Derivation and Expansion of Long-Term Neural Stem Cells from Human iPSCs

The human iPSC-derived lt-NESCs were produced from human skin fibroblasts as previously described [9 (link)]. Briefly, human fibroblasts were subjected to retroviral transduction with plasmids encoding for the viral glycoprotein VSV-G and the reprogramming factors Oct4, Sox2, KLF4, and c-MYC and split into plates with mouse embryonic fibroblasts. Colonies were then picked and expanded to establish human iPSC lines. Those lines were induced to differentiate to neural phenotype as previously described [10 (link)] through an embryoid body-production step. Neural rosettes were generated and carefully picked and grown in the presence of 10 ng/ml fibroblast growth factor (FGF) 2, 10 ng/ml epidermal growth factor (EGF; both from R&D Systems, Inc., Minneapolis, MN, USA), and 1 μl/ml B27 (Invitrogen, Carlsbad, CA, USA). The human iPSC-derived lt-NES cell line was routinely cultured and expanded on 0.1 mg/ml poly-L-ornithine- and 10 μg/ml laminin- (both from Sigma-Aldrich, St. Louis, MO, USA) coated plates in the same media supplemented with FGF, EGF and B27. The lt-NESCs were passaged at a ratio of 1:2 to 1:3 every second to third day using trypsin (Sigma-Aldrich).