Cell culture of human intestinal organoids and human airway
Human small intestinal tissue was obtained from the UMC Utrecht with informed consent of the patients. The patients wereoperated for a colorectal tumor, and a sample from non-transformed, normal mucosa was taken for this study. The study was approved by the UMC Utrecht (Utrecht, The Netherlands) ethical committee and was in accordance with the Declaration of Helsinki and according to Dutch law. This study is compliant with all relevant ethical regulations regarding research involving human participants.
Nasal inferior turbinate brushes were obtained from the Hadassah Medical Center, Jerusalem, with informed consent of the patient. Patients were diagnosed with primary ciliary dyskinesia, and tissue was obtained from healthy donors as a comparison. Healthy material was used for the RNA sequencing in this work. The study was approved by the ethical committee (Hadassah Medical Organization (HMO) IRB committee) and was in accordance with the Declaration of Helsinki and according to Israeli law under IRB approval number 075-16 HMO. This study is compliant with all relevant ethical regulations regarding research involving human participants.
Adult lung tissue was obtained from residual, tumor-free, material obtained at lung resection surgery for lung cancer. Tissue was rest material for which no informed consent was required. The Medical Ethical Committee of the Erasmus MC Rotterdam granted permission for this study (METC 2012-512).
Human small intestinal cells were isolated, processed and cultured as described previously55,56. Wnt surrogate was used (0,15 nM, U-Protein Express) instead of Wnt conditioned media. Differentiation of intestinal organoids was achieved as described previously56.
Nose tissue was dissociated and cultured as described previously57. Differentiation towards ciliated cells was performed by activating BMP signaling and inhibiting Notch signaling for 10 days (Van der Vaart et al., under review).
Isolation of human bronchial airway stem cells was performed using a protocol similar to Sachs and colleagues57. Small airway stem cells were isolated from distal human lung parenchyma as described before57. Tracheal stem cells were collected from tracheal aspirates of intubated preterm infants (28 weeks gestational age). Organoids were cultured as described before57. To obtain differentiated organoid-derived cultures, organoids were dissociated into single cells using TrypLE express (Gibco; #12604013). Cells were seeded on Transwell membranes (Corning) coated with rat tail collagen type I (Fisher Scientific). Single cells were seeded in AO growth medium: complete base medium (CBM; Stemcell Pneumacult-ALI; #05001) at a 1:1 ratio. After 2–4 days, confluent monolayers were cultured at air‐liquid interphase in CBM. Medium was changed every 5 days for 8 weeks.
Transfection of organoids for CRISPR-Cas9 experiments
sgRNAs targeting loci of interest were cloned into a SpCas9-EGFP vector (addgene plasmid #48138) using a protocol described before58. sgRNAs were designed using WTSI website (https://www.sanger.ac.uk/htgt/wge/). A full list of gRNAs and primers to generate SpCas9-EGFP expressing plasmids can be found in Supplementary Table 1. To generate homozygous frameshift mutations in genes of interest, organoids were transfected with SpCas9-EGFP containing the locus-specific sgRNA. Transient transfection using a NEPA21 electroporator was performed as described before59. 3–7 days after transfection, organoids were dissociated using TryplE (TryplE Express; Life Technologies) and sorted on a FACS-ARIA (BD Biosciences) for GFP positivity (Fig. s10) After sorting, Rho kinase inhibitor (Y-27632 dihydrochloride; 10 µM, Abmole) was added for 1 week to support single cell outgrowth.
Generation of stable genetically modified organoid lines
To generate clonal organoid lines with genotypes of preference, organoids were picked 2 weeks after sorting. Manually picked organoids were dissociated using TryplE (TryplE Express; Life Technologies) and plated in BME in pre-warmed cell culture plates. After two weeks, single cells grew into organoids and were split again to verify actively dividing stem cells. After the second split, 20 µL of organoid-BME suspension was directly taken from the plate and DNA was extracted from the organoids using the Zymogen Quick-DNA microprep kit according to protocol. Regions around sgRNA target sites were amplified using Q5 high fidelity polymerase (NEB) according to manufacturer’s protocol. CRISPR/Cas9-mediated indel formation was confirmed by sanger sequencing of these amplicons (Macrogen). Subsequently, sanger trace deconvolution was performed with the use of ICE v2 CRISPR analysis tool (synthego website) to call clonal organoid lines with homozygous frameshift mutations at the target site. Knockout clones were further expanded for viral infection experiments. Primers used for amplification and sanger sequencing can be found in Supplementary Table 1. For the generation of TMPRSS2/TMPRSS4 double mutants, TMPRSS4 was knocked out in TMPRSS2-clone 9. For the generation of CTSL/CTSB double mutants CTSL was knocked out in CTSB clone 3.
