Recombinant human bmp 4 protein

Retinal differentiation was conducted following the modified SFEBq method we recently reported (Kuwahara et al., 2019 (link); Yamasaki et al., 2021 ) (Nukaya et al.; WO19/017,492, 054,514, 054,515). In brief, subconfluent hESCs were treated with 5 μM SB431542 (TGFβ receptor inhibitor, Sigma-Aldrich) and 300 nM SAG (Smoothened agonist, Enzo Biochem) from 24 h prior to differentiation. Cells were then dissociated into single cells using TrypLE Select Enzyme (Thermo Fisher Scientific), suspended in 100 μL serum-free culture medium with 10 μM Y-27632, and cultured at 1.2 × 10⁴ cells per well in low cell adhesion 96-well V-bottomed plates (Sumitomo Bakelite). On differentiation day (DD) 3 after initiation of suspension culture, aggregates were treated with 1.5 nM recombinant human BMP4 protein (R&D Systems). Aggregates on DD14 were transitioned to RPE induction medium with 3 μM CHIR99021 (GSK3β inhibitor, Wako Pure Chemical Industries) and 5 nM SU5402 (FGF signaling pathway inhibitor, Wako Pure Chemical Industries) for 3–4 days in a 90-mm low adhesion culture dish (Sumitomo Bakelite). Subsequently, aggregates were cultured in the maturation culture (Yamasaki et al., 2021 ) (Nukaya et al. in preparation; WO2019017492A1, WO2019054514A1). The medium was exchanged every 3–4 days.