“…The development of HCOs was performed in three distinct phrases including gendoderm and hindgut differentiation and colonic organoid development ( Figure 1 ). Endoderm differentiation was performed in reference to the previous study with some modifications [ 22 ]. On day 1, 200,000 human embryonic stem cells (hESCs) were seeded in a 24-well plate and treated with B27 (1:100) (Gibco, Grand Island, NY, USA, 12587-010), Activin A (100 ng/mL, Peprotech, Rock Hill, NJ, USA, 120-14E), and mesoderm differentiation compound CHIR-99021 (3 µM, Sigma-Aldrich, St. Louis, MO, USA, SML1046-25MG), in which B27 is the key component to improve cell survival in definitive endoderm.…”