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Helicobacter pylori shows tropism to gastric differentiated pit cells dependent on urea chemotaxis


Gastric organoid generation and organoid-derived monolayer seeding

Human gastric tissue was obtained from four donors (age range 32–82 years) that underwent partial gastrectomy at the University Hospital of Würzburg. This study was approved by the ethical committee of the University of Würzburg (Approval 37/16) and informed consent was obtained from all the donors.

Generation and culture of gastric organoids were performed as described before21,60. For propagation, organoids were split every 12 days at a ratio of 1:10 using mechanical splitting. Culture medium was replaced every 2–3 days. Propagation culture medium contained: Advance Dulbecco’s modified Eagle medium/F12 (12634028, Thermo Fischer Scientific) supplemented with 10 mmol l−1 HEPES (15630056, Thermo Fischer Scientific), 1x GlutaMAX (35050-038, Thermo Fischer Scientific), 1xB27 (12587010, Thermo Fischer Scientific), N-Acetylcysteine 1 mM (A9165-25G, Sigma-Aldrich), Noggin conditioned medium 10%, R-spondin1 conditioned medium 10%, Wnt conditioned medium 50%, EGF 50 ng ml−1 (AF-100-15, Peprotech), FGF10 200 ng ml−1 (100-26, Peprotech), Gastrin 1 nM (3006, Tocris), TGFβi 2 µM (A-83-01, Tocris), Primocin 100 ng ml−1 (Ant-pm-1, Invivogen). The propagation culture medium was named ENRWFGTi_. After seeding RHOKi 10 µM (Y-27632, Sigma-Aldrich) was added.

Gastric organoid-derived monolayers were generated from 8-day-old organoids. Organoids were collected and mechanically disrupted. Organoid fragments were washed with Advanced DMEM/F12 containing GlutaMAX 1x (35050-038, Thermo Fischer Scientific), 10 mM HEPES (15630056, Thermo Fischer Scientific) and centrifuged at 400×g for 5 min. Organoid fragments were resuspended in TrypLE Express (12605028, Gibco) and incubated at 37 °C for 10 min. Single-cell suspension was washed and centrifuged at 400 × g for 5 min. Single cells were counted and seeded at 15,000 cells/well in 48-well plate (833923, Sarstedt) using the propagation culture media (ENRWFGTi_). After seeding RHOKi 10 µM (Y-27632, Sigma-Aldrich) was added. Monolayers were allowed to grow and differentiate for 14 days to reach 90–100% confluency. Six days prior to infection, Primocin was exchanged for H. pylori compatible antibiotics (vancomycin, nystatin and trimethoprim, concentration as described for the bacterial cultures).

To direct the culture toward the gland cell differentiation, nicotinamide 10 nM (N0636, Sigma-Aldrich) was added to the culture media on day 4 (for organoid-derived monolayers) or day 3 (for 3D organoids; ENRWFGTiNi). To induce differentiation into pit cells, Wnt was removed from the culture medium on day 10 (for organoid-derived monolayers) or day 9 (for 3D organoids; ENR_FGTi_).

Bacterial culture and infection

The clinical isolates H. pylori strain P12 and its derivate expressing GFP constitutively from a chromosomal locus were previously described61 and it was a kind gift of Thomas F. Meyer. The H. pylori isolate strain G27, ΔtlpB and tlpB complemented strain (tlpB*) were previously described36 and a kind gift from Manuel Amieva. We also received G27 and tlpB mutant from Cynthia Sharma52 and this showed the same results. H. pylori was grown on GC agar plates (CM0367B, Oxoid) containing 10% of heat-inactivated horse serum (S0900-500, Biowest) and supplemented with nystatin 0.2 µg ml−1 (N3503, Sigma-Aldrich), trimethoprim 0,25 µg ml−1 (T7883, Sigma-Aldrich), vancomycin 1 µg ml−1 (0242.3, Carl Roth) and kanamycin 8 μg ml−1 (C0378, Sigma-Aldrich). All bacterial cultures were incubated under microaerobic conditions (85% N2, 10% CO2, 5% O2) at 37 °C and in the case of liquid culture with orbital shaking (140 rpm).

