Figure 4: Frequency of stem and progenitor cells, using the NSA and N-CFCA, among the cells isolated by the shaking method, as well as immunophenotyping of the isolated neurons. (A): Representative sphere from isolated cells, 7 days after plating in the NSA culture. (B, C): Typical small and large (> 2.0mm) colonies that are respectively derived from a neural progenitor cell and a bona fide NSC, 21 days after plating in the N-CFCA. (D): The mean sphere forming frequency in the NSA (C) and the mean colony forming frequency in in the N-CFCA of the cells isolated based on the shaking method. No large colonies (> 2mm) (D) were formed by the cells isolated based on the shaking method and the colonies could only reach to a maximum size of 500 μm (B). (mean ± SEM; n=4 independent experiments). (E): Representative micrographs from differentiated purified neuronal cells 7 days after plating. Isolated neuronal cells were terminally differentiated in astrocyte conditioned medium supplemented with 20ng/ml of BMP-4 and 20ng/ml of BDNF and almost exclusively all of the MAP-2 IR cells were expressing GAD 65/67 and DARPP-32 after 7 days. Scale bars = 50 μm for A and E, and 500 μm for B and C. Abbreviations: NSA= Neurosphere assay, N-CFCA= neural- colony forming cell assay, BMP-4= Bone morphogentic protein-4, BDNF= Brain derived neurotrophic factor, GAD 65/67= Glutamic acid decarboxylase 65/67, DARPP-32= Dopamin and cAMP regulated phosphoprotein-32, Map-2= Microtubule associated protein-2.