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J. P. Wood, G. Chidlow, A. Ebneter, J. Crowston, R. J. Casson; A Comparison of Differentiation Protocols for RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):147.
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The RGC-5 cell line is being increasingly used for studies into ganglion cell response to pathological insults. Although originally described to be fully differentiated to a ganglion cell phenotype after treatment with succinyl concanavalin A (sConA), it has become apparent that these cells are now less responsive to this agent. Recently, the alternative compounds staurosporine (STSN) and trichostatin A (TCA) have also been described to terminally differentiate RGC-5 cells. We therefore compared the response of such cells to each of the three agents, in order to definitively ascertain which treatment rendered a cellular phenotype closest to true ganglion cells.
RGC-5 cells were passaged when approximately 70% confluent into appropriate vessels. After remaining in serum-free medium for 24 hours, cells were treated for 3 days with either STSN (300nM), TCA (500ng/ml), sConA (50microg/ml) or vehicle (controls). Cells were subsequently assayed for neuronal and ganglion cell markers by immunocytochemistry, immunoblotting and RT-PCR. In all cases, expression was compared with adult rat-derived ganglion cells maintained in culture for 14 days.
Control, vehicle-treated RGC-5 cells expressed trace levels of mRNA and proteins for the neuronal markers, tau, beta-tubulin, Neu-N, MAP2 and PGP9.5, as did the adult rat-derived ganglion cells. Although treatment with sConA had no effect on the expression of these markers, STSN and TCA both markedly increased the levels of beta-tubulin, PGP9.5 and MAP2, with the latter agent also increasing the levels of tau. The effect of TCA was much more marked than STSN in each case. No RGC-5 cells expressed the ganglion cell markers, Brn-3, Thy-1 or neurofilament (either 68kD or 200kD forms), or other retinal neurone markers such as PKC-alpha, calretinin or calbindin. Interestingly, all cells, whether treated or not, expressed high levels of the neuronal precursor protein, nestin. Expression levels of protein species investigated matched the levels of their appropriate mRNAs in all cases.
RGC-5 cells expressed low-levels of a number of neuronal but not ganglion cell specific proteins and mRNAs. These were unaffected by treatment with sConA, but STSN, and more particularly, TCA caused specific upregulation of such markers. In conclusion, although none of the tested agents led to expression of ganglion cell-specific marker proteins or mRNAs, treatment with TCA represented the best means by which to terminally differentiate RGC-5 cells into a true neuronal phenotype.
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