For infection, bacteria were grown on plates for 72 h, before passaging to a fresh plate and let to grow for an additional 24 h. A day before infection, scrapes from agar were used to inoculate 10 ml of brain heart infusion (BHI) broth supplemented with 10% heat-inactivated FBS (S0615/1109D, Merck Millipore) and antibiotics for 12 h. This culture was used to set up an overnight culture (14 h) for the infection. For this, the 12 h H. pylori culture was diluted to an OD600 of 0.05 in BHI broth and grown at 37 °C under microaerobic conditions (85% N2, 10% CO2, 5% O2) with shaking until OD600 0.5 (14 h). Bacteria were harvested by centrifugation and resuspended in the organoid culture medium. Bacteria were then added apically onto the organoid-derived monolayers with an MOI of 1 for 6 h unless otherwise indicated. After 6 h incubation, unbound bacteria were removed by 3 washes with PBS and cells were collected and further processed for western blot, flow cytometry, RNA isolation, microscopy, or scRNA-seq as described below. To quantify bacterial adhesion of the H. pylori strains G27MA, ΔtlpB and tlpB* to the organoid-derived monolayers by flow cytometry, bacteria were fluorescently labelled prior infection. Briefly, overnight bacterial cultures were obtained as described above, harvested by centrifugation, washed once with PBS and incubated with 1 ml of PBS containing 1 µM eFluor 670 (65-0840-85, Invitrogen™) for 10 min at 37 °C. Then, bacteria were centrifuged and incubated with BHI medium for 2 min at RT, washed twice with PBS and resuspended in organoid culture medium.

For infection of 3D human gastric organoids, organoids were microinjected with H. pylori as previously described21. Briefly, organoids were seeded in 50ul of Matrigel in a 4 well multidish (144444, Thermo Fisher Scientific) and on day 13, organoids were microinjected with an approximate MOI of 1. To achieve the final MOI, bacteria were resuspended in Advanced DMEM F12 at a density of 2 × 107 bacteria ml−1 (MOI 1) and organoids were injected with approximately 0.2 µl bacterial suspension using a micromanipulator and microinjector (M-152 and IM-5B, Narishige) under a stereomicroscope (Leica MZ75) inside a biosafety cabinet.

Flow cytometry and CFU analysis

After infection, cells were washed three times with PBS, incubated with TrypLE Express (12605028, Gibco) for 10 min, and washed twice with FACS buffer (PBS + 10% FBS). Cells were incubated with anti-PSCA Alexa 647 conjugated antibodies (1:50, sc-80654, Santa Cruz Biotechnologies) for 30 min at 4 °C in dark. Cells were then washed twice and resuspended in a solution of 50 ng ml−1 of propidium iodide (51-6621E, BD biosciences) in FACS buffer. Single cells were gated by using forward scatter area versus forward scatter peak linear. Dead cells were excluded by propidium iodide staining. PSCA and GFP (H. pylori P12-GFP) signals were measured in 10,000 cells. Cells were analysed using the AccuriTM C6 (BD Biosciences) and data were analysed with the AccuriTM C6 software (BD Biosciences) and FlowJoTM software package (BD Biosciences).

To quantify bound bacteria by CFU assay, after infection, cells were washed three times with PBS and lysed with 0.1% saponin (A4518-0100, AppliChem) in PBS. Cell lysates were serially diluted, plated in GC agar plates and left to grow for 4 days.

