Study Of The Differentiation Of Retinal Progenitor Cells Into Retinal Ganglion Cells

Part I Cultivation and identification of retinal progenitor cells Objectives: To isolate, cultivate and identificate RPCs of different gestation age.Methods: RPCs of E14 and E18 SD rats were isolated, cultivated in suspension in modified DMEM/F12 serum-free medium and then induced differentiation in vitro. Cells were observed under phase-contrast microscopy daily. RPCs were identified by immunocytochemistry, scanning electron microscope and transmission electron microscope.Results: Cultivated in DMEM/F12 serum-free medium, RPCs formed cell spheres. After plating, RPCs migrated outwards from cell spheres and differentiated. Scanning electron microscope demonstrated morphology of the cell spheres and differentiated cells. Transmission electron microscope demonstrated that there were RPCs-like cells in cell spheres and neuron- and glia-like cells after plating. Immunocytochemistry demonstrated that most cells in cell spheres expressed neural stem cell marker Nestin and cell division marker BrdU. After plating, RPCs could differentiate into various retinal cells, including Thy 1.1-positive RGCs. The percentage of RGCs of E14 was 16.91%±4.05% and that of E18 was 4.65%±1.88%. The differences were statistically significant(t=15.04, P<0.001).Conclusions: RPCs could be cultivated successfully in modified DMEM/F12 serum-free medium. The cultivated RPCs have the potential to proliferate undefinately and multi-differentiate. Early RPCs are inclined to differentiate into RGCs.Part II Influence of microenvironment on retinal progenitor cells differentiating into retinal ganglion cellsObjectives: To investigate the influence of optic nerve crush model on RPCs differentiating into RGCs.Methods: One eye of adult male SD rats was randomly selected in this experiment, crushing the optic nerve for the test group and exposing the optic nerve without crushing for the control group. Cultured RPCs of E14 SD rats were marked with BrdU and transplanted into the vitreous cavity. Animals were sacrificed at 1w, 2w, 4w following transplantation. Immunocytochemistry methods were used to examine the migration, incorporation and differentiation of the grafted cells.Results: The majority of the grafted RPCs in the control group remained in the vitreous cavity and were not observed migration and differentiation with time. However, when transplanted in the test group, these cells were observed migrating into different layersof the retina. The proportion of the grafted cells that migrated into the inner retina was significantly higher than that migrated into the outer retina and the most was in the GCL. With time, the proportion that migrated into the outer retina increased, but it was still far lower than that migrated into the inner retina. Immunocytochemistry methods demonstrated that the grafted cells in the different layer of the retina in the test group expressed different retina-specific markers. Thy 1.1-positive cells were primarily in the GCL and then the INL.Conclusions: Optic nerve crush model could induce RPCs to migrate into the GCL and differentiate into RGCs.Part III Role of Muller glia on retinal progenitor cells differentiating into retinal ganglion cellsObjectives: To determine whether Muller glia or MCM enhance the differentiation of RPCs into RGCs.Methods: E14 or E18, and P7 SD rat retinas were used for culturing RPCs and Muller glia respectively. Then RPCs were induced differentiation in vitro (Muller(-) group) or co-cultured with Muller glia (Muller(+) group). In order to examine whether the effects of Muller glia on neurogenesis depend on diffusible and membrane-associated factors, RPCs were re-suspended in MCM,plated onto coated coverslips (MCM group). The proliferating nature and progenitor properties of the cultured cells were determined by Immunocytochemistry and images were taken by a fluorescence microscope equipped with phase-contrast optics or confocal laser scanning microscope and analyzed by Leica Qwin V3.1 system.Results: Cultured in Muller(-), Muller(+) and MCM, the percentage of RPCs differentiating into RGCs was 16.91 %±4.05%, 47.25%±9.67% and 42.36%±10.52% at E14, 4.65% ±1.88%, 9.90%±3.19% and 8.69%±3.01% at E18, respectively. The differences between the percentage of RGCs cultured in Muller(+) or MCM and that in Muller(-) were statistically significant (P<0.001). The differences between the percentage of RGCs cultured in Muller(+) and that in MCM were not statistically significant (P>0.05). The percentage of proliferating cells cultured in Muller(-) and MCM was 32.13%±6.11% and 57.07%±9.31% at E14, 31.51%±6.32% and 56.18%±8.79% at E18. The differences between the percentage of proliferating cells cultured in MCM and that in Muller(-) were statistically significant (t_E14=12.28 , P<0.001 ; t_E18=12.49, P<0.001).Conclusions: Co-culture of RPCs and Muller glia could enhance RPCs proliferate and differentiate into RGCs. MCM may play therole.Part IV Regulation of Notch-1 on retinal progenitor cells differentiating into retinal ganglion cellsObjectives: To investigate the regulating role of Notch-1 on RPCs differentiating into RGCs.Methods: RPCs of E14 SD rats were cultured and then induced differentiation in the culture medium with Notch-1 antisense oligonucleotides (test group) or missense oligonucleotides (control group). Cells were observed under phase-contrast microscopy daily. RGCs were identified by Thy1.1 immunocytochemistry methods and analyzed by Leica Qwin V3.1 system.Results: RPCs cultured in such medium could differentiate into various retinal cells, including Thy 1.1-positive RGCs. The percentage of RGCs was 31.19%±6.90% in test group and 16.57%±4.31% in control group. The differences were statistically significant (t=9.84, P<0.001).Conclusions: Notch-1 may down-regulate RGCs differentiation. Inhibition of Notch-1 could promote RPCs to differentiate into RGCs.Part V Regulation of Math5 on retinal progenitor cells differentiating into retinal ganglion cellsObjectives: To investigate the regulating role of Math5 on RPCs differentiating into RGCs.Methods: RPCs of E14 SD rats were cultured and then transfected by constructed Math5-EGFP plasmid with EGFP plasmid as contrast. RPCs were observed under phase-contrast microscopy daily, especially their differentiation into RGCs. RGCs were identified by Thy1.1 immunocytochemistry methods and analyzed by Leica Qwin V3.1 system. Furthermore, fluorescence quantified RT-PCR was used to examine the expression of Math5-associated genes at different time point during the differentiation of RPCs.Results: Math5-EGFP/EGFP plasmid could transfect RPCs successfully and the transfection rate was 24.68%. After transfection, RPCs could still differentiate into various retinal cells, including Thy 1.1-positive RGCs. The percentage of RGCs was 30.85%±6.28% in A group (Math5-EGFP plasmid transfected), 15.84%±3.55% in B group (EGFP plasmid transfected) and 16.22%±3.60% in C group (no plasmid transfected). The differences between A group and B or C group were statistically significant (P<0.001) . The expression of Math5-associated genes, including Delta-1, Hes1 and Brn-3b was different at different timepoint during the differentiation of RPCs. Transfection by Math5-EGFP plasmid could not only change the expressing quantity of Delta-1, Hes1 and Brn-3b, but also change their expressing curve.Conclusions: Math5 may up-regulate RGCs differentiation. Overexpression of Math5 could promote RPCs to differentiate into RGCs and change the expression of Math5-associated genes.