NPC1 iPSCs were differentiated into neural stem cells (NSCs) by using a kit from Life Technologies. Briefly, iPSCs were digested with Dispase and re-seeded onto Geltrex-coated plate at 20% confluence. After cells attached, the medium was changed to Induction Medium containing the Neurobasal Medium plus Neural Induction Supplement (A15640SA, Life Technologies). At day 7 of neural induction, the initial NSCs were dissociated with Accutase and plated for further expansion in Neural Expansion Medium containing Neurobasal Medium and Advanced DMEM/F12 with 1x Neural Induction Supplement. NSCs were characterized by staining with antibodies against nestin and Sox2 that showed 99% positive cells with both neural markers.
For neuronal differentiation, dissociated NSCs were cultured on poly-L-ornithine and laminin coated 96-well plates in the Induction Medium with 5 μM Y27632, a ROCK inhibitor (Y0503, Sigma-Aldrich) for one day. The medium was changed to a differentiation medium containing Neurobasal Medium, 1x B27 (17504-044, Life Technologies), 1x glutamax (35050, Life Technologies), 200μM L-ascorbic acid (A8960, Sigma-Aldrich), 1μM cAMP (A6885, Sigma-Aldrich), 10ng/ml BDNF (10908-010, Life Technologies) and 10ng/ml GDNF (PHC7044, Life Technologies) that was changed every two days for 12 days.20 (link)
For neuronal differentiation, dissociated NSCs were cultured on poly-L-ornithine and laminin coated 96-well plates in the Induction Medium with 5 μM Y27632, a ROCK inhibitor (Y0503, Sigma-Aldrich) for one day. The medium was changed to a differentiation medium containing Neurobasal Medium, 1x B27 (17504-044, Life Technologies), 1x glutamax (35050, Life Technologies), 200μM L-ascorbic acid (A8960, Sigma-Aldrich), 1μM cAMP (A6885, Sigma-Aldrich), 10ng/ml BDNF (10908-010, Life Technologies) and 10ng/ml GDNF (PHC7044, Life Technologies) that was changed every two days for 12 days.20 (link)