RNA isolation and qRT-PCR

Isolated gastric units, organoids or cell monolayers were washed with PBS and harvested in RLT buffer (79216, Qiagen). Total RNA was extracted using the RNeasy Mini Kit (74106, Qiagen) according to the manufacturer’s instructions. RNA was then reverse-transcribed using hexameric random primers (48190011, Life Technologies) and M-MuLV reverse transcriptase (M0253, New England Biolabs) according to the manufacturer’s instructions. Quantitative real-time PCR analysis was performed according to the manufacturer’s instructions, in a CFX96 Touch Real-Time PCR detection system using Bio-Rad CFX Manager software (BioRad). The 10 μl PCR reaction included 1 μl of 1:5 diluted cDNA as template, 5 μl of SsoAdvanced Universal SYBR Green Supermix (172-5270, BioRad) and 1 μl of each primer (10 μM). Primers used in this study were: MUC5AC 5′-CTTCTCAACGTTTGACGGGAAGC-3′ and 5′-CTTGATCACCACCACCGTCTG-3′, MUC6 5′-GCCCCGGTATCTTCTCTCGG-3′ and 5′-ACACCTGCAGGGTGAGTACG-3′, PGC 5′-AGAGCCAGGCCTGCACCAGT-3′ and 5′-GCCCCTGTGGCCTGCAGAAG-3′, GAPDH 5′-CTCTCTGCTCCTCCTGTTCGAC-3′ and 5′-TGAGCGATGTGGCTCGGCT-3′, GKN1 5′-GGCCTGATGTACTCAGTCAACC-3′ and 5′-TTTAGTTCTCCACCGTGTCTCC-3′, GKN2 5′-TGCAGGATCATGCTCTTCTAC-3′ and 5′-TGGTCCATCTTCAGGATAAAG-3′, PSCA 5′- TGCTTGCCCTGTTGATGGCAG-3′ and 5′-CCAGAGCAGCAGGCCGAGTGCA-3′, PHGR1 5′- CCCTGCTCTGCACTCTCAG-3′ and 5′-CGCAGTGACCTGGAGGAT-3′, cagA 5′-TGGTGTGAATGGAACCCTAGT-3′ and 5′-CCCGCTGCTTGCCCTACACC-3′, ITLN1 5′-TCTGTTTGGCATCTACCAGAAAT-3′ and 5′-GATGCTGTTTTCTGGGCGTC-3′, OLFM4 5′-AGGTTCTGTGTCCCAGTTGT-3′ and 5′-CAAGCGTTCCACTCTGTCCA-3′ REG3G 5′-ATGCTGCTTTCCTGCCTCAT-3′ and 5′-GACAGCTGATCCGTGGAGAG-3′, LCN2 5′-CCAGGACAACCAATTCCAGG-3′ and 5′-GGCATACATCTTTTGCGGGT-3′, LL37 5′-CAAGAAGGACGGGCTGGTGAA-3′ and 5′-CACAACTGATGTCAAAGGAGCC-3′, HBD1 5′-TGAGATGGCCTCAGGTGGTAA-3′ and 5′-CACTTGGCCTTCCCTCTGTA-3′, HBD2 5′-CCAGCCATCAGCCATGAGGGTCTT-3′ and 5′-CATGTCGCACGTCTCTGATGAGGGAGC-3′. The 2−ΔΔCt method was used to calculate fold changes.

Immunohistochemistry

Organoids were fixed with 4% formaldehyde overnight at 4 °C. Paraffin sections and immunohistochemistry staining was performed according to a previously published protocol21. Briefly, organoids were permeabilized with 0.3% Triton X-100 and blocked with PBS 0.3% Triton X-100, 1% BSA, and 5% goat serum. MUC5AC (1:10, 45M1, Vision biosystems) or MUC6 (1:200, sc-16914, Santa Cruz Biotechnologies) antibodies were used to stain pit cells or gland cells, respectively. Images were taken using a standard light microscope (Nikon Eclipse E600). Cell length and area were quantified using ImageJ by counting 10 cells per organoid from 2 patients.

Confocal microscopy

To analyse organoid-derived monolayers, singularized cells were seeded on µ-Slide 8 Wells (80826, Ibidi) and expanded for 14 days. After infection, cells were fixed with 4% paraformaldehyde for 20 min at RT, permeabilized with 0.5% Triton X-100 in PBS for 15 min and then blocked with 1% BSA for 1 h. Primary antibodies were diluted in blocking solution and incubated overnight at 4 °C followed by 1 h at RT. Primary antibody anti-MUC5AC was used at 1:100 (MA5-12178, Thermo Fisher Scientific). The secondary antibody goat anti-mouse Alexa Fluor-488 (1:500, A-11001, Thermo Fisher Scientific) was also diluted in a blocking solution and incubated at RT for 1 h. Nuclei were counterstained with Hoechst 33342 (1:5,000; H3570, Life Technologies).