Genetically engineered organoid lines cannot be shared. Primers for the generation of these lines can be shared upon reasonable request to the corresponding authors.
Viruses and cell lines
Vero(ATCC, CCL 81) and VeroE6(ATCC, CRL 1586) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS), HEPES, sodium bicabonate, penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37 °C in a humidified CO2 incubator. Calu-3 cells were maintained in Opti-MEM I (1X) + GlutaMAX (Gibco) supplemented with 10% FCS, penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37 °C in a humidified CO2 incubator. SARS-CoV (isolate HKU 39849, genbank accession no. AY278491), SARS-CoV-2 (isolate Bavpat-1; European Virus Archive Global #026V-03883; kindly provided by Dr. C. Drosten) were propagated on VeroE6 cells in Opti-MEM I (1X) + GlutaMAX (Gibco), supplemented with penicillin (100 IU/mL) and streptomycin (100 IU/mL) at 37 °C in a humidified CO2 incubator. MERS-CoV (isolate EMC, genbank accession no. NC019843) was propagated on Vero cells in the same medium. Non-adapted SARS-CoV-2 Bavpat-1 and B.1.1.7 (genbank accession no. MW947280) were propagated in Calu-3 cells as described before39. Stocks were produced by infecting cells at a multiplicity of infection (MOI) of 0.01 and incubating the cells for 72 h. The culture supernatant was cleared by centrifugation and stored in aliquots at −80 °C. Stock titers were determined by preparing 10-fold serial dilutions in Opti-MEM I (1X) + GlutaMAX. Aliquots of each dilution were added to monolayers of 2 × 104 VeroE6 (for SARS-CoV and SARS-CoV-2) or Vero cells (for MERS-CoV) in the same medium in a 96-well plate. Plates were incubated at 37 °C for 5 days and then examined for cytopathic effect. The TCID50 was calculated according to the method of Spearman & Kärber. All work with infectious SARS-CoV, SARS-CoV-2 and MERS-CoV was performed in a Class II Biosafety Cabinet under BSL-3 conditions at Erasmus Medical Center.
SARS-CoV, SARS-CoV-2 and MERS-CoV infection
Infections were performed using a protocol similar to17. Briefly, organoids were harvested in cold Advanced DMEM (including HEPES, Glutamax and antibiotics, termed AdDF +++17), washed once in AdDF +++, and sheared using a flamed Pasteur pipette in AdDF +++. Differentiated organoids were broken using a 5-min incubation with TrypLE (TrypLE Express; Life Technologies). After shearing, organoids were washed once in AdDF +++ before infection was performed in expansion medium. For the experiment in Fig. s5b, organoids were gently harvested using a wide pipet tip to avoid shearing organoids. A multiplicity of infection (MOI) of 0.1 was used for SARS-CoV and SARS-CoV-2 and an MOI of 0.2 was used for MERS-CoV. After 2 h of virus adsorption at 37 °C 5% CO2, cultures were washed four times with 4 ml AdDF +++ to remove unbound virus. Organoids were re-embedded into 30 μL BME in 48-well tissue culture plates and cultured in 250 μL expansion or differentiation medium at 37 °C with 5% CO2. Each well contained ~200,000 cells per well. At indicated time points cells were harvested by resuspending the BME droplet containing organoids into 200 μL AdDF +++. Samples were stored at −80 °C, a process which lysed the organoids, releasing their contents into the medium upon thawing.
For testing the antiviral activity of chloroquine diphosphate (Sigma), camostat mesylate (Sigma) and E64D (Medchemexpress) in intestinal organoids, sheared organoids were preincubated with these compounds in AdDF +++ at the indicated concentrations for 30 min at 37 °C 5% CO2 before infection at an MOI of 0.1 with SARS-CoV-2. After virus adsorption for 2 h at 37 °C 5% CO2, organoids were washed and re-embedded in BME as indicated above. Medium containing the inhibitors was added to the wells for the duration of the experiment. Cells were harvested at indicated time points as described above and stored at −80 °C.
