Flow cytometric analysis of neural stem cells in the developing and adult mouse brain

Despite recent progress in the neural stem cell biology, their cellular characteristics have not been described well. We investigated various characteristics of neural stem cells (NSCs) in vivo during CNS development, using FACS to identify the NSCs. We first examined stage-dependent changes in the physical parameters, using forward scatter (FSC) and side scatter (SSC) profiles, of NSCs from the developing striatum, where they appear to be active throughout the life of mammals. NSCs were divided into several fractions according to their FSC/SSC profile. With development, their number decreased in the FSC^high fractions but increased in the FSC^low/SSC^high fraction, whereas NSCs were significantly concentrated in the fraction containing the largest cells (about 20 μm in diameter) at any stage, which were mostly the cells with the highest nestin-enhancer activity. Furthermore, we demonstrated that, at all stages examined, the "side population" (SP), defined as the Hoechst 33342 low/negative fraction, which is known to be a stem cell-enriched population in bone marrow, was also enriched for Notch1-positive immature neural cells (about 60%) from the developing striatum. However, these immature SP cells were not detected in the large-cell fraction, however, but were concentrated instead in the FSC^low/mid fractions. FACS analysis showed that SP cells from adults were included to some extent in the CD24^low/ PNA^low fraction, where NSCs were greatly concentrated. Collectively, the characteristics of NSCs were not uniform and changed developmentally.