Human iPS cell–derived respiratory organoids as a model for respiratory syncytial virus infection

Human iPS cells

The human iPS cell line, 1383D6 (provided by Dr. Masato Nakagawa, Kyoto University), was maintained on 0.5 μg/cm² recombinant human laminin 511 × 10⁸ fragments (iMatrix-511, Cat# 892 012; Nippi) with StemFit AK02N medium (Cat# RCAK02N; Ajinomoto Healthy Supply). Cell passage was performed every 6 d. For cell passaging, iPS cell colonies were treated with TrypLE Select Enzyme (Cat# 12563029; Thermo Fisher Scientific) for 10 min at 37°C and seeded with StemFit AK02N medium containing 5 μM Y-27632 (Cat# 034-24024; FUJIFILM Wako Pure Chemical).

Respiratory organoids

To start the differentiation, human iPS cell colonies were treated with TrypLE Select Enzyme (Cat# 12563029; Thermo Fisher Scientific) for 10 min at 37°C. After centrifugation, cells were seeded onto Matrigel Growth Factor Reduced Basement Membrane (Cat# 354230; Corning)-coated cell culture plates (2.0 × 10⁵ cells/4 cm²) and cultured for 2 d. The differentiation of the respiratory organoids was performed in serum-free differentiation (SFD) medium, composed of DMEM/F12 (3:1) (Cat# 044-29765; FUJIFILM Wako Pure Chemical and Cat# 11320033; Thermo Fisher Scientific) supplemented with N2 (Cat# 141-08941; FUJIFILM Wako Pure Chemical), B-27 supplement minus vitamin A (Cat# 12587001; Thermo Fisher Scientific), ascorbic acid (50 μg/ml, Cat# ST-72132; STEMCELL Technologies), 1× GlutaMAX (Cat# 35050-061; Thermo Fisher Scientific), 1% monothioglycerol (Cat# 195-15791; FUJIFILM Wako Pure Chemical), 0.05% BSA (Cat# 820024; Sigma-Aldrich), and 1× penicillin/streptomycin). During days 0–1 of differentiation, cells were cultured with SFD medium supplemented with 10 μM Y-27632 (Cat# 034-24024; FUJIFILM Wako Pure Chemical) and 100 ng/ml recombinant activin A (Cat# 338-AC-01M; R&D Systems). During days 1–3 of differentiation, cells were cultured with SFD medium supplemented with 10 μM Y-27632 (Cat# 034-24024; FUJIFILM Wako Pure Chemical), 100 ng/ml recombinant Activin A (Cat# 338-AC-01M; R&D Systems) and 1% FBS. Between days 3–5 of differentiation, cells were cultured in SFD medium supplemented with 1.5 μM dorsomorphin dihydrochloride (Cat# 047-33763; FUJIFILM Wako Pure Chemical) and 10 μM SB431542 (Cat# 037-24293; FUJIFILM Wako Pure Chemical) for 24 h, and then SFD medium supplemented with 10 μM SB431542 and 1 μM IWP2 (Cat# 04-0034; Stemolecule) for another 24 h. During days 5–12 of differentiation, cells were cultured with SFD medium supplemented with 3 μM CHIR99021 (Cat# 034-23103; FUJIFILM Wako Pure Chemical), 10 ng/ml human FGF10 (Cat# AF-100-26; PeproTech), 10 ng/ml human FGF7 (Cat# AF-100-19; PeproTech), 10 ng/ml human BMP4 (Cat# 120-05ET; PeproTech), 20 ng/ml human EGF (Cat# AF-100-15; PeproTech), and all-trans retinoic acid (Cat# R2625 ATRA; Sigma-Aldrich). On day 12 of differentiation, cells were dissociated and embedded in the Matrigel Growth Factor–Reduced Basement Membrane to generate organoids. During days 12–20 of the differentiation, organoids were cultured in SFD medium containing 3 μM CHIR99021, 10 ng/ml human FGF10, 10 ng/ml human FGF7, 10 ng/ml human BMP4, and 50 nM ATRA. On day 20 of differentiation, organoids were recovered from the Matrigel, and the resulting suspension of organoids (small free-floating clumps) was seeded onto Matrigel-coated cell culture plates. During days 20–30 of differentiation, organoids were cultured in SFD medium containing 50 nM dexamethasone (Cat# S1322; Selleck Chemicals), 0.1 mM 8-bromo-cAMP (Cat# 1140/50; Tocris Bioscience), and 0.1 mM IBMX (3-isobutyl-1-methylxanthine) (Cat# 095-03413;FUJIFILM Wako Pure Chemical).