SARS-CoV-2 entry inhibition assay by chloroquine in VeroE6 cells
Chloroquine was two-fold serially diluted in Opti-MEM I (1X) + GlutaMAX starting from a concentration of 100 µg/mL. A total of 100 μl of each dilution was added to a confluent 96-well plate of VeroE6 cells and pre-incubated at 37 °C 5% CO2 for 30 min. Next, cells were incubated with 400 plaque-forming units of virus in the same concentration range of chloroquine at 37 °C 5% CO2. After 8 h incubation, cells were fixed with formalin, permeabilized with 70% ethanol and stained with polyclonal rabbit anti-SARS-CoV nucleoprotein antibody (1:1000; 40588-T62, Sino Biological) followed by secondary Alexa488 conjugated donkey-anti-rabbit antibody (1:1000; Thermofisher Scientific, A-21206). Plates were scanned on the Amersham™ Typhoon™ Biomolecular Imager (channel Cy2; resolution 10 µm; GE Healthcare). Data was analyzed using ImageQuant TL 8.2 image analysis software (GE Healthcare).
Determination of virus titer using qRT-PCR
For determining the viral titer using qPCR, samples were thawed and centrifuged at 2000 g for 5 min. Sixty μL supernatant was lysed in 90 μL MagnaPure LC Lysis buffer (Roche) at RT for 10 min. RNA was extracted by incubating samples with 50 μL Agencourt AMPure XP beads (Beckman Coulter) for 15 min at RT, washing beads twice with 70% ethanol on a DynaMag-96 magnet (Invitrogen) and eluting in 30 μL MagnaPure LC elution buffer (Roche). Viral titers (TCID50 equivalents per mL) were determined by qRT-PCR using primer-probe sets described previously60,61,62 and comparing the Ct values to a standard curve derived from a titrated virus stock.
Immunostainings and western blot
Organoids were stained as described before55. Whole organoids were collected by gently dissolving the BME in ice-cold PBS, and subsequently fixed overnight at 4 °C in 4% paraformaldehyde (Sigma). Next, organoids were permeabilized and blocked in PBS containing 0,5% Triton X-100 (Sigma) and 2% normal donkey serum (Jackson ImunoResearch) for 30 min at room temperature. All stainings were performed in blocking buffer (2% normal donkey serum in PBS). For immunofluorescence, primary antibodies used were mouse anti-nucleoprotein (1:200; 40143-MM05, Sino Biological), mouse anti-dsRNA (1:200; Scicons, 10010200,), goat anti-ACE (1:100; R&D Systems, AF933), goat anti-DPP-4 (1:200; R&D systems, AF1180) and rabbit anti-MERS S1 (1:200; Sino Biological, 40069-T52). For immunofluorescence, organoids were incubated with the corresponding secondary antibodies; donkey anti-mouse Alexa647 (1:1000, Thermofisher Scientific, A-31571), donkey anti-rabbit Alexa488 (1:1000, Thermofisher Scientific, A-21206), donkey anti-rabbit Alexa568 (1:1000, Thermofisher Scientific, A-10042), donkey anti-goat Alexa488 (1:1000, Thermofisher Scientific, A-11055), donkey anti-goat Alexa568 (1:1000, Thermofisher Scientific, A-11057), donkey anti-goat Alexa647 (1:1000,Thermofisher Scientific, A-21447) and donkey anti-mouse Alexa568 (1:1000, Thermofisher Scientific, A-10037). or Phalloidin-Alexa488 (1:1000; Thermofisher Scientific, A12379) in blocking buffer containing 4ʹ,6-diamidino-2-phenylindole (DAPI; 1;1000; Invitrogen). After staining, organoids were transfected to a glass slide embedded in Vectashield (Vector labs). Stained organoids were imaged using a SP8 confocal microscope (Leica) or a Zeiss LSM700, and image analysis and presentation was performed using ImageJ and Leica LAS X Version 1.1 software.
Immunohistochemistry was performed as described before63. Antigen retrieval for TMPRSS2 staining was achieved by boiling for 20 min in citrate buffer. Primary antibody used was rabbit anti-TMPRSS2 (1:100; Abcam, ab109131) followed by Goat-anti-rabbit conjugated to horseradish peroxidase (1:2000, Dako, P0448) and then visualized (Powervision, Leica).
