Mnoggin

Intestinal organoids were derived from crypt fragments, which were mechanically released from the small intestine of a six-week-old C57BL/6 mouse and enriched by size exclusion of villi fragments^{35 (link)}. Organoids grew embedded in matrigel (Corning) in Advanced DMEM/F-12 medium supplemented with GlutaMAX, B-27, N-2 (ThermoFisher), 10 mM HEPES, 1 mM N-acetylcysteine (Sigma-Aldrich), 0.1 µg/ml mEGF (ImmunoTools), 0.1 µg/ml mNoggin (PeproTech), 20% R-spondin1-conditioned medium and antibiotics at 37 °C in a 10% CO₂ atmosphere, and were passaged weekly. R-spondin1 medium was prepared from 293 T cells stably expressing HA-mR-Spondin1-Fc^{53 (link)}. For the organoid growth assay, the medium of freshly passaged organoids was supplemented with 0, 10, or 50 µM GBZ, and after 120 h small (0–1 buds), medium (2–4 buds), and large (>4 buds) organoids were counted. The assay was performed in technical octuplicates.

Crypt isolation was performed as described previously [23 (link)]. Briefly, Intestines were harvested and washed with 1× PBS. Villi were scraped and the remaining tissue were cut into 2 cm pieces and incubated in 2 mM EDTA/PBS for 30 min at 4 °C. Crypts were then collected by shaking and were cultured in Matrigel (BD Bioscience; Heidelberg, Germany; #356231) overlaid with medium containing 50 ng/mL EGF (Life technologies; Darmstadt, Germany; PMG8043), 100 ng/mL mNoggin (Peprotech, #250-38, Cranbury, NJ, USA), 1 μg/mL mR-spondin1 (R&D systems, #2474-RS-050, Minneapolis, MN, USA) (ENR) in the presence of 10 μM Rock-inhibitor (Sigma, Y0503, Setagaya City, Tokyo). Crypts were plated in 8-well Ibidi treat^® dishes and imaging was performed on a Leica DMI 6000 confocal microscope equipped with an incubation system and image analysis was completed using Leica LAS AF software (Frankfurt, Germany).

Type I collagen gel (cellMatrix type 1-A) was purchased from Nitta Gelatin, Inc. (Osaka, Japan). Ham's F-12 nutrient mix, advanced DMEM/F12, N-2, B-27, gentamicin/amphotericin B, HEPES, and GlutaMAX-I were obtained from Gibco (Waltham, MA, USA). Matrigel was acquired from BD Biosciences (Franklin Lakes, NJ, USA). mEGF and mNoggin were purchased from PeproTech (Rocky Hill, NJ, USA). CHIR99021, [Leu15]-Gastrin 1, SB202190, nicotinamide, N-acetylcysteine, and thiazovivin were obtained from Sigma-Aldrich (St. Louis, MO, USA). A83-01 was acquired from Tocris Bioscience (Bristol, UK). Y-27632 and Gentle Cell Dissociation Reagent were purchased from STEMCELL Technologies (Vancouver, Canada). Penicillin and streptomycin were acquired from Welgene Inc. (Gyeongsan-si, Republic of Korea). FGF10 was purchased from ATGen (Seongnam, Republic of Korea).

Intestinal organoids were obtained as previously described^{63 (link)}: small intestines of mice were dissected and dissociated in 8 mM EDTA/PBS for 5 min at RT followed by 20 min incubation in ice-cold 2 mM EDTA/PBS at 4 °C. The epithelia were fractionated by shaking in ice-cold PBS. 250 crypts were seeded in 20 µl of growth factor-reduced Matrigel (BD Matrigel #356231) and cultured in basic crypt medium (60/40 Advanced DMEM/F12 supplemented with N2 and B27, GlutaMax, N-Acetylcysteine and Penicillin/Streptomycin (Invitrogen)) containing 50 ng/ml mEGF (Gibco), 100 ng/ml mNoggin (Peprotech) and 500 ng/ml hR-spondin1 (Peprotech). Organoids were split every 4–6 days by mechanical disruption. Cre-mediated recombination was induced by the addition of 800 nM 4-hydroxy-tamoxifen for 2 days. β-cat^GOF organoids were selected by R-spondin1 withdrawal. All analyses were performed at 7–10 days after mutagenesis. Recombinant human BMP4 (Peprotech) was added for 24 h in Noggin-free crypt medium supplemented with EGF and R-spondin1. For serial re-plating, β-cat^GOF; Mll1^−/− organoids were dissociated into single cells with TrypLE Express (ThermoFisher Scientific) for 5 min at 37 °C, and 2000 cells were seeded in 20 µl of growth factor-reduced Matrigel and cultured in crypt medium supplemented with mEGF, mNoggin, and hR-spondin1.