Fig. 1. Protocol used for analyzing the differentiation potential of neurospheres. Fully dissociated neurospheres were grown in serum-free neurosphere culture medium, containing EGF (20 ng/ml) for various times, generally not exceeding 3 days in order to minimize necrosis, which might affect the core of larger spheres, and the generation of new stem cells, which might be at the origin of a `subclone' whose developmental potential could interfere with interpretation. At t=0, spheres (50-100) were deposited on coverslips coated with polyornithine and allowed to differentiate in neurosphere culture medium containing 2 ng/ml EGF, in order to reduce proliferation. After various times of differentiation, spheres were fixed and processed for immunocytology, and analyzed by confocal microscopy (the observation plane being at the basis of the spheres where the cells differentiate).

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