FTY720 induces differentiation of neurospheres both into neurons and oligodendrocytes.
a Schematic view of the protocol used for differentiation of SVZ-derived neurospheres into neurons or oligodendrocytes. SVZ from young rats (P2–P4) was dissociated to obtain proliferating NSCs. Cells were maintained for 7 days in vitro (DIV) in the presence of the mitogenic factors EGF, bFGF and PEDF (proliferation phase). After 7 DIV, neurospheres were differentiated into neurons up to 14 days, in the presence of NGF and BDNF or pre-committed to an oligodendrocytic phenotype for 3 days and differentiated for up to 4 days in presence of NT3 and CNTF (differentiation phase). FTY720 was added to the cultures during the differentiation phase at different concentrations (10 and 100 nM). At DIV 14, cells were fixed and immunofluorescence was performed using anti-β-III tubulin (b, c) or CNPase (e, f) as markers of mature neurons or oligodendrocytes, respectively (b, c and e, f: representative photomicrographs of the staining). Total nuclei were stained with propidium iodide (PI). Fluorescence intensity both from neurons (β-III tubulin) or oligodendrocytes (CNPase) and total nuclei (PI) was measured using a fluorescence plate reader with appropriate excitation and emission filters. Differentiation was evaluated as a ratio of β-III tubulin or CNPase fluorescence intensity over total nuclei fluorescence intensity (d, g). Data are expressed as mean ± SEM of 6–7 independent experiments. Statistical analysis: paired, two-tailed Student t test; *p < 0.05 and **p < 0.01 vs control