The healthy KUTE-4 female skin fibroblast-derived human iPSC (hiPSC) line (available from the European Collection of Authenticated Cell Cultures (karyotyped, passage 24-36)) was cultured on plates coated with 40µl/ml vitronectin in PBS (StemCell Technologies). E8 (Gibco) media was changed daily, pockets of differentiation were actively removed, and round, pluripotent colonies were passaged with Versene (Gibco) every 4-6 days, when 60-70% confluent, or before circular colonies began merging.
KUTE-4 hiPSC were differentiated into human small intestine organoids (HIO) following established protocols18 (link). In short, hiPSC were patterned toward definitive endoderm in RPMI with daily increasing B27 (0.2%, 1%, 2%) and 100ng/ml ActivinA (R&D) for 3.5 days, then patterned towards midgut in RPMI+2%B27 with 3μM CHIR99021 (Wnt agonist, TOCRIS) and 500ng/ml recombinant FGF4 (R&D) for 4days. At this point, CDX2 colonies were picked using a 200µl pipette tip, replated in 35µl Matrigel, then matured in basal media with hEGF 100ng/ml, R&D) rh-Rspondin (500ng/ml, R&D), rh-Noggin (100ng/ml, R&D), and 2ng/ml IL-2 supernatant for at least 35 days prior to establishing co-cultures with hILC1 or encapsulation in synthetic hydrogels for aILC1 characterization.
KUTE-4 hiPSC were differentiated into human small intestine organoids (HIO) following established protocols18 (link). In short, hiPSC were patterned toward definitive endoderm in RPMI with daily increasing B27 (0.2%, 1%, 2%) and 100ng/ml ActivinA (R&D) for 3.5 days, then patterned towards midgut in RPMI+2%B27 with 3μM CHIR99021 (Wnt agonist, TOCRIS) and 500ng/ml recombinant FGF4 (R&D) for 4days. At this point, CDX2 colonies were picked using a 200µl pipette tip, replated in 35µl Matrigel, then matured in basal media with hEGF 100ng/ml, R&D) rh-Rspondin (500ng/ml, R&D), rh-Noggin (100ng/ml, R&D), and 2ng/ml IL-2 supernatant for at least 35 days prior to establishing co-cultures with hILC1 or encapsulation in synthetic hydrogels for aILC1 characterization.