Differentiation of human ES and iPS cells to neural progenitors in 2-D adherent culture was performed as follows: 2 million human ES or iPS were passaged onto matrigel-coated dishes using PBS w/o Ca2+/Mg2+, filtered through a 40μm mesh to remove mEFS, and cultured directly in NGD medium containing dorsomorphin (2.5mM, Stemgent), bFGF (10ng/mL, Thermo) and Insulin (additional 10ng/mL) for 3 days until super-confluent. bFGF and Insulin were subsequently removed, and NGD + dorsomorphin was replaced every day for 10 days. Cells were subsequently passaged 1:1 with PBS−/− when rosette lawns were observed throughout the culture. Rho-associated protein kinase (ROCK) inhibitor Y27632 (10mM, Stemgent) was added to the medium during each of the first 3 passages. Initial passaging at no more than 1:2 ratio, followed by 1:3 to 1:6 every 5 days. Neural progenitors were expanded and maintained in NGD medium with 10ng/ml bFGF, and additional 10ng/mL Insulin (NGM medium).
