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A. H. Dahlmann-Noor, S. Vijay, J. S. Ellis, P. T. Khaw; Matrix Environment and Cell-Cycle Inhibitor Hydroxyurea Independently Influence Expression of Neuronal Characteristics by RGC-5 Cell Line. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2111.
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1) In stem cells the extracellular environment can determine differentiation. We tested whether matrix environment influences expression of neuronal characteristics in RGC-5 cells.2) Staurosporine and succinyl concanavalin A induce neuronal differentiation in RGC-5, while other cell cycle inhibitors lack this effect. We tested whether hydroxyurea (HU) +/- neural growth factor (NGF), known to induce differentiation a neuroblastoma cell line, induces RGC5 to assume a neuronal phenotype
RGC-5 culture 1) in or on collagen or Matrigel (Matr) or collagen/Matr 50:50 mix, 2) on glass coverslips coated with either poly-L-lysine (PLL) or 1:100 dilute Matr. Immunocytochemistry for neuronal markers neurofilaments (RT-97) and MAP-2. Epifluorescence microscopy. Image analysis with ImageJ: longest cell diameter, pixel intensity for MAP-2 immunopositivity
1) 3D matrix environment: RGC-5 remain rounded and fail to produce extensions when seeded into Matr or collagen/Matr mix or on top of Matr. Seeding into or on top of collagen induces spindle-shaped morphology. Longest extensions are produced when seeded on top of collagen/Matr mix (top fig). 2) 2D culture (PLL, Matr): untreated RGC-5 display a rounded to oval shape with short filopodial protrusions. Occasional cells show long processes. HU increases the proportion of elongated cells with two or more processes. NGF slightly increases the proportion of elongated cells with processes. Co-administration of HU and NGF does not increase greatest cell length beyond that observed for HU alone. Combined HU and NGF increases expression of neuronal marker MAP-2 (bottom figs)
1) The extracellular matrix has a profound influence on RGC-5 morphology. Culture in/on collagen or on a collagen/Matr mix induces a neuronal phenotype with long extensions. 2) HU and NGF combined induce expression of neuronal characteristics by RGC-5, adding to the range of reported cell cycle inhibitors with this effect
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