Generation of RPE cells from iPSCs

RPE cells were generated from iPSCs following the previously reported method [26 (link)]. Briefly, iPSCs were grown to 80% confluence in mTeSR^TMPlus medium and switched to RPE differentiation medium containing Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA), 20% knockout serum replacement (KSR; Thermo Fisher Scientific, Waltham, MA, USA), 1× non-essential amino acids (NEAA; Sigma, San Luis, MO, USA), 50 μM β-mercaptoethanol (Gibco), 1% penicillin–streptomycin (Gibco) and 10 mM nicotinamide (MedChemExpress, Princeton, NJ, USA) for 0–7 days. Then, nicotinamide was replaced with 100 ng/mL Activin A (Peprotech, Rocky Hill, NJ, USA) for another week. On day 14 of differentiation, Activin A was removed from the medium, and 3 μM CHIR99021 (MedChemExpress) was added. On day 42, RPE cells were collected by incubating in TrypLE Express (Gibco) for 30 min at 37 °C and then seeded on a Matrigel-coated 6-well plate with a density of approximately 1 × 10⁶ cells/well. Meanwhile, RPE differentiation medium was replaced with RPE maintenance medium consisting of DMEM, 4% KSR, 1× NEAA, 1% penicillin–streptomycin and 50 μM β-mercaptoethanol without molecule compound. At this point, the cells were defined as passage 1 (P1). Only RPE cells at P2 and P3 were used in this study.