Differentiation of hESC and hiPSC into RPE Cells

Two hESC lines (Regea 08/023; 46, XY, Regea 08/017; 46,XX) [50 (link)] and one human induced pluripotent stem cell (hiPSC) line, (UTA.04511.WTS 46, XY) [51 (link)] were used for this study. Cell lines were cultured on top of mitomycin-treated (10 μg/ml,Sigma-Aldrich) (i.e. mitotically inactivated) human foreskin fibroblasts feeder cells (CRL-2429TM, ATCC, Manassas, VA, USA). The undifferentiated cells were cultured similarly as in Sorkio et al. [52 (link)] and after one week of culture the differentiation was induced by reducing the KO-SR concentration to 15%, removing the bFGF and commencing the floating culture as previously described in Vaajasaari et al. [53 (link)]. Floating aggregates were fed thrice a week and grown for 70–195 days. The pigmented areas of floating aggregates were manually dissected, dissociated with 1x Trypsin-EDTA and replated on collagen IV from human placenta (5 μg/cm², Sigma-Aldrich). Adherently cultured cells were imaged for the fusiform morphology after 8 days (range 6–9 days), for the epithelioid morphology after 9 days (range 8–9) and for the cobblestone morphology after 19 days (range 17–24) of culturing.