Y 27632

The hiPSCs (253G1) were provided by the RIKEN BRC through the Project for Realization of Regenerative Medicine and the National Bio‐Resource Project of the MEXT, Japan. The hiPSCs were cultured under feeder‐free conditions according to a previous report 13. Briefly, hiPSCs were seeded on plates coated with iMatrix‐511 (Nippi, Tokyo, Japan). hiPSCs were incubated in StemFit medium supplemented with Y‐27632 (10 µm; Wako, Osaka, Japan) for the first 24 h, and the medium was replaced with StemFit medium without Y‐27632. The StemFit medium was replaced with fresh medium every other day until culture day 7. After day 7, hiPSCs were used for differentiation protocols to obtain EPO cells.

Crypts were isolated from the small intestine of male WT mice aged 7–11 weeks or small intestinal tumors of female APC^min mice aged 21 weeks as previously described (27 (link)). One mouse was used for each isolation. The crypts were embedded in Matrigel (Corning). Advanced DMEM/F12 (Gibco) containing 50 ng/mL mEGF (Peprotech), 100 ng/mL mNoggin (R&D Systems), 100 U/mL penicillin/streptomycin (Nacalai), 10 mM HEPES (Nacalai), 2 mM GlutaMAX-1 (Gibco), 1 mM N-acetylcysteine (Sigma), 1×N2 supplement (Gibco), 1×B27 supplement (Gibco) and 10 μM Y-27632 (Wako) were added to each well (complete medium). Organoids from APC^min mice were passaged at least twice before co-culture with IECs.

All human cell protocols used in this study were approved by the Takeda Institutional Ethical Committee and followed the guidelines of the Declaration of Helsinki. Human iPSCs (Clone XCL‐1; XCell Science, Novato, CA, USA) were cultured on laminin‐coated plates in StemFit (Ajinomoto Healthy Supply Co., Inc, Tokyo, Japan) containing penicillin and streptomycin. The cells were passaged every 6–7 days using 0.5 mm EDTA, seeded at a density of 2.0 × 10⁴ cells·well⁻¹ on a six‐well plate, and maintained in the presence of 10 μm Y‐27632 (FujiFilm Wako Pure Chemical). The culture medium was replaced with StemFit containing penicillin and streptomycin in the absence of Y‐27632, 24 h after passaging.

Immortalized NPCs (1 × 10⁶ cells) supplemented with 1 × 10⁴ hiPSCs (1%) were seeded onto hESC-qualified Matrigel-coated 6-well plates in mTeSR Plus, and hiPSC-NPC preparations (2.5 × 10⁶ cells) supplemented with 2.5 × 10⁴, 250, and 50 hiPSCs (1%, 0.01%, and 0.002%, respectively) were seeded onto hESC-qualified Matrigel-coated 60-mm dishes in STEMdiff neural progenitor medium (Veritas) with 10 µM Y27632 (Fujifilm Wako Pure Chemical Corporation). Cells were transduced with the Ad vector at 3 or 18 pfu/cell for 24 h, followed by adding 10 nM AP1903. Twenty-four hours after treatment, cells were harvested by treatment with Accutase, and the cell suspension was incubated with an anti-TRA-1-60 monoclonal antibody conjugated with DyLight 650 (Thermo Fisher Scientific, #MA1-023-D650; 1:100) and with a FITC conjugated rBC2LCN (Fujifilm Wako Pure Chemical Corporation, #186-02,993; 1:100) on ice for 1 h, followed by washing with PBS. In the experiments using hiPSC-NPC preparations, the cells were fixed using a Foxp3/Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) on ice for 30 min. The stained cells were determined and analyzed using a cytoFLEX S (Beckman Coulter, Brea, CA, USA). The measurements were stopped when 2 × 10⁴ cells in the gates 1 and 2 shown in Supplementary Figs. S3, S8, and S9 were counted.

According to the previous study [34 (link),35 (link)], tet-on inducible iPSCs of NIL factors were established using the following vectors: PB-Neo-TRE3G-hNEUROG2-P2A-hLHX3-T2A-hISL1, pCMV-HyPBase-PGK-Puro and PB-CAG-rtTA3G-IH. These vectors were co-transfected into dissociated iPSCs using Gene Juice Transfection Reagent (Merck, Darmstadt, Land Hessen, Germany), as described previously. Transfectants were cultured in StemFit/AK02N containing 20 μM Y27632 (Wako, Chuo-ku, Tokyo, Japan), 50–150 μg/mL hygromycin (Nacalai), and 100–1000 μg/mL G418 (Nacalai) on iMatrix 511-treated 6-well plate. All surviving colonies were expanded without selection for a single clone. They were stored appropriately by StemCellBanker (ZENOAQ, Fukushima, Japan) and used for this study.