Stemdiff neural induction medium smadi

NPCs were differentiated using the STEMdiff™ SMADi Neural Induction Kit (STEMCELL Technologies) as monolayer cultures, according to the manufacturer’s recommendation. Two million iPSCs per well were plated on Matrigel^®-coated six-well plates (Corning) in a single-cell suspension using STEMdiff™ Neural Induction Medium + SMADi (STEMCELL Technologies), supplemented with 10 μM ROCK inhibitor (Stemgent). Daily medium changes were performed with STEMdiff™ Neural Induction Medium + SMADi (STEMCELL Technologies). Cells were passaged after 7 days using Accutase (STEMCELL Technologies) and plated at a density of 2 × 10⁶ cells per well. After 7 days of growth, cells were passaged for a total of two passages. Mature NPCs were then plated at a density of 1.2 × 10⁶ cells per well on Matrigel^®-coated six-well plates (Corning) and cultured in STEMdiff™ Neural Progenitor Medium (STEMCELL Technologies) with daily medium changes.

Primed PSCs were differentiated into expandable NPCs by using the STEMdiff SMADi Neural Induction Kit (Stem Cell Technologies) as previously described [34 (link)–36 (link)]. In brief, primed PSCs were maintained on a Matrigel (Corning)-coated plate in mTeSR1 media (Stem Cell Technologies) prior to the NPC induction. The cells were harvested using Accutase (EMD Millipore) and transferred at 3 x 10⁶ cells to a well of an AgrreWell800 plate (Stem Cell Technologies) in STEMdiff Neural Induction Medium + SMADi (Stem Cell Technologies) supplemented with 10 μM Y-27632. Five days later, uniformly sized aggregates were collected using a 37 μm Reversible Strainer (Stem Cell Technologies) and plated onto a Matrigel-coated 6-well plate in STEMdiff Neural Induction Medium + SMADi. Seven days later, neural rosette structures were selectively removed by using STEMdiff Neural Rosette Selection Reagent (Stem Cell Technologies) and plated onto a new Matrigel-coated 6-well plate in STEMdiff Neural Induction Medium + SMADi. After that, the cells were passaged every 2–3 days until day 30 post-differentiation. The established NPCs were maintained on a Matrigel-coated plate in STEMdiff Neural Progenitor Medium (Stem Cell Technologies) and passaged every 3–4 days.

All iPSCs were characterized as previously described (Brennand et al., 2011 (link)). EBs were formed by mechanical dissociation of iPSC colonies using collagenase and plating onto low-adherence dishes. For EB differentiation, floating EBs were treated in STEMdiff™ Neural Induction Medium + SMADi (StemCell Technologies) for 20 days. To obtain neural progenitor cells, EBs were then plated onto polyornithine/laminin (Sigma)-coated dishes in DMEM/F12 plus 1% N2 and 1% B27. Rosettes were manually collected and dissociated with accutase (Chemicon) after 1 week and plated onto PLO (50 µg/ml) and laminin-coated dishes in neural progenitor cell media (DMEM/F12, 1% N2, 1% B27 (Invitrogen), and 20 ng/ml  EGF, and 20 ng/ml  FGF2 (Invitrogen)). To obtain hippocampal mature neurons, neural progenitor cells were plated onto dPGA or PLO (50 µg/ml) and laminin (1 µg/ml) coated-dishes and differentiated in BrainPhys neuronal medium (StemCell Technologies) supplemented with 1 × N2, 1 × B27, 20 ng/ml BDNF (Peprotech), 1 mM dibutyrl-cyclicAMP (Sigma), 200 nM ascorbic acid (Sigma), 1 μg/ml laminin and 620 ng/ml Wnt3a (R&D) for 2 weeks. After 2 weeks post differentiation, media was replaced by STEMdiff™ Forebrain Neuron Maturation Kit (StemCell Technologies) in BrainPhys media until use. All cells used in the present study were verified as free from mycoplasma contamination.