For Western blot of CTSL, organoid proteins were solubilized using a standard RIPA buffer for 30 min on ice in the presence of protease inhibitors. Samples were run on a 4–15% PAA gel (BioRad) under reducing conditions. Proteins were electrophoresed to PVDF membranes from Immobilon. Both primary antibodies, mouse anti-CTSL (±25 kDa, 1:100, Thermofisher Scientific, #BMS1032) and mouse anti-ITGB4 (±180 kDa, 1:100, Origene; AM33010PU-N), were incubated O/N at 4 C in PBS/10% milk protein/0.1% Tween20. The secondary rabbit anti-mouse HRP-conjugate (1:2000, Dako, P0260) was incubated for 2 h at 4 C in the same buffer. The mouse IgG2a antibody against ITGB4, 58XB4, was a gift from A. Sonnenberg (NKI, Amsterdam, The Netherlands). HRP activity was visualized with ECL (GE-Healthcare).
Bulk RNA sequencing
Single Cell Discoveries (Utrecht, The Netherlands) performed library preparation, using an adapted version of the CEL-seq protocol, as we have done previously17. After library generation, paired-end sequencing was performed on the Illumina Nextseq500 platform using barcoded 1 × 75 nt read setup. Read 1 was used to identify the Illumina library index and CEL-Seq sample barcode. Read 2 was aligned to the CRCh38 human RefSeq transcriptome, with the addition SARS-CoV-2 (Ref-SKU: 026V-03883) or MERS (NC_038294.1) genomes, using BWA using standard settings64. Reads that mapped equally well to multiple locations were discarded. Mapping and generation of count tables was performed using the in-house MapAndGo script, filtering to exclude reads with identical library- and molecule-barcodes. RNA sequencing data from expanding and differentiated human intestinal organoids, infected with SARS-CoV-2, was used from a previous publication17. Normalization using the median of ratios method and differential gene expression analysis was performed using the DESeq2 package64. SARS- and MERS- mapping reads were removed before normalization to avoid biasing organoid transcript counts. To generate heatmaps, row z scores of selected genes were calculated from the samples selected.
Organoid preparation for single cell sequencing analysis
Human ileal organoids were differentiated as previously described56. A control condition was kept in human organoid expansion medium to obtain stem- and progenitor cells for comparison.
Dissociation of organoids to single cells was performed by a 10-min incubation with TrypLE (TrypLE Express; Life Technologies) supported by repeated mechanical disruption using a narrowed glass pipette. Viable cells were sorted using a BD FACS Aria (BD Biosciences) using DAPI exclusion. Individual cells were collected in 384-well plates with ERCC spike-ins (Agilent), reverse transcription primers and dNTPs (both Promega). Single cell sequencing was performed according to the Sort-seq method65. Sequencing libraries were generated with TruSeq small RNA primers (Illumina) and sequenced paired-end at 60 and 26 bp read length, respectively, on the Illumina NextSeq.
Single cell RNA sequencing analysis from intestinal organoids and tissue
Reads derived from 1344 cells (192 expansion medium, 1152 differentiation medium) were mapped to the human GRCh37 genome assembly. Sort-seq read counts were filtered to exclude reads with identical library-, cell- and molecule barcodes. UMI counts were adjusted using Poisson counting statistics65. Cells with fewer than 1000 unique transcripts were excluded from further analysis. This resulted in 944 remaining cells (126 from expansion, 818 from differentiation medium)
Subsequently, RaceID3 was used for k-medoids-based clustering (knn = 5; cln = 20) of cells and differential gene expression analysis between clusters using the standard settings described at https://github.com/dgrun/RaceID3_StemID2_package. Cell types were annotated by cluster based on the expression of marker genes (OLFM4 for stem cells, FABP1 for enterocytes, FCGBP for goblet cells. Lack of these and expression of cell cycle markers including PCNA defined proliferating progenitor cells).
For comparison with tissue-derived cells, we reanalyzed a previously published dataset32 of primary human ileal cell types. To compare with the organoid data set, cells were required to be annotated as stem cell, progenitor cell, goblet cell or enterocyte and exhibit >3000 unique transcripts. This resulted in 2137 included cells, which were subsequently clustered using the standard settings of RaceID3 (cln = 16). Cells were assigned an identity based on their annotation from32.
Quantification and statistics
No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to the sample allocation during experiments and outcome assessment. All data are presented as mean ± standard error of the mean (SEM), unless stated otherwise. Value of n is always displayed in the figure as individual data points, and in the legends.
Statistical analysis was performed with the GraphPad Prism 9 software. We compared differences in virus replication and organoid growth by one-way ANOVA followed by a multiple-comparison test (Original FDR method of Benjamini and Hochberg; Q = 0.05) on log10 transformed values. Statistics were applied if N ≥ 3. Exact p values for all figures can be found in Supplementary dataset 5.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
