the above groups respectively; The retinal stem cells were observed daily under the inverted phase contrast microscope. At7days and14days, double labeling immunofluorescence of ganglion cell-specific markers Thy1.1and Brn3b was detected and calculated the percentage of ganglion cells; The statistical software SPSS13.0was used, and the one-way ANOVA and Student’s t-test was selected for statistical analysis, the P<0.05was considered significant statistically. The expression of Atoh7, Brn-3b, Isl-1and Notch were detected by RT-PCR and Western-blot.Results:MOI=10, FACS analysis showed that the transfection efficiency was75.41%. After transfection for7days, the percentage of ganglion cells was similar between cells transfected with PGC-FU-GFP and cells untransfected (13.30±13.25%versus9.10±3.21%), significantly lower than that in cells transfected with PGC-FU-Atoh7-GFP (50.40±8.22%)(F=61.642, P<0.001). At14days after transfection, immunocytochemical analysis showed that the percentage of ganglion cells was8.40±2.32%(group B),8.20±3.19%(group C) and49.90±10.16%(group A)(F=58.366, P<0.001) These results showed that at7and14days, the efficiency of Atoh7to promote the differentiation of retinal stem cells into retinal ganglion cells was similar (50.40±8.22%versus49.90±10.16%).The differences between corresponding group A and group B or C were not statistically significant(P>0.05). RT-PCR and Western-blot analysis showed that Atoh7, Brn-3b and Isl-1were up-regulated in addition to Notch gene down.Conclusion:Atoh7can up-regulate the differentiation of retinal stem cells derived from Muller cells into retinal ganglion cells in vitro and efficiency of differentiation was maximum after transfection for7 days. Atoh7regulates the expression of Atoh7, Brn-3b and Isl-l,and down-regulates negative pathway of Notch.Chapter3The signaling pathways of Atoh7regulates the differentiation of retinal stem cells derived from Muller cells into retinal ganglion cellsObjective:To investigate the signaling pathways of Atoh7on retinal stem cells derived from Muller cells dedifferentiating into ganglion cells in vitro.Methods:Retinal stem cells derived from Muller cells were divided into three groups randomly:group al:Brn-3b siRNA group, group a2: Control siRNA group (scrambled sequence), group a3:Control group (without any handling); group b1:Isl-1siRNA group, group b2:Control siRNA group (scrambled sequence), group b3:Control group (without any handling); group c1:Notch signal pathway inhibitor (GSI) group, group c2:Control group (without any handling). The stem cells were transfected with the indicated siRNA or treated with GSI. After72h, the cells were transfected with lentivirus PGC-FU-Atoh7-GFP. At7days after transfection, immunofluorescence staining was performed to detect the percentage of ganglion cells in total differentiated cells. The statistical software SPSS13.0was used, and the one-way ANOVA and Student’s t-test was selected for statistical analysis, the P<0.05was considered significant statistically. The expression of Atoh7, Brn-3b, Isl-1and Notch were detected by RT-PCR and Western-blot.Results:Statistics showed that the percentage of the ganglion cells was:group A (F=58.366, P<0.001):23.30%±4.45%(group al), 50.60%±7.04%(group a2) and49.70%±7.36%(group a3); group B (F=107.771, P<0.001):25.90%±3.35%(group b1),49.90%±5.38%(group b2) and49.20%±4.64%(group b3); group C (t=3.211, P<0.001):58.20%±6.46%(group c1) and49.40%±5.78%(group c2). The difference was also statistically significant in c1group comparison with a3and b3group (F=6.533, P=0.005). Western-blot and RT-PCR showed that the protein and mRNA expression of Brn-3b, Isl-1and Notch were reduced in the Brn-3bsiRNA group, the Isl-1siRNA group and the Notch signal pathway inhibitor group with the time. Atoh7up-regulates the expression of Atoh7, Brn-3b and Isl-1, and down-regulates the expression of Notch.Conclusion:Brn-3bsiRNA and Isl-1siRNA were able to inhibit retinal stem cells derived from Muller cell differentiation into retinal ganglion cells. However, Notch was able to promote retinal stem cells derived from Muller cell differentiation into retinal ganglion cells. Atoh7and gamma-secretase inhibitors (GSI) were synergistic in the inhibition process of retinal stem cells derived from Muller cells differentiation into retinal ganglion cells. Up-regulation of Atoh7, Brn-3b and Isl-1and down-regulation of Notch jointly promote retinal stem cells derived from Muller cell differentiation into retinal ganglion cells. Chapter4Atoh7promotes the differentiation of Muller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucomaObjective:This study aimed to develop novel protocol to promote the differentiation of retinal Miiller cells into ganglion cells in vivo in a rat model of glaucoma.Methods:Ocular hypertension was induced using laser photocoagulation and identified. Retinal Miiller cells were induced to differentiate into retinal stem cells in vitro; The retinal stem cells were divided into three groups randomly:group A was transfected by PGC-FU-Atoh7-GFP lentivirus; group B was transfected by PGC-FU-GFP vector; group C was non-transfection as contrast. After24h of transfection, the neurospheres were dissociated into single stem cells with Accutase. The stem cells of each group were collected at a concentration of1x104cells/μl. Vitreous cavity was injected with5μl of retinal stem cells,5μl BDNF (1ng/ml),30nmol BrdU,100ng RA (1μM). At days7and14, Eyeball frozen sections and immunohistochemistry were performed for immunocytochemical analysis. The cell proliferation of retinal stem cells was detected by BrdU staining. The expression of Nestin、Ki-67、 Atoh7and Brn-3b were detected by RT-PCR and Western-blot.Results:The IOP of glaucomatous eyes was elevated significantly compared with those of controlateral eyes from day3to day28. IOP was gradually increased with the time and reached the maximum in7-14days. IOP began to decline about day28and returned to normal level about2months later. TUNEL staining of retinal ganglion cells in rat chronic ocular hypertension glaucoma model showed that apoptosis of ganglion cells was detected at7days after laser photocoagulation and with the number of apoptotic cells was increasing with HIOP. At day60, IOP was decreased to the normal level, but the number of apoptotic cells was still increasing. The nuclei of Nestin and Ki-67positive staining was migrated from the vitreous chamber to retinal depth with the time. Western-blot and RT-PCR analysis showed that the amount of protein and mRNA of Nestin and Ki-67at14days were more than at7days. Western-blot and RT-PCR analysis showed that the expression of Atoh7and Brn-3b at both mRNA and protein levels were significantly increased in group A. Immunocytochemical staining of retinal tissue sections showed that the proportion of Brn-3b positive cells was21.3%±2.0%in group A, but only7.9%±1.1%and7.8%±1.7%of positive cells expressed Brn-3b in group B and C.Conclusion:Atoh7promotes the differentiation of Muller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma. Nestin and Ki-67staining nuclei over time to the retina from the vitreous chamber depth migration.