To analyse human gastric tissue, tissue samples from human stomach biopsies were fixed in formalin 10% (09122, Noegen) and embedded in paraffin. Embedded sections (5 μm) were deparaffinized in xylene and then hydrated in graded alcohol. Citrate buffer (10 mM, pH 6) was used for antigen retrieval using a steamer. Then slides were washed in PBS and permeabilized using 0.5% Triton X-100 in PBS for 30 min, followed by blocking with 1% BSA in PBS 0.5% Tween 20 for 30 min. Primary antibodies used for this study were: anti-GKN1 (1:500, HPA047684, Atlas antibodies-for double staining with anti-MUC5AC antibody; 1:50, AF7287, R&D Systems- for double staining with anti-H. pylori antibody), anti-GKN2 (1:500, ab188866, Abcam), anti-MUC5AC (1:100, 45M1, Vision Biosystems). Samples were incubated with the primary antibody in a blocking solution overnight at 4 °C followed by an additional hour at RT. Secondary antibodies were incubated for 1 h at RT. The following secondary antibodies were used: donkey anti-mouse Alexa Fluor 594 (1:250, A-21203, Thermo Fischer Scientific), goat anti-rabbit Alexa Fluor 488 (1:500, 4412S, Cell Signaling Technology), donkey anti-sheep Alexa Fluor 594 (1:500, A-11016, Thermo Fischer Scientific). Nuclei were counterstained with Hoechst 33342 (1:5,000; Life Technologies, H3570). Slides were mounted in Mowiol (8138.1, Sigma-Aldrich).

Confocal microscopy images, shown as maximum projected Z-stack images, were acquired with a Leica SP5 laser scanning confocal microscope and LAS AF Lite software (Leica Microsystems).

Data mining from “The Human Protein Atlas”

IHC images of human stomach tissue samples stained for MUC5AC, GKN1, GKN2, PSCA, PHGR1, and CEACAM5 were obtained by datamining the human protein atlas database (www.proteinatlas.org)33.

scRNA-seq library preparation and sequencing

For scRNA-seq of H. pylori-infected 2D monolayers, cells were washed three times with PBS after infection, incubated with TrypLE Express (12605028, Gibco) for 10 min and washed twice with FACS buffer (PBS + 10% FBS) and resuspended in PBS. The cell suspension was then filtered with a 30 µm filter and sorted based on the signal intensity from the FITC-A channel. Mock-infected cells were also sorted. Dead cells marked with propidium-iodide (1:1000, 556463, BD Biosciences) were excluded.

For scRNA-seq of ex vivo isolated epithelium, gastric units were isolated from gastric biopsies as described before for the organoid cultures. Gastric units were then incubated with TrypLE Express (12605028, Gibco) in order to get a single-cell suspension. The cell suspension was then washed with FACS buffer and stained with EpCAM antibody (1:350, sc-25308, Santa Cruz Biotechnologies) for 30 min on ice. After three washes with FACS buffer, cells were filtered as described above and submitted to sorting. Only EpCAM-positive cells were sorted. Dead cells marked with propidium-iodide were excluded.

For scRNA-seq of 3D organoids, the organoids were taken from the Matrigel dome and dissociated mechanically and enzymatically as described above. Single-cell suspensions were filtered with a 30 µm filter. Dead cells marked with propidium-iodide were excluded.

