Neural Rosette Induction from Embryonic Bodies

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)].