NPCs were acutely dissociated from P8 CNP-hEGFR and WT SVZ mice and then processed for purification using anti-prominin-1 microbeads antibodies (Miltenyi Biotec, Auburn CA) following manufacturer recommendations. Purified NPCs were cultured at high density in a monolayer in the presence of EGF and bFGF, as described above. After 48 hrs, FACS-purified WT GFAP-GFP+LeX+ cells were plated on top of NPCs for 5 days in EGF- and bFGF-containing medium. Finally, GFAP-GFP+ cells were purified from the co-culture by FACS and assayed in neurosphere cultures to determine proliferation and self-renewal. FACS-purified GFAP-GFP+ cells were seeded at a density of 5 viable cells/μl on uncoated 12mm-well plates (BDFalcon, Franklin Lakes, NJ), and maintained in SCM for 6DIV with daily addition of EGF and bFGF. Primary neurosphere colonies were subcloned by mechanical dissociation in SCM with EGF and bFGF. Cells were re-plated at a density of 5 cells/μl on uncoated 24-well plates. Stem cell self-renewal was assessed after further 6 DIV.
