Gibco DMEM/F-12, HEPES

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Human iPSC differentiation to retinal organoids: Human iPSCs were differentiated to retinal organoids based on a previously described protocol with minor modifications [12]. Briefly, hiPSCs were initially washed with Phosphate buffered saline (PBS), and dissociated into single cells using Accutase (Gibco, A1110501). The hiPSCs were seeded at a density of 7,000 cells/well onto U-bottom 96-well plates (Helena, 92697T) manually pre-coated with Lipidure solution (AMSbio, AMS.52000011GB1G) in mTeSR™1 with 10 μM Y-27632 ROCK inhibitor (Chemdea). After 2 days, 200 μl of differentiation medium consisting of 41% Iscove’s modified Dulbecco’s medium (IMDM, Gibco, 12440–053), 41% Hams F12 (Gibco, 31765–029), 15% KnockOut serum replacement (KOSR) (Gibco, 10828–028), 1% Glutamax (Gibco, 35050–038), 1% chemically defined lipid concentrate (Thermo, 11905031), 225 μM monothioglycerol (Sigma, M6145), and 1% P/S (Gibco, 15140–122) was added per well followed by half medium changes two days later. Half of the differentiation medium was changed every 2 days. On day 6 of differentiation, 2.25 nM BMP4 (R&D, 314-BP) was added to the media and the medium was replaced every 3 days thereafter. On day 18 the medium was changed to reversal medium using DMEM/F12, 1% Glutamax, 1% N2 supplement (Thermo, A1370701), 4 μM CHIR99021 (Sigma-Aldrich, SML1046-5MG), 2.5 μM SU5402 (Tocris, 3300), and 1% P/S, where the medium was changed every 2 days. At day 24, the medium was replaced to maintenance medium consisting of DMEM/F12 (Gibco, 31330–038), 5% Foetal Bovine Serum (FBS), 1% N2 supplement, 0.1 mM taurine (Sigma, T8691), 0.25 μM retinoic acid (Sigma, R2625), 0.25 μg / ml Fungizone (Gibco, 15290–02), 1% P/S (Gibco, 15140–122), medium was changed every 3–4 days thereafter.

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