Cultures of FACS-purified cells have been previously described19. FACS-purified NSCs were seeded at a density of 10 viable cells/μl on uncoated 24-well plates (BD Falcon, Franklin Lakes, NJ), and grown in SCM for 6 days in vitro (DIV) with daily addition of 20ng/ml EGF and 10ng/ml bFGF (Upstate, Charlottesville, VA)19,20. Primary neurosphere colonies were subcloned to assay NSC self-renewal by mechanical dissociation and replated at a density of 10cells/μl on uncoated 24-well plates. It is important to note that in Supplementary Figure 6 neurosphere analysis was performed starting with the 2nd passage. This was done to overcome the experimental limitations due to low number of cells recovered after FACS.
