4.2. Induction of hiPSC Differentiation to RGCs

Induction of hiPSC differentiation employed a procedure based on SFEB methods [52]. In brief, hiPSCs were dissociated to single cells in Accutase (eBioscience, San Diego, CA, USA) and were resuspended in retinal differentiation medium (RDM, G-MEM supplemented with 20% KSR, 0.1 mM nonessential amino acids, 1 mM pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/mL penicillin, and 100 μg/mL streptomycin) containing Y-27632 (20 μM; Merck Millipore, Darmstadt, Germany), IWR-1e (3 μM, Merck Millipore, Darmstadt, Germany), and 0.5% Matrigel (BD Bioscience, San Jose, CA, USA). After separation from the feeder cells by decantation (the feeder cells adhered to the gelatin-coated bottom of the dish), the floating hiPSCs collected from the medium were seeded into 10% Pluronic-coated V-bottomed 96-well plates (Costar, Cambridge, MA, USA) at 9000 cells per well. On day 12 (D12), the aggregates were transferred to 10% Pluronic-coated 24-well plates (Costar), and the medium was replaced with RDM containing 0.5% Matrigel and 1% FBS. On Day 15, CHIR99021 (3 μM, Merck Millipore, Darmstadt, Germany) and SAG (100 nM, Merck Millipore, Darmstadt, Germany) were added to the medium. On Day 18, the aggregates were transferred to retinal maturation medium (RMM, DMEM/F12 Glutamax medium containing the N₂ supplement, 100 U/mL penicillin, and 100 mg/mL streptomycin) and were then cultured in the absence of FBS. Then we added retinoic acid (0.5 μM, Sigma, St. Louis, MO, USA) and 1%FBS on D24. The concentration of FBS was increased stepwise from 1% up to 10% over the course of the adhesion culture period. On Day 27, the adhesion culture started when the aggregates were transferred to 3% Matrigel-coated PBG scaffolds or cover glass in 24-well plates (Costar, Cambridge, MA, USA) in RMN medium containing FBS and 100 ng/mL BDNF (R&D Systems, Minneapolis, MN, USA) [18]. Cells were adhesive cultured until Day 34, and then cells were harvested for further observation.