Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells

Bacterial strains and culture conditions.S. Typhimurium SL1344, a well-characterized isolate used routinely in laboratory experiments (18), was used in these studies. In order to examine iHOs during an attenuated bacterial infection, we used an S. Typhimurium SL1344 strain defective in the invA gene of Salmonella pathogenicity island 1 (SPI-1). S. Typhimurium SL1344 carrying an invA deletion was constructed by moving the invA::Km^r deletion from S. Typhimurium SL3261 to SL1344 by P22 transduction. invA mutant S. Typhimurium has been described previously (19). Bacteria were grown in Luria-Bertani broth overnight at 37°C with shaking. Before infection, bacterial cells were resuspended at 2 × 10⁷ CFU ml⁻¹ in Dulbecco's phosphate-buffered saline (Sigma-Aldrich), with 1 × 10⁶ bacteria added per well of a 24-well plate of organoids. For microinjection, bacterial cultures were diluted 1:1 with phenol red (Sigma-Aldrich).

hIPSC culture.hIPSCs were grown under feeder-free conditions as previously described (20). To allow adherence of hIPSC colonies, tissue culture plastic (Corning) was precoated with porcine gelatin (1 g/liter; Sigma-Aldrich) dissolved in water for embryo transfer (Sigma-Aldrich) for 30 min, followed by MEF medium consisting of Dulbecco's modified Eagle's medium (DMEM; Invitrogen), 10% fetal bovine serum (Biosera), 1% 200 mM l-glutamine (Invitrogen), and 1% penicillin-streptomycin (10,000 U ml⁻¹; Invitrogen). hIPSCs were maintained feeder free at 37°C and 5% CO₂ in chemically defined, serum-free IPSC maintenance medium (CDM-PVA) consisting of 50% DMEM/F-12 (Invitrogen), 50% Iscove's modified Dulbecco's medium (Invitrogen), 5 ml of chemically defined lipid concentrate (Invitrogen), 450 μM monothioglycerol (Sigma), 7 μg ml⁻¹ insulin (Roche), 15 μg ml⁻¹ transferrin (Roche), 1 mg ml⁻¹ PVA (Sigma), and 10,000 U ml⁻¹ penicillin-streptomycin (Invitrogen) supplemented with activin A (10 ng ml⁻¹; R&D Systems) and basic fibroblast growth factor (bFGF, 12 ng ml⁻¹; R&D Systems). The medium was changed daily, and hIPSCs were passaged by using a mixture of collagenase and dispase at a ratio of 1:1 every 5 days. The hIPSC line used in this study was A1ATD-1 (21).

Growth and passaging of intestinal organoids.Before beginning differentiation, hIPSCs were passaged and grown for 2 days (days 0 and 1) under CDM-PVA maintenance conditions (described above). hIPSCs were differentiated into endoderm, hindgut, and then intestinal organoids by using a previously published protocol (22). For differentiation into endoderm, the medium was changed to CDM-PVA supplemented with 100 ng ml⁻¹ activin A (R&D Systems), 100 ng ml⁻¹ bFGF (R&D Systems), 10 ng ml⁻¹ bone morphogenetic protein 4 (BMP-4; R&D Systems), 10 μM phosphoinositol 3-kinase inhibitor LY294002 (Promega), and 3 μM CHIR99021 (Stratech Scientific Ltd.), which inhibits GSK3, resulting in WNT activation (days 2 and 3, day 3 without CHIR99021). To pattern the definitive endoderm to hindgut, the medium was changed to RPMI/B-27 medium consisting of RPMI 1640 medium plus GlutaMAX, 10 ml of 50× B27 supplement, 5 ml of nonessential amino acids (Gibco), and 10,000 U ml⁻¹ penicillin-streptomycin (Invitrogen) supplemented with 100 ng ml⁻¹ activin A and 100 ng ml⁻¹ bFGF (days 4 and 5, day 5 without bFGF and with activin A at 50 ng ml⁻¹). The medium was then changed to RPMI/B27 supplemented with 3 μM CHIR99021 and 3 μM retinoic acid (Sigma) (days 6 to 9). To generate three-dimensional (3D) intestinal organoids, hindgut tissue was washed with phosphate-buffered saline (PBS), incubated at 37°C with collagenase, and removed from the plate via gentle cell scraping. Tissue was manually broken up into smaller pieces, washed twice by centrifugation, and then resuspended in Matrigel (growth factor reduced phenol red free; Corning). Sixty-microliter droplets were added to each well of a 24-well plate, incubated for 15 to 20 min at 37°C, and then covered with 800 μl of iHO base growth medium: Advanced DMEM/F12 (Gibco), 10 ml of 50× B-27 serum-free supplement (Life Technologies), 5 ml of 100× N-2 supplement (Life Technologies), 1% 200 mM l-glutamine, 10,000 U ml⁻¹ penicillin-streptomycin, supplemented with 500 ng ml⁻¹ R-spondin 1 (R&D), 100 ng ml⁻¹ Noggin (R&D), 100 ng ml⁻¹ epidermal growth factor (EGF; R&D), 3 μM CHIR99021, 10 μM Y-27632 dihydrochloride monohydrate (Sigma), and 2.5 μM prostaglandin E₂ (Sigma). The medium was changed every 2 to 3 days. iHOs were passaged every 4 to 6 days by dissolving Matrigel with Cell Recovery Solution (Corning), washing by centrifugation, manual disruption of the organoid ultrastructure via pipetting, and resuspension in fresh Matrigel overlaid with iHO base growth medium.

