Crl 2429

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.

Two hESC lines, Regea08/023 and Regea08/017, that were derived and characterized in our laboratory as described previously [26 (link)], were used in this study. The undifferentiated hESC lines were cultured on top of mitotically inactivated, mitomycin (10 μg/ml, Sigma-Aldrich, St. Louis, USA) treated, human foreskin fibroblasts feeder cells (CRL-2429TM, ATCC, Manassas, VA, USA) [27 (link)].
The hESCs were differentiated towards RPE in a RPEbasic medium as cell aggregates for 56–93 days. The selection of pigmented areas from floating aggregates was done manually, isolated areas were dissociated with 1× Trypsin–EDTA (Lonza, Walkersville, USA), plated with methods described previously, [28 ] and cultured in average for 85 days (ranging from 28 to 130) to expand amount of cells.

Healthy fibroblast donor was recruited from Kuopio University Hospital (Kuopio, Finland; Approved by the committee on Research Ethics of Northern Savo Hospital district (license no 64/2014). Written informed consent was obtained from the donor. Skin biopsy derived fibroblasts were reprogrammed with CytoTune® iPS Sendai Reprogramming kit (Thermo Scientific, MA, USA) as previously described (Holmqvist et al., 2016 (link)), with slight modifications. Briefly, fibroblasts (1 × 10⁵) were transduced with 3 or 4 separate vectors including the four Yamanaka factors OCT-3/4, KLF-4, SOX-2 and c-MYC. One week after transduction, 0.75 × 10⁵ cells were seeded on the top of mitotically inactivated (10 μg/ml mitomycin-C for 2.5 h in 37°C) human foreskin fibroblast feeder cells (CRL-2429, ATCC, Manassas, VA) growing in 10 cm petri dish. First colonies started to appear a week later, and they were re-seeded by picking up individual colonies. The pluripotency of created hiPSC line was assessed as in our earlier studies (Qu et al., 2013 (link)).