NPs were dissociated from the neural rosettes with Accutase and expanded by culturing on Matrigel-coated plates at >0.1 x 106 cells/cm2 or seeded at 0.33 x 106 cells/ml in Ultra-Low Attachment plates (Corning) for suspension culture in NDM supplemented with 10 ng/ml hLIF (Millipore) and 20 ng/ml bFGF (R&D Systems). After two passages, NPs were dissociated as single-cell suspensions and cryopreserved for future use in 50% NDM, 40% Knockout serum replacement and 10% dimethyl sulfoxide (Sigma).
