Sb431542 sb

The highly efficient dual inhibition of SMAD signaling was used for neural induction of the iPSCs [16 (link)]. Briefly, the iPSCs cultured in growth-factor-reduced (GFR) Matrigel (BD Biosciences, San Diego, CA, USA)-coated dishes were collected and suspended in low-attachment dishes (Primesurface, Sumitomo Bakelite, Tokyo, Japan) to form embryoid bodies (EBs). For neural induction, the EBs were subsequently cultured in DMEM/F12 containing 5 % B27 supplement (Life Technologies), 5 % N2 supplement (Life Technologies), 20 ng/ml recombinant human (rh) basic fibroblast growth factor (bFGF) (Wako, Osaka, Japan), 10 μM SB431542 (SB; Sigma-Aldrich), and 1 μM dorsomorphin (DSM; Wako) for 2 weeks under 5 % O₂. To obtain neural progenitor cells (NPCs) as neurospheres, the cell aggregates were mechanically dissociated and cultured in DMEM/F12 containing 2 % B27 supplement (Life Technologies), 20 ng/ml rh-basic fibroblast growth factor (bFGF; PeproTech, Rocky Hill NJ, USA), 20 ng/ml rh-epidermal growth factor (EGF; PeproTech), 10 ng/ml rh-leukemia inhibitory factor (LIF; Millipore, Billerica, MA, USA), and 5 μg/ml heparin (Sigma-Aldrich). The neurospheres were passaged before the center of the spheres darkened, typically every 12–14 days as described previously [17 (link)].

mTeSR1 medium (STEMCELL Technology, Vancouver, Canada) was used for the feeder-free culture of PSCs. The induction and maintenance of hNCCs were performed using previously reported CDM [23] (link), which contains Iscove's modified Dulbecco's medium/Ham's F-12 1∶1, 1x chemically defined lipid concentrate (GIBCO, Grand Island, NY, USA), 15 µg/ml apo-transferrin (Sigma, St. Louis, MO, USA), 450 µM monothioglycerol (Sigma), 5 mg/ml purified BSA (99% purified by crystallization; Sigma), 7 µg/ml Insulin (WAKO), and penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA). Culture dishes were coated with growth factor-reduced Matrigel (BD, Bedford, MA, USA) or fibronectin (Millipore, Bedford, CA, USA). EGF (R&D, Minneapolis, USA) and FGF2 were used to maintain hNCCs [24] (link). SB431542 (SB) (Sigma), CHIR99021 (CHIR) (WAKO), BMP4 (R&D), DMH1 (Tocris, Bristol, UK), LDN193189 (Stemgent, Cambridge, MA, USA), and recombinant human Noggin (R&D) were used to modulate growth factor signals. Retinoic acid (RA) (Sigma) was used to modulate hNCCs.

For induction of neural rosettes, Embryonic bodies (EBs) were cultured in suspension for 4 days in hESC media excluding bFGF but supplemented with 5mM dorsomorphin (DM) (Calbiochem, Darmstadt, Germany) and 5 to 10 μM SB431542 (SB) (Sigma, St. Louis, MO, USA). On day 4, EBs were attached in Matrigel-coated culture dishes (BD Biosciences, San Jose, CA, USA) in DMEM/F12 N2 supplemented media (N2 media) with 20 ng/ml bFGF (R&D Systems, Minneapolis, USA) and 19 to 21 μg/ml human insulin solution (Sigma, St. Louis, MO) for another 5 days. The emerged rosette structures were mechanically isolated using pulled glass pipettes within the EB colonies, and isolated neural rosette clumps were replaced in Matrigel-coated dishes. Replated neural rosettes were then expanded for an additional 6 to 7 days at 90% confluence [12 (link)].