H9 hesc line

This study was approved by the Korean Public IRB (IRB number: P01-201409-ES-01-09, P01-201609-31-002). The H9 hESC line (WiCell Research Institute, Madison, WI, USA) and two independently derived hiPSC lines were routinely cultured as described previously.^{22 (link)} The 3D hIOs were differentiated as described previously.^{22 (link)} hPSCs were induced into definitive endodermal cells by treatment with 100 ng/mL Activin A (R&D Systems, Minneapolis, MN, USA) for 3 d in RPMI 1640 (Thermo Fisher Scientific, Waltham, MA, USA) and increasing concentrations of fetal bovine serum (FBS, 0%, 0.2%, 2% Thermo Fisher Scientific). To induce differentiation into hindgut (HG) stage cells, definitive endodermal cells were treated with 500 μg/mL FGF4 (R&D Systems) and 3 μM CHIR 99021 (Tocris Bioscience, Minneapolis, MN, USA) in RPMI 1640 medium containing 2% FBS for 4 d. Following HG induction, 3D HG spheroids spontaneously formed. 3D HG spheroids were placed in Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and cultured in hIO medium containing 100 µg/mL EGF (R&D Systems), 500 µg/mL R-spondin1 (R&D Systems), 100 µg/mL Noggin (R&D Systems), and 1X B27 supplement in advanced DMEM F12 (Thermo Fisher Scientific).

The H9 hESC line from WiCell Inc. was used in this study. Expansion of hESCs was performed on feeder cells as described previously 16. The differentiation of H9 CRX‐GFP reporter lines towards retinal lineages was performed at least three times under three‐dimensional (3D) culture conditions, using bacteriological Petri dishes (BD Biosciences) and IGF‐1 supplemented media as described in our recent publication 12.

The H9 hESC line was purchased from WiCell Research Institute (Madison, WI, USA). The generation of hiPSC and culture methods of hESC and hiPSCs have been described in a previous report^{40 (link)}. Briefly, hESCs and iPSCs were maintained on Matrigel (BD Biosciences) in mTesR1 medium without feeders, and routinely passaged every week.