Differentiation of Human Intestinal Organoids

Human intestinal organoids (HIO) were generated and maintained as previously described [36 (link),37 (link)]. For definitive endoderm (DE) induction, IPSCs were passaged with Accutase (Invitrogen, Waltham, MA, USA) and plated at a density of 100,000 cells per well in a Matrigel-coated, Nunclon^® surface 24-well plate (Thermo fisher Scientific, Waltham, MA, USA). On the first day of splitting, 10 μM Y27632 compound (Sigma) was added to the mTeSR1 media. After 24 h, media were changed to mTeSR1 and cells were grown for an additional 24 h. Then, the cells were treated with 100 ng/mL of Activin A for 3 days as previously described for DE formation [6 (link)]. DE was then treated with hindgut induction medium (RPMI 1640, 2 mM L-glutamine, 2% decomplemented FBS, penicillin-streptomycin and 100 ng/mL Activin A) for 4 days with 500 ng/mL FGF4 (R&D) and 3 μM Chiron 99021 (Tocris) to induce the formation of mid/hindgut floating spheroids and mid/hindgut endoderm which remained in the monolayer.