HyClone Dulbecco's Modified Eagle Medium (DMEM)/ F12 1:1: Liquid

Protocol tips

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	Use of an extra medium to make a 1:1 mixture, Neurobasal (Thermo Fisher Scientific) and DMEM/F12

Protocol tips
Use of an extra medium to make a 1:1 mixture, Neurobasal (Thermo Fisher Scientific) and DMEM/F12

Publication protocol

Then the expanded pNPCs were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific). 10,000 cells were cultured in suspension in the presence of ROCK inhibitor Y-27632 (10 μM) in ultra-low-attachment 96-well plates to form uniform organoids and then those organoids were transferred to ultra-low-attachment 6-well plates. To pattern these organoids to the fate of ventral forebrain, we treated them with sonic hedgehog (SHH) (50 ng/mL, Peprotech) and purmorphamine (1 μM, Cayman Chem), an agonist of sonic hedgehog signal pathway from week 3 to 5 (Figure 1A). The media were replenished every day. Starting from week 5, the cell culture plates were kept on orbital shaker with speed of 80 rpm/min. Maximum 8 organoids were cultured in each well of the ultra-low-attachment 6-well plate in neuronal differentiation medium containing a 1:1 mixture of Neurobasal and DMEM/F12, supplemented with 1 × N2 (Thermo Fisher Scientific), 1 × B27 (Thermo Fisher Scientific), BDNF (20 ng/ml, Peprotech), GDNF (20 ng/ml, Peprotech), dibutyryl-cyclic AMP (1mM, Sigma), and ascorbic acid (200 nM, Sigma) and media was replenished every other day. For 2D neuronal differentiation, 5,000 dissociated cells were plated onto poly-L-ornithine (0.002%, Sigma) and laminin (10 μg ml−1) pre-coated coverslips in the same neuronal differentiation medium. The neurons cultured in the neuronal differentiation medium for 4–6 weeks were used for experiments.

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