All animal experiments were approved by the Finnish National Animal Experiment Board (Permit: ESAVI/5824/2018). Maf lacZ mice were caged in standard light-dark conditions at the pathogen-free animal facility of the faculty of Medicine and Health Technology. Food, water ad libitum was followed in a regularly timed schedule. B6.129-Maftm1Gsb/J heterozygous mice were purchased from Jackson laboratories (Cat number: 004158 | MaflacZ). To generate Maf wildtype line, heterozygous line, and homozygous line, B6.129-Maftm1Gsb/J heterozygous were mated with, B6.129-Maftm1Gsb/J heterozygous, F1 generation was backcrossed with heterozygous mice. Littermates with Maf wildtype were used as control. Maf genotypes were confirmed by qPCR. After experiments, Maf wildtype and Maf knockout mice were euthanized ethically in a controlled CO2 chamber
5.2.β-. galactosidase staining of Peyer’s patches and organoids
Ileal PP’s were isolated from the ileal section of the small intestine and transferred to a 10cm dish with 30ml of cold PBS. Excess fat was cut from the tissues and embedded into paraffin blocks. Organoids grown in RANKL was washed with PBS and made into paraffin blocks. The blocks were cut at 10um sections and mounted on a slide. X-gal (5-Bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside, Goldbio) was dissolved in dimethylformamide at 50 mg/ml. Paraffin blocks were fixed with 4% PFA for 10 minutes. The slides were washed 3 times with 3 changes of PBS for 5 minutes wash and the final rinse in distilled water. After drying the slides were incubated in X-gal working solution at 37 C for 24 hours in a chamber with adequate moisture content. The sections were washed in PBS solution 2 times for 5 minutes each. After rinsing with distilled water, the sections were counter-stained with nuclear fast red for 3-5 minutes followed by further rinsing and washing in distilled water for 2 minutes. The sections were finally dehydrated for 3 minutes each in 70%, 95%, and twice with 100% ethanol and thrice with xylene. The sections were mounted Permount and covered with a coverslip and examined by digital slide scanner Hamamatsu Nanozoomer.
5.3. Mouse Intestinal Organoid culture
Mouse intestinal crypts were isolated and cultured in an in vitro setting as previously described 15,41 Crypts were isolated as soon as the stillborn pups were delivered. Isolated duodenums were washed in PBS and longitudinally cut. Villi were gently scraped with a glass slide. Following washes with PBS, the tissue was cut into 2mm pieces and pipetted up and down with a 10 ml pipette. After repeated changes of the PBS till the suspension was clear, the tissues were suspended in 10mM EDTA in PBS for 20 minutes rocking at room temperature. The crypts were separated from the rest of the tissues using a 70-μm cell strainer (Fisher Scientific). The crypts were counted and cultured on a 24 well plate by embedding them in 30ul of cold Matrigel (Corning). Organoids were cultured in an optimal medium consisting of advanced DMEM/F12 (Thermo Fisher Scientific) that contained HEPES (10mM, Sigma-Aldrich), Glutamax (2mM, Thermo Fisher Scientific), Penicillin-streptomycin (100U/ml, Sigma-Aldrich), B-27 supplement minus Vitamin A (Thermo Fisher Scientific), N-2 supplement (Thermo Fisher Scientific), N-acetylcysteine (1 mM; Sigma-Aldrich), recombinant murine EGF (50 ng/ml; Thermo Fisher Scientific), recombinant murine Noggin (100 ng/mL; PeproTech), recombinant mouse R-spondin1 (1 μg/mL; R&D Systems). Media were changed every 2 days. For M cell differentiation, recombinant mouse RANKL (100ng/ml, Peprotech) was added to the media and incubated for 4 days.
5.4. CRISPR–Cas9 gene knockout of intestinal organoids
Guide RNAs for gene encoding RANK were designed using CRISPR design tool (http://crispr.mit.edu)42. These were cloned into the vector lentiCRISPR v2 (Addgene, 52961). The cloned product was transfected into HEK 293FT cells (ThermoFisher R7007). The supernatant was collected after 48 h and the Lenti-X concentrator (Clontech) was added to the suspension. The 293FT cell line was tested for mycoplasma. Intestinal organoids were cultured in ENCY (EGF, Noggin, Chir-99021, and Y-27632) prior to transduction. After the organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) and supplemented with 1,000 U/ml DnaseI at 32 °C for 5 mins, the cells were washed once with Advanced DMEM and resuspended in a transduction medium (ENR media supplemented with 1mM nicotinamide, Y-27632, Chir99021, 8 μg/ml polybrene (Sigma-Aldrich)) and mixed with the previously collected concentrated virus. The mixture was centrifuged at 600 x g 32 °C for 1 hr followed by 3 hr incubation at 37°C, after which they were collected and plated on 60% Matrigel with enriched transduction medium without polybrene. On day 2 and day 4, RANK transduced organoids were selected with 2 μg/ml of puromycin (Sigma-Aldrich). Surviving clones were expanded in cultured with ENR medium. RANK KO organoids were confirmed by western blot to validate the expression of deleted gene.
5.5. Immunofluorescence of Organoids
Maf WT and Maf KO Intestinal crypt organoids were analyzed by whole-mount immunostaining. The organoids were cultured for 4 hours in an 8-well chamber plate in the presence and absence of RANKL 100ng/ml following fixation with 4% PFA for 15 minutes, followed by permeabilization with 0.1% Triton X-100 for another 15 minutes. The organoids were stained with Gp2 (MBL, D278-3) antibodies overnight at +4 degrees Celsius. This was followed by 2-hour incubation of anti-Rabbit secondary for Gp2 (and Anti-Rat for the secondary antibody. Gp2 expressing cells were analyzed by Nikon A1R+ Laser Scanning Confocal Microscope after mounting with ProLong Diamond with Dapi mounting solution (Molecular Probes P36962).