Neural Differentiation of Pluripotent Stem Cells

Neural differentiation of H9 hESCs, H1 hESCs and iPSCs was performed using the monolayer culture protocol following the STEMdiff Neural Induction Medium (Stem Cell Technologies) method based on ref. 61 (link). Undifferentiated pluripotent stem cells were rinsed once with PBS and then we added 1 ml of Gentle Dissociation Reagent (Stem Cell Technologies) for 10 min. After the incubation period, we gently dislodged pluripotent cells and add 2 ml of Dulbecco's Modified Eagle Medium (DMEM)-F12+10 μM ROCK inhibitor (Abcam). Then, we centrifuged cells at 300g for 10 min. Cells were resuspended on STEMdiff Neural Induction Medium+10 μM ROCK inhibitor and plated on polyornithine (15 μg ml⁻¹)/laminin (10 μg ml⁻¹)-coated plates (200,000 cells cm⁻²). For neuronal differentiation, NPCs were dissociated with Accutase (Stem Cell Technologies) and plated into neuronal differentiation medium (DMEM/F12, N2, B27 (ThermoFisher Scientific), 1 μg ml⁻¹ laminin (ThermoFisher Scientific), 20 ng ml⁻¹ brain-derived neurotrophic factor (BDNF) (Peprotech), 20 ng ml⁻¹ glial cell-derived neurotrophic factor (GDNF) (Peprotech), 1 mM dibutyryl-cyclic AMP (Sigma) and 200 nM ascorbic acid (Sigma)) onto polyornithine/laminin-coated plates as described in ref. 8 (link). Cells were differentiated for 1–2 months, with weekly feeding of neuronal differentiation medium.