Stimulation experiments.After at least 6 weeks in culture A1ATD-1 iHOs were challenged with S. Typhimurium SL1344 (1 × 10⁶ CFU ml⁻¹) or left uninfected. Stimulated organoids were incubated for 3 h at 37°C and 5% CO₂, and then RNA was purified with the RNeasy minikit (Qiagen).

Microinjection.After at least 6 weeks in culture, iHOs were prepared for microinjection 4 days prior to infection by passaging as described above. Disaggregated iHOs were resuspended in 200 μl of Matrigel, plated into glass bottom Willco Wells microinjection dishes (INTRACEL), and covered with iHO base growth medium. The Eppendorf TransferMan NK2-FemtoJet express system was used to inject bacteria into organoids. All injections were carried out in a chamber at 37°C and 5% CO₂. After injection, organoids were incubated for 3 h at 37°C and 5% CO₂ and then fixed for microscopy. For RNA sequencing (RNA-Seq) analysis, 30 organoids per microinjection plate were injected with either PBS or S. Typhimurium SL1344 mixed 1:1 with phenol red dye. iHOs were incubated for 3 h at 37°C and 5% CO₂, Matrigel was dissolved with Cell Recovery Solution (Corning), and phenol red-marked iHOs were isolated. RNA was then purified with the RNeasy minikit (Qiagen).

RT-qPCR.RNA isolated from whole iHOs before or after injection was reverse transcribed with the QuantiTect reverse transcription (RT) kit (Qiagen) according to the manufacturer's protocol. All RT-quantitative PCR (qPCR) experiments were performed with TaqMan gene expression assays and TaqMan gene expression master mix (Applied Biosystems) on the Applied Biosystems StepOne real-time PCR system. RT-qPCR data were analyzed via the comparative C_T method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control.

RNA-Seq and analysis.RNA was prepared from iHOs microinjected with S. Typhimurium SL1344 and iHOs microinjected with PBS (as described above) for three biological replicates per condition. Multiplexed mRNA libraries were prepared by using the Illumina TruSeq protocol and sequenced via paired-end sequencing with the Illumina-C HiSeq 2500 platform. Each lane of Illumina sequence was assessed for quality on the basis of GC content, average base quality, and adapter contamination. RNA-Seq reads were aligned with TopHat (23) version 2.0.8 with the human reference version GRCh37 used for the 1000 Genomes project. The read counts per gene were generated with featureCounts version 1.4.5-p1. The annotation for featureCounts came from ENSEMBL 75. Read counts were used to represent gene expression levels. R version 3.1.2 was used to import count data, and the DESeq2 package was used to normalize the data and detect differentially expressed genes (24). Significantly differentially expressed genes were selected with a cutoff false-discovery rate of less than 0.05 and a log change of >2.0-fold. Heat maps and principal-component analysis (PCA) plots were constructed from rlog-transformed data with the ggplot2 R library (http://cran.r-project.org/web/packages/ggplot2/index.html). For the 250 most significantly upregulated genes, enriched gene ontology terms were identified by using InnateDB with the category Biological Processes selected.

Anti-human cytokine/chemokine multiplex bead assays.Triplicate 25-μl samples of iHO culture supernatants from unstimulated iHOs and iHOs stimulated with S. Typhimurium SL1344 by addition to the culture medium were analyzed for cytokine/chemokine concentrations. Millipore customized anti-human Milliplex magnetic bead kits were used in accordance with the manufacturer's instruction, and a multiplex selection of the analytes tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8 was measured. Briefly, 25-μl aliquots of supernatants or dilutions of a mixed cytokine standard were captured on anticytokine antibody-coated beads overnight. On the following day, after washing with a 96-well plate-formatted magnet, detection reagents were added and allowed to bind to any captured cytokine present on the beads. Following further washes with the 96-well magnet, data were acquired on a Luminex FlexMap3D and analyzed with Luminex X-exponent software by extrapolation from the standard-curve dilutions. Results are expressed in picograms per milliliter.

