the media was changed every other day. After 2–4 days in suspension, NE cells were collected and plated onto laminin (20µg/mL in DMEM/F12) coated 6-well cell culture plates to allow the NE cells to adhere to the surface. Cells were cultured in neural induction media, and half of the media was changed every other day. NE cells plated on laminin were cultured for 10–14 days. During this time, formation of neural tube-like rosettes was observed in the culture. NE cells were collected by mechanical detachment, centrifuged, and re-suspended in neural induction media and incubated in 75mL low adhesion flasks for 7–14 days with half of the media changed every other day. NE cells were then collected, pelleted, and resuspended in neural differentiation media [Neurobasal media (Invitrogen) supplemented with 1× non-essential amino acids, 4× B27 Supplement (Invitrogen), 1µM dibutyryl-cAMP (Enzo Life Sciences, Farmingdale, NY, USA), and 200µM ascorbic acid (Sigma-Aldrich)]. NE cells were then mechanically dissociated by pipetting and plated onto 13mm glass coverslips (Fisher Scientific, Pittsburg, PA, USA) pre-coated with polyornithine (0.1mg/mL in water) and Matrigel (0.67mg/mL in DMEM/F12) (BD Biosciences, Bedford, MA, USA) coated at a density of 100,000 cells per coverslip. Cells were allowed to adhere to coverslips
