Knockout sr xenofree cts

The study was approved by the Ethical Committee of the CHA University Bundang CHA Hospital, Republic of Korea (application number: KNC12005). Human adult dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen) and 1x penicillin/streptomycin (P/S) (all from Invitrogen, Carlsbad, CA, USA).
Human iPS cells were cultured on vitronectin XF (Primorigen Biosciences, Madison, USA) coated culture dishes using our recently established xeno-free/feeder-free hPSC culture medium with minor modifications [17 (link)]. Briefly, the medium consisted of DMEM/F12, 15% KnockOut SR XenoFree CTS, 1x nonessential amino acids (NEAA), 1x GlutaMAX, 0.1 mM β-mercaptoethanol, 1x P/S (all from Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF) (CHA Biotech Co., Daejeon, Korea), 10 nM trichostatin A (TSA) (Sigma-Aldrich, St. Louis, MO, USA), 5 uM Gö6983 (Tocris, Ellisville, MO, USA), and 1 mM dorsomorphin dihydrochloride (Tocris).

To test their pluripotency, the iPS cell colonies were mechanically detached and cultured in suspension in Petri dishes (SPL Lifesciences, Pocheon, Korea) in embryoid body (EB) medium (DMEM/F12, 10% KnockOut SR XenoFree CTS, 1x NEAA, 1x P/S, and 0.1 mM β-mercaptoethanol; all from Invitrogen). After 5–10 days of 3-dimensional culturing, the EBs were attached to Matrigel (BD Biosciences, Bedford, MA, USA) coated slides and further cultured for 15 days in differentiation medium (DMEM/F12 supplemented with 1% NEAA, 1x P/S, 0.1 mM β-mercaptoethanol, and 10% FBS for endoderm and mesoderm or DMEM/F12 supplemented with 1x NEAA, 1% P/S, 0.1 mM β-mercaptoethanol, 1x N2 supplement, and 10 ng of bFGF for ectoderm). Several representative markers specific for derivatives of these three germ layers were used for immunostaining after differentiation. The antibodies used in this study are listed in Supplementary Table  2.

We cultured Q-CTS-hESC-2 cell line [10 (link), 31 (link)] using xeno-free Essential 8™ Medium (A1517001, Gibco) to induce differentiation into RPE cells as previously described [10 (link)]. Briefly, the differentiation from hESC to RPE cells employs procedures such as super-confluence, acquired pigment foci, and excision. The culture medium for the RPE cells that diffused from the excised pigment foci contained 78% KO-DMEM CTS (Invitrogen), 20% Knock Out SR xenofree CTS (Invitrogen), 1% CTS glutaMAX-1 supplement (Invitrogen), 1% MEM NEAA (Invitrogen), and 1% 2-Mercaptoethanol (Procell). hESC-derived RPE cells were cultured in cell culture dishes at 37 °C in an incubator with 5% CO₂/95% air, and the medium replaced every 2 days. Proliferating cultures were passaged 1:4, after being digested with CTS™ TrypLE™ Select Enzym (Gibco).