Jagged 1 peptide

Small intestinal tissue was decellularised as previously described by¹⁵ . Pieces of decellularised small intestinal ECM (dECM) were washed 10 min in PBS, plated on a culture dish, and stained with Col-F Collagen Binding Reagent dye (Col-F) (BioSite, 260-6346) overnight at 4°C. dECM was then washed with fresh PBS, and primed for approximately 30 minutes with live imaging medium consisting of Advanced DMEM/F12, 1x penicillin/streptomycin, 1x Glutamax (Thermo Fisher Scientific), 10mM HEPES (Thermo Fisher Scientific), 1x B27 (Life Technologies), 1x N2 (Life Technologies), 1mM N-acetylcysteine (Sigma), 50ng/ml of murine recombinant Epidermal growth factor (R&D), 100ng/ml recombinant murine Noggin (Peprotech), 10μM Y-27632 (Sigma) and 1 μM Jagged-1 peptide (Anaspec). After priming, excess medium was discarded to allow seeding of Lgr5⁺ cells.

Single colonic epithelial cells isolated by flow cytometry were cultured as described (Sato et al., 2009 (link)) in Matrigel droplets (BD Biosciences, 356231) supplemented with 1 μM Jagged-1 peptide (AnaSpec, 61298) and covered with 0.5 mL Wnt3a (70%) and R-spo1 (30%) conditioned medium supplemented with antibiotics, N-2 and B-27 supplements and rEGF as described (Sato et al., 2009 (link)). In various experiments, 10 μg/ml NOV (R&D Systems, 1976-NV-050), 10 ng/ml FGF18 (R&D Systems, 8988-F18–050), or IL-11 (Peprotech, 220–11, 0.1 μg, 0.2 μg or 0.4 μg) was added to the culture medium. For passaging, organoids were disaggregated manually by pipetting, then transferred to fresh Matrigel droplets for further culture as above. Organoids were visualized using a Celigo S Imaging Cytometer (Nexcelom) to capture at least 5 focal planes, then counted on the resulting images, ensuring that each structure was counted only once. For histology, organoids were fixed for 15 min in 4% PFA in PBS at room temperature, while still in Matrigel, then incubated in 5 U Dispase (Stem Cell Technologies, 07913) at 37°C for 30 min to remove Matrigel, and fixed overnight at 4°C in 4% PFA in PBS. Organoids were then embedded in OCT compound (Tissue-Tek, 4583), frozen at −80°C, and 8 μm sections were cut for staining with hematoxylin and eosin.

For organoid gene transduction, we utilized the modified method based on the previous reference (Onuma et al., 2013 (link)). FACS sorted Lgr5^high single cells (~5000) from PAF WT and PAF KO mice were incubated with media containing retroviruses expressing c-Myc with RFP (MSCV-c-Myc-IRES-RFP, Addgene [#35395]), or wild-type PAF and mutPIP-PAF (Jung et al., 2013 (link)) for 6h at 37 °C with polybr ene (7 μg/ml) and Jagged-1 peptide (1 μM, AnaSpec). Then, infected cells were seeded on 50 μL Matrigel/well in a 12-well plate with conventional ENR media. RFP+ cells were able to observe 2 days after infection. Only RFP+ cells were considered as transfected cells. For selecting nt-PAF or mutPIP-PAF infected organoids, blasticidin (10 μg/ml) was treated.

Following crypt isolation from the whole small intestine of both male and female mice, the single-cell suspension was re-suspended in Matrigel (BD Bioscience) with 1μM Jagged-1 peptide (Ana-Spec). Roughly 300 crypts embedded in 25μl of Matrigel were seeded onto each well of a 24-well plate. Once solidified, the Matrigel was incubated in 600μl culture medium (Advanced DMEM/F12, Invitrogen) with streptomycin/penicillin and glutamatax and supplemented with EGF (100 ng/mL, Peprotech), R-Spondin-1 (600ng/mL, R&D), Noggin (100ng/mL, Prepotech), Y-276432 dihydrochloride monohydrate (10μM, Tochris), N-acetyl-1-cysteine (1μM, Sigma-Aldrich), N2 (1X, Life Technologies), B27 (IX, Life Technologies) and Wnt3A (25ng/mL, R&D Systems). Fresh media was replaced on day 3, and organoids were passaged by dissociation with TrypLE and re-suspended in new Matrigel on day 6 with a 1:3 split ratio.