Neural induction supplement

Confluent hESC cultures were dissociated into cellular clumps with 1 mg/ml collagenase, washed once with phosphate buffered saline (PBS) through centrifugation and then digested into a single cell suspension through treatment with StemPro™ Accutase Cell Dissociation Reagent (Cat. no. A11105, Life Technologies Inc.), for 5 to 10 min followed with gentle pipetting. The dissociated hESC were then reconstituted in mTeSR1™ medium supplemented with 10 μM ROCK Inhibitor Y27632 (Cat. no. Y0503, Sigma-Aldrich Inc.), and plated into 6-well culture plates pre-coated with Geltrex™, at a density of 2.5 × 10⁵–3 × 10⁵ cells per well. The following day, induction of hESC into NSC was carried out by replacing the culture milieu with Neurobasal™ medium (Cat No. 21103-049, Life Technologies Inc.) supplemented with 2 % (v/v) Neural Induction Supplement™ (Cat No. A16477-01, Life Technologies Inc.) and 1 %(v/v) penicillin-streptomycin antibiotic solution. Cultures became confluent after 6 to 7 days of culture in a 5 % CO₂ incubator set at 37 °C, and were serially passaged onto Geltrex™ pre-coated 6-well plates with StemPro Accutase Cell Dissociation Reagent™, utilizing similar aforementioned seeding densities. NSC from the third passage onwards were utilized for differentiation into mature neurons, as well as for further experiments.

Induced Pluripotent Stem cells (iPSc) derived from human fibroblasts, obtained and characterized previously [24 (link)], were cultured on Geltrex coated dishes in Essential 8 medium (both from Life Technologies) at low oxygen conditions (5% O2/5% CO2).
Induced Neural Stem cells (iNSc) were generated from iPSc using PSC Neural Induction Medium (Gibco) according to the manufacturer’s protocol and propagated as an adherent culture on Geltrex coated dishes in Neural Expansion Medium (Neurobasal Medium: Advanced DMEM/F12 (1:1), supplemented with Neural Induction Supplement (1:50; all from Life Technologies). Cells were maintained at 37°C in humidified 5% CO2 incubator.
IDH-mutant glioma neurospheres generated from xenograft-forming tumor (MGG152) were a kind gift from Dr. Daniel P. Cahill, Massachusetts General Hospital [12 (link), 16 (link)]. Neurospheres were cultured in DMEM/F12 medium supplemented with B27 (1:50; Life Technologies), human recombinant EGF (20 ng/mL; ABM), human recombinant basic FGF2 (10 ng/mL; ABM) and heparin (2 μg/mL; Sigma Aldrich). Cells were maintained at 37°C in humidified 5% CO2 incubator.

For cell size, proliferation, and neurite outgrowth, assays were performed as previously described^{17 (link)} . Briefly, cell size and proliferation were determined from live cells (3 biological replicates/treatment condition) by trypan blue exclusion using the Countess II automated cell counter (ThermoFisher, Waltham, MA). For neurite outgrowth, NPCs (6250 per cm²) were seeded on Poly-D-lysine coated wells (0.1 mg/ml; Sigma, St. Louis, MO) and Fibronectin (5 μg/ml, Corning, Corning, NY) in growth factor-depleted neural expansion medium (30% NEM) containing 1:1 of neurobasal media and advanced DMEM/F12 (ThermoFisher, Waltham, MA), 1X penicillin/streptomycin and 0.3X neural induction supplement (ThermoFisher, Waltham, MA). Cells were grown in presence of DMSO, 50 nM rapamycin, or 10 nM of RMC-6272 for 48 h and fixed with 4% paraformaldehyde (PFA; Microscopy Sciences, Hatfield, PA) for 20 min prior to immunostaining. Cover slips from 3 biological replicates were analyzed. For each cell line, images from 4 independent fields/condition (~50 cells/field) were acquired for analysis. Processes that were at least two times the length of the cell body were considered as neurites. The average neurite number per cell and the average neurite length per cell were analyzed using HCA-Vision software V.2.2.0 (CSIRO, Canberra, Australia).

All six human hiPSC lines were adapted to E8 medium (Thermo Fisher Scientific, Carlsbad, CA, USA) for at least four consecutive passages, and cells were later split. After 24 h of splitting the cells, we maintained them in Pluripotent Stem Cells (PSC) Neural Induction Medium (Thermo Fisher Scientific, Carlsbad, CA, USA), which contained Neurobasal medium and PSC supplement, according to the manufacturer’s protocol. Medium was changed every other day for 7 days, during which initial NSCs split and expand with Neural Induction Medium (NEM, Advanced DMEM/F12 and Neurobasal medium (1:1) with Neural Induction Supplement; Thermo Fisher Scientific, Carlsbad, CA, USA).

The neural induction of iPSC lines into NSCs was performed with PSC Neural Induction Medium (Thermo Fisher Scientific) according to the manufacturer’s instructions. For neural induction, iPSC lines were cultured in Essential 8 medium (Thermo Fisher Scientific) under feeder-free conditions on Matrigel (Corning, NY, USA). On day 0 of neural induction, ~24 h after the cells were split, the culture medium was replaced with PSC Neural Induction Medium containing neurobasal medium and PSC neural induction supplement. The neural induction medium was exchanged every other day from day 0 to day 4 of neural induction and every day after day 4 of neural induction. On day 7 of neural induction, NSCs (P0) were harvested and expanded in neural expansion medium containing 50% neurobasal medium (Thermo Fisher Scientific), 50% advanced DMEM/F12 (Thermo Fisher Scientific), and neural induction supplement (Thermo Fisher Scientific) on Matrigel. Expanded NSCs after passage 6 were used for subsequent assays.

iPSCs were cultured in Induction medium (Neurobasal and 2% Neural Induction Supplement [Thermo Fisher Scientific]) for 7 days. On day eight, cells were harvested by Accutase (Euroclone) and seeded on Matrigel‐coated dishes in Expansion medium (50% Neurobasal, 50% DMEM/F12 and 4% Neural Induction Supplement) with addition of 10 μmol/L RHO‐associated kinase (ROCK) inhibitor Y27632 (Selleckchem). Cells were allowed to grow and expanded for at least five passages, until they showed a homogeneous neural morphology. Expression of NSCs‐specific marker nestin (1:100, Chemicon) was assessed by immunofluorescence.