Neural Differentiation of hESCs and hiPSCs

This study was performed in conformity with ‘The Guidelines for Derivation and Utilization of Human Embryonic Stem Cells' of the Ministry of Education, Culture, Sports, Science and Technology of Japan, after approval by the institutional review board. hESCs (Kh-ES1 (ref. 19 (link)) passages 30–40) and hiPSCs (1039A1 (ref. 20 (link)) passages 15–25) were maintained on iMatrix-511(Nippi)-coated six-well plate at a density of 3 × 10⁴ cells per well with StemFit medium. When we began neural differentiation, these cells were dissociated into single cells with TrypLE select (Invitrogen) and were then replated on iMatrix-511-coated six-well plate at a density of 4 × 10⁵ cells per well with StemFit medium. Three days later, the medium was changed to a differentiation medium containing GMEM supplemented with 8% KSR, 0.1 mM non-essential amino acids solution, 2-ME, 1 mM Pyruvate and 2 mM L-Glu. In addition, 500 nM A83-01 (Wako) and 100 nM LDN193189 (STEMGENT) were added until day 7 and day 12, respectively; 2 μM Purmorphamine (Wako) and 100 ng ml⁻¹ FGF8b (Wako) were added from day 1 to day 7; and 3 μM CHIR99021 (STEMGENT) was added from day 3 to day 12. We changed the medium every day11 .