h and were characterized for their neural stem cell capacity with nestin and BrdU staining every 3–6 months. The long-term DRG NSC cultures were maintained in the above-mentioned medium in the presence of EGF and basic fibroblast growth factor (bFGF) (10 ng/ml each) to further screen for EGF/bFGF-responsive NSCs. In brief, DRG NSC were maintained in free-floating neurosphere culture, in the above medium (changed twice/week), continuously for 3–4 years; after this time samples from the main DRG NSC lines were periodically frozen. During the biweekly medium changes throughout the years, no dissociation (trypsin digestion and cell transferring) was performed. Although we have not performed karotyping to verify spontaneous transformation, we noted no signs of tumor formation in our in vivo or in vitro assay. To assure long-term in vivo use of these cells, karyotying should be performed to assure this potential nature.
