To generate colonic organoids, we modified previously reported protocols (14 (link)-16 (link, link)). Briefly, 2.4 million hESCs were plated in a 24-well plate and allowed to reach 70% confluency as day 0. From day 0 to day 3, definitive endoderm (DE) was induced using RPMI 1640 medium (Welgene, Korea) containing 2% fetal bovine serum (FBS) (Welgene, Korea), 3 uM CHIR99021 (Sigma Aldrich, USA), and 100 ng/ml Activin A (Peprotech, USA). From day 4 to day 7, hindgut cells were differentiated using DMEM/F-12 medium (Welgene, Korea) containing 2% FBS, 1% B27 (Thermo Fisher, USA), 3 uM CHIR99021, and 500 ng/ml fibroblast growth factor 4 (FGF4) (Peprotech, USA), and then the resulting cell aggregates were embedded in Geltrex (Thermo Fisher, USA) after detachment using dispase (Thermo Fisher, USA). From day 8, DMEM/F-12 medium containing 1% B-27, 3 uM CHIR99021, 100 nM LDN193189 (STEMGENT, USA), and 100 ng/ml epidermal growth factor (EGF) (Peprotech, USA) was applied continuously, and from day 14 to day 20, 20 ng/ml BMP4 (Peprotech, USA) was temporarily added to the same medium composition to generate colonic organoids.