Stemdiff neural induction medium

Neural induction is completed using the dual SMAD inhibition neural induction kit (StemCell Tech). High quality iPSCs are selected and passaged using ReLeSR and centrifuged to pellet. Cells are then resuspended into a single cell suspension in STEMdiff Neural Induction Medium (StemCell Tech) + 10 μM Y-27632. Cells are replated onto matrigel coated plates at a density of about approximately 1 × 10⁶ cells/mL in a 6-well plate. Media is replaced the following day, without the addition of Y-27632, and is replaced every day until cells are confluent, approximately 6–9 days later. The NSCs are then dissociated again using Accutase (StemCell Tech) as a cell dissociation reagent, and replated in STEMdiff Neural Induction Medium + 10 μM Y-27632. Cells are cultured as described above until at least passage 3. Cells can then be characterized, cryopreserved, or further expanded.
Immunocytochemistry staining for Nestin and Sox2 has been used to confirm NSC identity. These are well established markers for human NSCs. Evaluated cell lines are >90% Nestin positive, therefore are primarily NSCs and thus suitable for downstream analysis.

Neural conversion of iPSCs was performed using STEMdiff^TM Neural Induction Medium (#05831, StemCell Technologies Inc.) in combination with a monolayer culture method according to the manufacturer’s technical bulletin (#28044). In brief, single-cell suspensions were prepared by treatment of hES cells with accutase, and 1.5–2.0 × 10⁶ cells were seeded per well of a six-well plate in 4 mL STEMdiff^TM Neural Induction Medium supplemented with 10 μM Y-27632 ROCK inhibitor. Neural induction was performed for indicated time periods with daily medium change.

Neural induction of hES H1 cells expressing different shRNA or protein constructs was performed using STEMdiff^TM Neural Induction Medium (#05831, StemCell Technologies Inc.) in combination with a monolayer culture method according to the manufacturer’s technical bulletin (#28044) and as previously described (Werner et al., 2015 (link)). In brief, single cell suspensions were prepared by treatment of hES cells with accutase (1.25 × 10⁶ cells were seeded per well of a six-well plate in STEMdiff Neural Induction Medium supplemented with 10 μM Y-27632 ROCK inhibitor. Neural induction was performed for indicated time periods with daily medium change. To determine the effect of CK2 or PP1/PP2A inactivation on neural conversion, indicated concentrations of CX4945 or okadaic acid were added throughout the differentiation experiment. For NOLC1 rescue experiments, to induce expression of NOLC1 variants, 1 μg/mL doxycycline was added to indicated conditions for 12 hr before the start and until the end of the differentiation experiment.