Cell culture

Procedure of iPSC-derived RPE differentiation

The iPSC-lines used in this study were maintained in Primate ES medium (ReproCELL) supplemented with 5ng/ml basic fibroblast growth factor (bFGF) (Kamao et al., 2014). RPE differentiation was induced in the following time course. For each step, the culture medium was changed every 2-3 days. 

Day -1: pre-treat iPSCs in Primate ES medium + 20% KnockOut Serum Replacement (KSR, Invitrogen) for bFGF depletion

Day 0: plate on a gelatin-coated dish in basal medium (GMEM [Sigma] + 1mM sodium pyruvate + 0.1 mM non-essential amino acids [Sigma] + 0.1 mM 2-mercaptoethanol [Sigma]) supplemented with 20% KSR, Y-27632 (10 μM, Wako), SB431542 (5 μM, Sigma) and CKI7 (3 μM, Sigma)

Day 5: change the medium to basal medium + 15% KSR + Y-27632 + SB431542 + CKI7

Day 11: change the medium to basal medium + 10% KSR + Y-27632 + SB431542 + CKI7

Day 19: change the medium to basal medium + 10% KSR

Day 31 or maybe Day 40 (*1): when pigmented cells appear (*2), change the medium to SFRM (DMEM/F12 [7:3] supplemented with B27 [Invitrogen], 2 mM L-glutamine VIII [Sigma])

Day 38 or maybe Day 45 or Day 55 (*1): transfer the pigmented colonies onto a laminin (Sigma)-coated dish in SFRM supplemented with 10 ng/ml bFGF and SB431542 (0.5 μM) until confluent

(*1) optimal duration may depend on the iPSC-line used

(*2) place the dish on a white paper and verify with eyes