iPSC-derived RPE Cell Differentiation

The study was approved by the ethical committees of the Institute of Biomedical Research and Innovation Hospital and the RIKEN Center for Developmental Biology, Japan. Human iPSC-lines 201B7 (HPS4290) and 253G1 (HPS0002) (Nakagawa et al., 2008 (link)) were provided by the RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. iPSCs were cultured and differentiated into RPE cells as described previously (Kamao et al., 2014 (link)). Briefly, to differentiate human iPSCs into RPE cells, human iPSCs were cultured on gelatin-coated dishes in differentiation medium (GMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 1 mM sodium pyruvate, 0.1 mM non-essential amino acids (Sigma-Aldrich), and 0.1 mM 2-mercaptoethanol (Sigma-Aldrich)) with 20% KnockOut Serum Replacement (KSR; Invitrogen, Waltham, MA) for four days, 15% KSR for six days, and 10% KSR for 20 days. Y-27632 (10 μM; FUJIFILM Wako, Osaka, Japan), SB431542 (5 μM; Sigma-Aldrich), and CKI7 (3 μM; Sigma-Aldrich) were added for the initial 18 days. After the emergence of pigmented cells, the medium was switched to SFRM (DMEM/F12 [7:3] supplemented with B27 (Invitrogen), 2 mM L-glutamine).
Lonza-RPE cells (line #476621; Lonza, Basel, Switzerland) were maintained in SFRM as well.
Human dermal fibroblasts were obtained from a healthy donor and cultured as described previously (Sugita et al., 2015 (link)).