Rostral and Caudal NPC Differentiation from hESCs

H9 hESCs were transferred to vitronectin-coated 4-well plates in N2B27 medium (1:1 mix of neurobasal medium and DMEM/F12 medium, 1% insulin/transferrin/selenium, 1 × N2 supplement, 1 × retinol-free B27 supplement, 0.3% glucose, and 1% p/s (Thermo Fisher Scientific)) for 11 days of differentiation. As previously described [35 (link)], for rostral neural progenitor cell (NPC) differentiation, SB431542 (SB, 10 µM; Tocris, Bristol, UK) and LDN193189 (LDN, 0.1 µM; STEMCELL Technologies) were added to the media for 4 days. The cells were further cultured with SB, LDN, and FGF2 (20 ng/ml; STEMCELL Technologies) until day seven. Between day 7 and 11, the cell media was only supplemented with FGF2 (20 ng/ml). For caudal NPC differentiation, SB431542 (10 µM) and CHIR99021 (CHIR; 3 µM; STEMCELL Technologies) were added to the media from days zero to four. The cells were further cultured from days 4 to 11 in the absence of SB and CHIR, but with FGF2 (20 ng/ml). RNA was harvested at days 0 (before differentiation), 4, and 11.