Single cells were sorted by FACSAria III sorter (BD Biosciences) using FACSDiva software (BD Biosciences) and were collected in 1xPBS containing 0.04% w/v BSA (400 μg ml−1) at a concentration of 200-400 cells μl−1. Chromium™ Controller was used for partitioning single cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs) and Single Cell 3’ reagent kit v2 for reverse transcription, cDNA amplification and library construction (10× Genomics). The detailed protocol was provided by 10× Genomics. SimpliAmp Thermal Cycler was used for amplification and incubation steps (Applied Biosystems). Libraries were quantified by QubitTM 3.0 Fluorometer (Thermo Fischer Scientific) and quality was checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent Technologies). Sequencing was performed in paired-end mode using NextSeq 500, HiSeq 2500, and NovaSeq 6000 sequencer (Illumina) to reach a mean of 120,000 reads per single cell.

Data analysis of scRNA-seq

After sequencing, data were demultiplexed and mapped to the GRCh38 human reference genome and feature-barcode matrices were generated using Cell Ranger. To aggregate the naive, bystander and infected libraries of gastric unit libraries, cellranger aggr command was used with default settings and depth normalization of data. The organoid-derived monolayer infection data were analysed using RaceID, and Seurat R package in the Rstudio environment. Low-quality cells were identified based on the common quality control metrics including the number of expressed features, number of UMIs and percentage of mitochondrial genes and were removed from the downstream data analysis. The common workflow of single-cell RNA-seq data analysis was used to perform the downstream data analysis. Briefly, data were subjected to normalization, log transformation, and feature selection, and highly variable features were used to conduct dimension reduction and clustering. Highly variable features were detected using default settings and based on the PCElbowPlot (function in Seurat package), 10 first principal components were selected for dimension reduction and clustering. Clusters were defined at the resolution of 0.85, 1.4, and 0.6 for gastric units, 3D organoid and 2D monolayer cells, respectively. Afterward, identified clusters were projected on two-dimensional space using either UMAP or t-SNE visualizations. FindAllMarkers command from Seurat package and clustdiffgenes command from RaceID package with default settings were used to perform differential expression analysis. EnhancedVolcano was used to visualize differentially expressed genes using volcano plot.

Cluster assignations

A defined set of established marker genes were used to define cell cluster identities (Fig. 1). We first used the ex vivo data to define the cell identities and took this as a basis to name the populations in the in vitro models. MUC6 and PGC are classical necks and chief cell markers respectively. In addition, PGA3-5 and LIPF were identified as neck cell markers18,21,62,63,64. In our data, we do not have clusters of cells that are separated by these markers. We, therefore, named the cluster expressing all these markers “Neck/chief cells”. They likely contain the 2 separated cell identities but they might not be separated at the cell numbers in our dataset. “Neck/chief cell progenitors” express the same markers at lower levels, similarly as suggested in other transcriptomic datasets63. “Parietal cells” were defined by classical markers ATP4B and ATP4A21,63. “Enteroendocrine cells” were defined by chromogranin A21,62.“Pit cells” were defined by high levels of MUC5AC, TFF1, GKN1 GKN2, which are markers also in histology21,30,31 and other transcriptomic studies62,63. “Pit cell progenitors” express lower levels of MUC5AC and TFF1 as well as GKN1 and GKN263. “Proliferating cells” were defined by high levels of PCNA and MKI6763,65.

In the in vitro models, “Neck/cell progenitors” were further separated into 2 populations. Only one population expresses high levels of LIPF. We named these two populations “Neck/cell progenitor populations I and II”. The proliferating cells and early pit progenitors were not separated in the in vitro models, therefore we named this cluster “Proliferating cells and early progenitors”. In the 2D models, the “Pit cell progenitors” were further separated into 2 populations. “Pit cell progenitors I” cell cluster was marked by high MT gene expression. A similar expression signature has been observed in other transcriptomic datasets63. “Pit cell progenitor cells II” cluster was marked by TESC expression highlighted as pit cell marker by another transcriptomic analysis62.

Mass cytometry (CyTOF)

Mass cytometry was performed as described before66. In brief, we used the following preconjugated antibodies (Fluidigm) as per manufacturers recommendation: p-H2AX [S139] (1:100; 3147016A, 147-Sm), cPARP (1:100; 3143011A, 143Nd). For antibodies not available as metal-conjugates, we used the Maxpar Antibody Labelling Kit (Fluidigm) according to the manufacturer’s instructions for the addition of the respective metal tags: YAP D8H1X (1:100; 14074, Cell Signaling Technologies, 150Nd), PSCA (1:50; sc-80654, Santa Cruz Technology, 159Tb).

