Neurospheres differentiated into glial cells when cultured in differentiation medium in vitro . A) cGFP-positive neurospheres isolated from β-actin GFP-transgenic mice expressing cytoplasmic GFP (cGFP), maintained 12 days in neurosphere medium in vitro were observed as free-floating aggregates consisting various sized spheres. Most cells within the sphere expressed cGFP. Upon withdrawal of growth factors and culture in differentiation medium for the myelinating culture, on PLL-coated coverslips for 3 days, the majority of neurospheres acquired antigenic properties of astrocytes, as shown with antibody to GFAP (red) (B) or oligodendrocytes as illustrated with the oligodendroglial marker O4 (red) (C). In B and C, the endogenous GFP signal is enhanced with an antibody to GFP. Representative images from at least 3 separate experiments.