hiPSCs grown in HUES media on mEFs were incubated with Collagenase (1 mg/ml in DMEM) at 37°C for one to two hours until colonies lifted from the plate and were transferred to a nonadherent plate (Corning). Embryoid Bodies (EBs) were grown in suspension in N2 media (DMEM/F12-Glutamax (Invitrogen), 1x N2(Invitrogen)). After seven days, EBs were plated in N2 media with 1 µg/ml Laminin (Invitrogen) onto polyornithine (PORN)/Laminin-coated plates. Visible rosettes formed within one week and were manually dissected onto PORN/Laminin-coated plates. Rosettes were cultured in NPC media (DMEM/F12, 1x N2, 1x B27-RA (Invitrogen), 1 µg/ml Laminin and 20 ng/ml FGF2) and dissociated in TrypLE (Invitrogen) for three minutes at 37°C. NPCs are maintained at high density, grown on PORN/Laminin-coated plates in NPC media and split approximately 1:4 every week with Accutase (Millipore).