Urea concentration measurement

The urea concentration in the conditioned-organoid media was measured using a Urea Assay kit (ab83362, Abcam) and following the manufacturer’s instructions. Samples were collected after 24 h, centrifuged for 15 min at 1500 × g to remove cell debris, and diluted 1/100 in the urea kit buffer prior to quantification.

H. pylori survival assay

A bacterial survival assay was performed as previously described40 with minor adjustments. Briefly, organoid-derived monolayers were infected with H. pylori strain P12 (WT, kanamycin sensitive) at MOI 1 as described above. After 6 h of infection, the culture medium was collected and centrifuged at full speed for 10 min to remove bacteria and cell debris. H. pylori strain P12-GFP (kanamycin-resistant) was grown to OD 0.5 as described above, harvested, washed once with PBS, and resuspended in PBS 1% BHI at a density of 1 × 106 bacteria ml−1. An aliquot of 5 µl of the bacterial suspension was mixed with 25 µl of conditioned medium collected on the same day and incubated for 2 h at 37 °C. Then, bacterial solutions were plated onto GC agar plates as described before (supplemented with kanamycin 8 μg ml−1; C0378, Sigma-Aldrich) and colonies were counted.

H. pylori chemotaxis assay

Chemotaxis assays were performed using the method described previously35 with minor changes. H. pylori strains were grown as described before to an OD of 0.5. Then, bacteria were harvested by centrifugation, washed once with PBS, and resuspended in PBS to a density of 1 × 107 bacteria ml−1. An aliquot of 100 µl of the bacterial suspension was loaded into a 200 µl pipette tip. Organoid-derived monolayer conditioned media (100 µl) was drawn up through a 25 G needle into a 1 ml syringe, and the point of the syringe was submerged into the pipette tip containing the bacterial solution. After incubation horizontally for 2.5 h at 37 °C, the content of the syringe was transferred to a 1.5 ml tube, serially diluted in PBS and plated onto GC agar plates. To saturate the urea sensing potential of H. pylori, 500 mM urea was added to the conditioned media. In addition, syringes containing only 1 mM urea or PBS were used as a positive or negative control, respectively.

Western blotting

Cells were washed with PBS and lysed with Laemmli’s buffer, sonicated, and separated on SDS–PAGE gels followed by electro-transfer to nitrocellulose membranes. Antibodies used for western blot analysis were: anti-CagA (1:2000, sc-25766, Santa Cruz Biotechnology), anti-P-Tyr (1:1000, sc-7020, Santa Cruz Biotechnology), anti-GKN1 (1:500, HPA047684, Atlas antibodies), anti-GKN2 (1:11,000, ab188866, Abcam), anti-MUC5AC (1:1000, MA5-12178, Thermo Fisher Scientific), anti-MUC6 (1:500, AM10120SU-N, Acris), anti-α-tubulin (1:3000, T6074, Sigma-Aldrich), anti-β-actin (1:3000, A2228, Sigma-Aldrich), anti-mouse HRP (1:10,000; NA931, GE Healthcare), anti-rabbit HRP (1:10,000; NA934, GE Healthcare). HPR signals were detected using a solution of luminol (0.25 mg ml−1, A8511-5G, Sigma-Aldrich) and p-coumaric acid (1.1 mg ml−1, sc-215648A, Santa Cruz Biotechnologies) with an ImageQuant LAS 4000 CCD camera (GE Healthcare). Quantification of western blots was performed with ImageJ. Uncropped images of immunoblots are included as Source Data.

