Human intestinal organoids were generated following previously established protocols (Spence et al, 2011 (link)) using multiple pluripotent stem cell (PSC) lines (Table EV2). Briefly, PSCs were differentiated through the definitive endoderm stage via the addition of Activin A (Cell Guidance Systems) between day‐0 (D0) and D2, BMP4 (R&D Systems) at D1 and CHIR99021 (Stemgent)/FGF4 (R&D Systems) from D3 to D6. The addition of CHIR99021/FGF4 prompted the formation of midgut tube spheroids from the definitive endoderm monolayer. At D7 midgut tube spheroids were collected and suspended in a Matrigel dome (Corning). Spheroids were then grown in gut media: DMEM/F12 (Gibco) supplemented with N‐2 (Gibco), B‐27 (Gibco), HEPES solution (Millipore‐Sigma), recombinant murine epidermal growth factor (Peprotech), L‐Glutamine (Gibco), Penicillin/Streptomycin (Gibco). At D21, sample density was reduced to ≈1–3 per 50 µl Matrigel bubble. Samples were grown for an additional 14 days in gut media, resulting in terminal HIO cultures (Spence et al, 2011 (link)). Individual HIOs were considered as biological replicates for all experiments. All experimentation using human tissues described herein was approved by an IRB at CCHMC (IRB#2014‐0427). Informed consent for tissue collection, storage, and use of the samples was obtained from the donors at CCHMC.