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Justin Chew, Chenying Guo, Maximilian Staudt, Dongfeng Chen; <b>Using the RGC-5 Cell Model for the Study of Retinal Ganglion Cell Development and Axon Regeneration</b>. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3493.
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To characterize and optimize the culture conditions for the RGC-5 cell line as a model for the study of retinal ganglion cell (RGC) development and axon regeneration.
RGC-5 cells were cultured in High Glucose DMEM at a cell density of 4 x 104 cells/mL. Cells were induced to undergo terminal differentiation under four different conditions: 10 nM staurosporine + human non-pigmented epithelium (HNPE)-conditioned medium, 50 nM staurosporine + HNPE-conditioned medium, 50 nM staurosporine, and 316 nM staurosporine. Optimal cell differentiation conditions were assessed by gene expression profiles and quantification of RGC neurite outgrowth. Genes examined included mature RGC markers, such as beta3-tubulin, Map2ab, Thy 1.2, Tau, and Brn3b. Additionally, expression levels of insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R were examined. Gene expression was quantified using qRT-PCR and western blot analysis, and induction of expression of RGC markers was observed through immunohistochemical staining.
The optimal differentiation condition for RGC-5 was found to be 316 nM staurosporine, which caused cells to adopt a cell morphology and gene expression profile most similar to primary RGCs. Differentiation events were found to occur largely in the first 12-24 hours after addition of staurosporine, with neurite growth occurring in conjunction with an observed upregulation of mature RGC markers. All measured RGC markers were upregulated 3.5 to 4-fold within two hours after addition of staurosporine, with the exception of Map2ab, which was not significantly upregulated. IGF-1 was found to be drastically upregulated over 20-fold during the first two hours after addition of staurosporine.
RGC-5 may serve as a valid RGC model through induction of differentiation with staurosporine. Upregulation of RGC markers suggests that differentiated RGC-5 resembles primary RGCs genetically. Furthermore, the drastic upregulation of IGF-1 in tandem with neurite outgrowth agrees with previous literature describing it as an important axonal growth factor.
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