Homo sapiens
Protocols: All work with hESCs was reviewed and approved by the University of Michigan human pluripotent stem cell research oversight committee. The hESC cell line H9 (WA09; National Institutes of Health stem registry 0062) was obtained from the WiCell Research Institute (Madison, WI). Karyotypically normal cell lines were used for all experiments.In brief, hESCs were grown in feeder-free conditions on Matrigel (Corning, NY) with mTesrR1 medium (Stem Cell Technologies, Vancouver, BC, Canada), passaged into 24-well plates, and grown to approximately 70% confluency. Media then was changed to RPMI1640 media plus Activin A (ACTA) (100 ng/mL; R&D Systems, Minneapolis, MN) for 1 day. The same media was used on days 2 and 3 of differentiation, except that 0.2% and 2% fetal bovine serum (HyClone, GE Life Sciences, Logan, UT) was added on the respective days. For induction of caudal type homeobox2 (CDX2), endoderm was treated for 4 days with RPMI1640 with 2% fetal bovine serum (HyClone) and supplemented with fibroblast growth factor 4 (500 ng/mL), generated as previously described,28 plus 2 umol/L Chir99021 (Tocris Bioscience, Bristol, United Kingdom). Media was changed daily. For organoid experiments, spheroids that had formed on day 4 of CDX2 induction were transferred to a droplet of Matrigel and grown in advanced Dulbecco's modified Eagle medium/F12 supplemented with 1× B27 (Thermo Fisher, Waltham, MA), epidermal growth factor (EGF) (R&D Systems; 100 ng/mL), Noggin (5% conditioned medium), and R-Spondin2 (5% conditioned medium). Media was changed every 3-4 days. RNA was isolated using the mirVana RNA isolation kit, following the “Total RNA” isolation protocol (Thermo-Fisher Scientific, Waltham MA). RNA sequencing library preparation and sequencing was carried out by the University of Michigan DNA Sequencing Core and Genomic Analysis Services (https://seqcore.brcf.med.umich.edu/).