Immunohistochemistry.For immunohistochemical analysis, iHOs suspended in Matrigel were fixed in 3% paraformaldehyde and 0.125% glutaraldehyde in PBS for 1 h at room temperature. They were then cooled to 4°C and dehydrated through an ethanol series (25, 50, 70, and 100% for 30 min each), infiltrated with glycol methacrylate (GMA) JB-4 and benzoyl peroxide (JB-4 embedding kit; Sigma-Aldrich), polymerized in gelatin capsules at 4°C for 24 to 48 h, and stored at −20°C. One- to 2-μm sections prepared on a Leica UCT ultramicrotome were mounted on slides precoated with 0.01% (wt/vol) poly-l-lysine hydrobromide. For immunoperoxidase staining, samples were blocked for 30 min with 0.1% sodium azide and 100 μl of 30% hydrogen peroxide and for 30 min with RPMI 1640 culture medium (Invitrogen) with 5% fetal calf serum and 1% BSA and then washed with TBS buffer (0.6 g of Tris, 8 g of sodium chloride, 1 liter of distilled H₂O [dH₂O], 3 to 4 ml of 1 M HCl to pH 7.6) three times for 15 min each. Samples were incubated overnight at 4°C with primary antibodies for mucin 2 (MUC2; ABCAM ab11197), lysozyme (LYZ; ABCAM ab2408), and chromogranin A (CHGA; ABCAM ab36997) in TBS buffer at antibody-dependent dilutions (MUC2, 1:200; LYZ, 1:50; CHGA, 1:50). Bound primary antibodies were detected with the EnVision+ System-HRP (AEC) (Dako), and nuclei were visualized by counterstaining with Mayer's hematoxylin. Finally, sections were covered with GeneTex Clear Mount (Sapphire Bioscience) and baked at 80°C for 5 min to set. For immunofluorescence staining for LAMP-1 (ABCAM ab24170), followed by donkey anti-rabbit 647 (ABCAM ab150075) and Salmonella common structural antigen 1 (CSA-1), fluorescein isothiocyanate-labeled (Insight Biotechnology Limited 02-91-99) sections were similarly processed by omitting the first block and diluting all of the antibodies in PBS (LAMP-1, 1:50; donkey anti-rabbit 647, 1:100; CSA-1, 1:20). Sections were mounted in Prolong-Gold with added 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen).

Transmission electron microscopic analysis of infected iHOs.Infected organoids were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (1 liter of dH₂O, 21.4 g of sodium cacodylate, 1 g of MgCl₂, 0.5 g of CaCl₂, adjusted to pH 7.42 with HCl), postfixed in 1% osmium tetroxide diluted in sodium cacodylate buffer, dehydrated with an ethanol series, and then embedded with the Epoxy Embedding Medium kit (Sigma-Aldrich). After embedding, samples were cured at 65°C for 48 h. Semithin (0.5-μm) sections were cut on a Leica UCT ultramicrotome and stained with toluidine blue on a microscope slide with suitable areas selected for ultrathin 50-nm sectioning. Ultrathin sections were collected on copper grids and contrasted with uranyl acetate and lead citrate before viewing on an FEI 120-kV Spirit BioTWIN transmission electron microscope. Images were taken on an F4.15 Tietz charge-coupled device camera.

Invasion assays.Microinjection was carried out as described above. To assess the invasion of iHO epithelial cells by bacteria, we modified the commonly used gentamicin protection assay (25) for use in iHOs. Forty iHOs per microinjection dish were injected with either wild-type or invA mutant S. Typhimurium SL1344. Injected iHOs were incubated for 90 min at 37°C and 5% CO₂. iHOs were isolated from Matrigel with Cell Recovery Solution (Corning), centrifuged, washed once with PBS, subjected to manual disaggregation of the organoid ultrastructure, resuspended in iHO base growth medium supplemented with 0.1 mg ml⁻¹ gentamicin (Sigma), and incubated at 37°C for 1 h. iHO aggregates were then centrifuged and washed once with PBS (Sigma). Cells were lysed with 1% Triton X-100 in PBS. Lysates were serially diluted in PBS, and 20-μl spots were plated onto prewarmed LB plates for CFU counting. Protection assays were performed with three biological replicates.

Statistical analysis.For statistical comparisons, we used unpaired, two-tailed t tests done with the Prism 6.0b software (GraphPad).

Nucleotide sequence accession numbers.RNA-Seq data are stored in the European Genome-Phenome Archive under study accession number EGAS00001001253. Data will be made available to all researchers upon request to the Data Access Committee (DAC) for the Wellcome Trust Sanger Institute, accession number EGAC00001000205. The named person of contact for the DAC for the Wellcome Trust Sanger Institute is Giselle Kerry (gh2@sanger.ac.uk). The restriction on data access is required for human donor protection.