Generation of MUC5AC and PSCA knockout organoid lines

Plasmid constructs

Human gastric organoids were edited by the CRISPR-Cas9 system using a protocol previously described67. Briefly, a guide RNA (sgRNA; 5′-GCCCTCTCTCCTATCGCCCG-3′ for MUC5AC, 5′-TGTTGATGGCAGGCTTGGCC-3′ for PSCA) was designed to target the second exon of MUC5AC using CRISPOR68 and the first exon of PSCA using Benchling (www.benchling.com). For MUC5AC, overhangs were added to the gRNA sequence and cloned into AflII (R0520S, New England Biolabs) linearized gRNA_Cloning plasmid (41824, Addgene). A plasmid expressing hCas9 was acquired from Addgene (41815, Addgene). For PSCA, overhangs were added to the gRNA sequence and cloned into BbsI-HF (R0539S, New England Biolabs) linearized pSPCas9(BB)−2A-Puro V2.0 plasmid (62988, Addgene) which readily expresses Cas9.

Organoid transfection

The transfection protocol was adapted from Fujii et al.69. Human gastric organoids were cultured in 24-well plates as described above. Two days before the electroporation, Primocin was removed and Noggin-conditioned media was replaced with recombinant Noggin (100 ng ml−1, 250-38, Peprotech). A day before the electroporation, human gastric organoids were treated with 1.25% DMSO. On the day of the electroporation, organoids were shredded mechanically and made into single cells using TrypLE Express (12605036, Gibco).

A total of 5 × 105 cells were resuspended in 100 μl of BTXpress buffer (45-0805, BTX Molecular Delivery Systems) supplemented with RHOKi (Y-27632; 10 μM, M1817, AbMole). Cells were electroporated with 4 μg hCas9 plasmid (41815, Addgene) and 4 μg of gRNA plasmid (41824, Addgene) or 45 µg of pSPCas9(BB)−2A-Puro V2.0 plasmid (62988, Addgene) (for MUC5AC KO or PSCA KO, respectively) in a nucleofection cuvette (EC-002S, Nepagene) using the NEPA 21 Super Electroporator (Nepagene) with following settings: Poring pulse (175 V, pulse length—5 ms, pulse interval—50 ms, number of pulses—2, decay rate—10% and positive polarity), Transfer pulse (20 V, pulse length—50 ms, pulse interval—50 ms, number of pulses 5, decay rate—40% and positive/negative polarity). After electroporation, cells were washed with Opti-MEM (31985070, Gibco) containing RHOKi (Y-27632; 10 μM, M1817, AbMole) and seeded in Matrigel (356231, Corning). The organoid medium was supplemented with RHOKi (Y-27632; 10 μM, M1817, AbMole), nicotinamide (10 mM, 72340, Sigma Aldrich), prostaglandin E2 (10 mM, 2296, TOCRIS bioscience) and GSK-3 inhibitor (CHIR99021, 1386, Axon MedChem). CHIR99021 was removed after 24 h. MUC5AC-KO organoids were selected with puromycin (2 µg ml−1, sc-108071B, Santa Cruz Biotechnologies) for 48 h five days after electroporation. PSCA-KO organoids were selected with 1 μg ml−1 puromycin on days 4–7 after electroporation. Individual organoids were split and seeded in 25 µl Matrigel (356231, Corning). Organoid lines were maintained and expanded until molecular characterization. Genomic DNA from potential KO organoid lines was isolated and used to amplify a fraction of MUC5AC or PSCA containing the gRNA (5′-GTGGTCTGGTCCCACTATGCTG-3′, 5′-GTGGAGGGTGGAATCTGACA-3′ for MUC5AC, 5′-ATGGCCCTGGGTAGGCTCTGTC-3′, 5’-GAAGCTGCAGTGCTGGGACTGG-3′ for PSCA). Sanger sequencing was used to confirm proper KO at the targeted locus. MUC5AC KO phenotype was confirmed by western blot and immunostaining. PSCA KO phenotype was confirmed by flow cytometry.

Statistical analysis

Data are presented as mean ± standard deviation (SD), with the number of experiments performed and organoid lines indicated in figure legends. Statistical analysis was performed using Prism Software (v6.01, GraphPad). Statistical significance was defined as p  <  0.05. Statistical analyses are detailed in Supplementary Data 6.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.



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