Differentiation of hiPSCs into Neural Progenitors

The differentiation protocol was adapted from Hörner et al. [40 (link)]. Briefly, when hiPSCs reached 75% confluence, mTeSR™1 medium was replaced with differentiation medium composed by Neurobasal medium and Ko-DMEMF12 (Thermo Fisher SCIEN-TIFIC) (1:1) complemented with 1× B27 (Thermo Fisher SCIENTIFIC, Waltham, MA, USA), 1× N2 (Thermo Fisher SCIENTIFIC), 1% (non-essential amino acids(Thermo Fisher SCIENTIFIC), 1% Glutamax (Thermo Fisher SCIENTIFIC), extemporaneously supplemented with 2 µM SB-431542 (Tocris, Bioscience, Minneapolis, MN, USA), 2 µM dorsomorphin (Sigma-Aldrich, Merck, Saint-Louis, MO, USA), 3 µM CHIR 99,021 (Sigma-Aldrich, Merck), and 0.1 mM µM Ascorbic Acid (Tocris, Bio-Techne). The medium was changed daily for 5 days. To obtain NPs, cells were then dissociated using Accutase (STEMCELL Technologies, Saint Égrève, France) and plated at 1:6 ratio on Geltrex-coated 6-well plates. The culture medium was then supplemented with 2 µM SB-431542, 2 µM dorsomorphin, 1 µM CHIR 99021, 0.1 µM Ascorbic Acid, 0.1 µM retinoic acid (Sigma-Aldrich, Merck), 0.5 µM purmorphamin (Abcam, Waltham, MA, USA). The medium was changed daily for 5 days.