Forebrain neural progenitor cells (fbNPCs) differentiation from hiPSCs (dual SMAD inhibition)

Human induced pluripotent stem cells (hiPSCs) are rinsed once with DPBS (GIBCO) and dissociated when 70-90% confluent with 0.5 mM EDTA (75 ml/cm2; GIBCO) at 37°C for 0h 7m 0s

Dissociated cells are then carefully washed away from the wells and collected in wash medium (9.5mL DMEM/F-12 (31330-038; GIBCO) and 0.5mL knockout serum replacement (GIBCO)).

The cells are then centrifuged at 400x g,0h 0m 0s for 0h 5m 0sand resuspended in iPS brew medium (StemMACS iPS-Brew XF and 0.5% penicillin/streptomycin (GIBCO)).

The protocol is based on the one described in Nolbrant et al., 2017 and Grassi et al., 2019.

Day 0 of differentiation. The resuspended cells are then plated on LN111-coated (1.14µg/cm2; Biolamina) coated Nunc multidishes at a density of 10000 cells/cm² and grown in N2 medium (1:1 DMEM/F-12 (21331020; GIBCO) and Neurobasal (21103049; GIBCO) supplemented with 1% N2 (GIBCO), 2 mM L-glutamine (GIBCO), and 0.2% penicillin/streptomycin). 10 μM of Y27632 (Rock inhibitor; Miltenyi) are also added to the cells when plating. Finally, 10 μM SB431542 (Axon) and 100 ng/ml noggin (Miltenyi) for dual SMAD inhibition are supplemented to the media.

The media, supplemented with dual SMAD inhibitors, is changed every 2-3 days until day 9 of differentiation . The amount of media is slightly increased at every feeding as the cells' confluency increases.

Day 9 of differentiation. The cells are fed with N2 media without dual SMAD inhibitors.

Day 11 of differentiation. The cells are dissociated, centrifuged, and resuspended as described above (hiPSCs dissociation SECTION). They are then replated on LN111-coated Nunc multidishes at a density of 800000 cells/cm² in B27 medium (Neurobasal supplemented with 1% B27 without vitamin A (GIBCO), 2 mM L-glutamine and 0.2% penicillin/streptomycin Y27632 (10 μM), BDNF (20 ng/ml; R&D), and L-ascorbic acid (0.2 mM; Sigma)). 10 μM of Y27632 are also added. The cells are kept in the same medium until day 14 of differentiation, when they are harvested for downstream analyses (e.g. bulk RNA sequencing).