Recombinant human r spondin 1

H2B-mCherry murine intestinal organoids were a gift from Norman Sachs and Joep Beumer (Hubrecht Institute, The Netherlands). Organoids were cultured in basement membrane extract (BME, Trevingen) and overlaid with growth medium consisting of murine recombinant epidermal growth factor (EGF 50 ng/ml, Life Technologies), murine recombinant Noggin (100 ng/ml, Peprotech), human recombinant R-spondin 1 (500 ng/ml, Peprotech), n-acetylcysteine (1 mM, Sigma-Aldrich), N2 supplement (1×, Life Technologies) and B27 supplement (1×, Life Technologies), Glutamax (2 mM, Life Technologies), HEPES (10 mM, Life Technologies), and Penicilin/Streptomycin (100 U/ml 100 μg/ml, Life Technologies) in Advanced DMEM/F-12 (Life Technologies). Organoid passaging was performed by mechanically dissociating crypts using a narrowed glass pipette.

Small intestinal organoids were cultured according to the established protocol^{1 (link)}. Approximately 200 crypts were mixed with 20 μl of Matrigel (Corning) and plated in 24-well plates. After polymerization of Matrigel, 500 μl of crypt culture medium (Advanced DMEM/F12 (Gibco), 1 × Glutamax (Gibco), 10 mM HEPES (Gibco), N2 supplement (1:100) (Gibco), B27 supplement (1:50) (Gibco), 0.5 U/mL penicillin/streptomycin (Gibco), 50 ng/ml mouse recombinant epithelial growth factor (PeproTech), 100 ng/ml mouse recombinant Noggin (PeproTech), and 500 ng/ml human recombinant R-spondin1 (PeproTech)) was added.

Hoechst 33342, Ko143 and YHO-13177 were purchased from Med Chem Express (Monmouth Junction, NJ, USA). Recombinant human R-spondin 1 and recombinant mouse noggin were obtained from Pepro Tech Inc. (Rocky Hill, NJ, USA). Mouse recombinant EGF, advanced DMEM/F12, B-27 supplement and N-2 supplement were supplied by Invitrogen (Carlsbad, CA, USA). Matrigel (GFR, phenol-free) was bought from Corning Inc. (Bedford, MA, USA). Anti-BCRP antibody and goat anti-rabbit antibody were purchased from Abcam (Cambridge, UK). Trizol, PrimeScript™ RT reagent Kit and SYBR^® Premix Ex Taq™ II MIX were supplied by Takara (Dalian, China).

Isolated crypts were resuspended in Matrigel (Corning) and plated in 48-well culture plates (Corning). After incubation at 37 °C for 15 min, 250 μL of maintenance medium (50% Wnt3a-conditioned medium (ATCC#CRL-2647, Manassas, VA, USA) and 50% of 2× basal medium, supplemented with recombinant human EGF (Sigma-Aldrich), recombinant human noggin (R&D Systems, Minneapolis, MN, USA), recombinant human R-spondin1 (PeproTech, Cranbury, NJ, USA), nicotinamide (Sigma-Aldrich), p160ROCK inhibitor (Selleck Chemicals, Houston, TX, USA), p38 MAP kinase inhibitor (SB202190, Sigma-Aldrich), and Prostaglandin E2 (PGE2, Cayman Chemical, Ann Arbor, MI, USA), were added to the wells. A GSK3 inhibitor (Stemgent, Cambridge, MA, USA) was added to the medium during the first 2 days.
Depending on their shape, organoids can be classified into spheroids and enteroids. Spheroids are defined as round- or oval-shaped organoids with a thin wall composed of a single layer of undifferentiated cells. Enteroids are defined by the presence of visually sharp borders (buddings) along their basolateral (anti-luminal) side or irregularly thickened walls, which consist of all components of epithelial cells [28 (link)] (Figure 10).

Advanced Dulbecco’s modified Eagle’s medium/F12, Antibiotic-Antimycotic (Anti-Anti), and B-27 supplement were from Thermo Fisher Scientific (Waltham, MA); recombinant human R-spondin-1, recombinant human EGF, recombinant human HGF, recombinant human FGF10, and recombinant human noggin were from PeproTech (Cranbury, NJ):, gastrin, N-acetylcysteine, Y27632, nicotinamide and A83-01 were from Millipore-Sigma (Burlington, MA); human recombinant Wnt3A, Forskolin and human recombinant IL-17A were from R&D Systems (Minneapolis, MN). Matrigel (354230) and cell recovery solution were from Corning (Kennebunk, ME, USA).

After 2D pre-culture, cells were detached by 0.5% trypsin-EGTA solution (Sigma) from the plate and washed with DMEM and 10% FBS. The cell pellet (5000 cells per a well of 24-well culture plate) was mixed with extracellular matrices that consisted of Matrigel (BD Bioscience, San Jose, CA, USA) and Collagen Type-1 (Nitta Gelatin, Japan). In Fig. 2 (b), 2500 cells per well of 24-well culture plates were used. After 10 min incubation, gels become solid and the culture medium was added. The culture medium was a 1∶1 mixture of H-CFU-C medium and DMEM/F-12 supplemented with 2% B27 supplement, 0.25 μmol A-83-01 (Wako Pure Chem., Osaka, Japan), 10 μmol Y-27632 (Wako Pure Chem.), 20 ng/ml EGF (Peprotech), 40 ng/ml HGF, 40 ng/ml recombinant human Wnt-3a (R&D Systems), and 100 ng/ml recombinant human R-spondin 1 (Peprotech). Media was changed at every 3 days. At 8 and 12 days, the number of cysts with >50 μm diameter was counted.

This study was approved by the Institut Pasteur’s ethical and medical committee under the agreement N°2012-37. Surgically resected human colonic tissues were obtained from the Henri Mondor Hospital. All samples were obtained from patients who provided informed consent before surgery. Normal epithelia were isolated and cultured according to the protocol described by Sato et al., with minor modifications^{36 (link)}. Organoids were cultured with Advanced DMEM/F12 (ThermoFisher) supplemented with Hepes (ThermoFisher), 2 mM GlutaMAX (ThermoFisher), 100 U/mL penicillin and 100 μg/mL streptomycin (ThermoFisher), 1× N2 and B27 supplements (ThermoFisher), 1 mM N-acetyl-L-cysteine (Sigma), 10 μM Y-27632 (Sigma), 500 nM A83-01 (Tocris), 10 μM SB202190 (Sigma), 10 mM nicotinamide (Sigma), 10 nM gastrin I (Sigma), 100 ng/mL recombinant human Noggin (R&D Systems), 50 ng/mL recombinant human EGF (R&D Systems), 1 μg/mL recombinant human R-Spondin-1 (Peprotech), 100 ng/mL recombinant human Wnt-3A (R&D Systems), 3 μM CHIR99021 (Sigma), and 10% (vol/vol) FBS (ThermoFisher), at 37 °C in 5% CO₂. Oraganoids were cultured in 48-well plates, 100 crypts per well. RNA was isolated using the RNeasy Mini kit and the RNase free DNase kit (Qiagen). Gene expression was analyzed using primers purchased from Sigma. Data were normalized to the B2M housekeeping gene expression.