RSV

Isolated RSV (RSV/Sendai/28-30 [subgroup A]) was used in this study (GISAID accession number: EPI_ISL_19696283). RSV was replicated in HEp-2 cells (CCL-23; ATCC) and stored at −80°C. HEp-2 cells were cultured with EMEM (Cat# 051-07615; FUJIFILM Wako Pure Chemical) supplemented with 10% FBS and 1% penicillin/streptomycin.

Viral titration

Viral titers were measured by a median tissue culture infectious dose (TCID50) assay. Cells were seeded into 96-well cell culture plates (Cat# 167008; Thermo Fisher Scientific). Samples were serially diluted 10-fold from 10⁻¹ to 10⁻⁸ in cell culture medium, transferred onto the cells, and incubated at 37°C for 96 h. Cytopathic effects were evaluated under a microscope. TCID50/mL was calculated using the Reed-Muench method.

Antiviral drug and antibody assay using respiratory organoids

The human iPS cell–derived respiratory organoids were infected with 0.1 TCID50/cell (8 × 10⁴ TCID/well) RSV. Infected respiratory organoids were cultured with the medium containing serially diluted nirsevimab (Cat# MA5-42214; Thermo Fisher Scientific), suptavumab (Cat# HY-P99586; MedChemExpress), clesrovimab (Cat# HY-P99804; MedChemExpress), palivizumab (Cat# HY-P9944; MedChemExpress), or ribavirin (Cat# 182-02331; FUJIFILM Wako Pure Chemical). At 96 h after the infection, TCID50 assay was performed.

Immunofluorescence staining

For immunofluorescence staining of respiratory organoids, cells were fixed with 4% PFA in PBS at 4°C. Respiratory organoids were harvested and prepared as paraffin sections (∼15 μm). Paraffin was removed using xylene and afterward rehydrated with different percentages of ethanol. Antigen retrieval was performed with 0.1%-tTBS (10×) (pH 7.4) (Cat# 12750-81; Nacalai Tesque). For blocking non-specific staining, the slides were incubated in Blocking One (Cat# 03953-066; Nacalai Tesque) for 10 min at room temperature. Primary antibody incubation was performed overnight at 4°C. Stained slides were washed thrice with 1× PBS (Cat# 14249-24; Nacalai Tesque) the next day. Alexa Fluor 488- or 594-conjugated secondary antibody incubation was performed at room temperature for 45 minutes, and slides were washed afterward three times for 5 min. Sections were washed, mounted with ProLong Glass Antifade Mountant with NucBlue Stain (Cat# P36985; Thermo Fisher Scientific) and DAPI (Cat# 12593-64; Nacalai Tesque), and analyzed using an inverted laser scanning confocal microscopy system (FV3000; Evident). Antibodies used are summarized in Table S3.

Measurement of cytokines in cell culture supernatants

For measuring cytokine concentrations, the cell culture supernatants of respiratory organoids were analyzed using the Human Anti-Virus Response Panel (Cat# 740349; BioLegend). Flow cytometry was performed according to the manufacturer’s instructions using MACSQuant Analyzer 10 Flow Cytometer (Miltenyi Biotec).

Hematoxylin and eosin and sirius red staining

Respiratory organoids were fixed with 4% PFA (Cat# 163-20145; FUJIFILM Wako Pure Chemical) for 15 min, harvested, and used to prepare paraffin sections. Paraffin-embedded tissue sectioning and histological staining were performed by the Applied Medical Research Laboratory.

Capillary-based immunoassay

Respiratory organoids were lysed in RIPA buffer (Cat# 89900; Thermo Fisher Scientific) containing a protease inhibitor mixture (Cat# P8340; Sigma-Aldrich). After the centrifugation, the supernatants were collected. Antibody-based protein quantification was performed using a Jess system (ProteinSimple) with the 12–230 kD Separation Module (Cat#SM-W001; ProteinSimple) as instructed. Antibodies used for this analysis are summarized in Table S3. Data were analyzed and visualized using Compass for Simple Western software (ProteinSimple). Jess protein images are shown throughout the manuscript.

Ultrathin section transmission electron microscopy

Respiratory organoids were fixed with 2.5% glutaraldehyde in 0.1M cacodylate buffer and post-fixed with 1% osmium tetroxide in the same buffer at 4°C. Then, they were dehydrated in a series of ethanol gradients and embedded in epoxy resin. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and examined using an electron microscope (HT-7700; Hitachi High-Tech Corporation) at 80 kV.

Bulk RNA sequencing

Total RNA was isolated from RSV-infected respiratory organoids. RNA integrity was assessed with a 2100 Bioanalyzer (Agilent Technologies). RNA-seq libraries were constructed using a TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer’s instructions. Sequencing was performed on an Illumina NextSeq 2000 with the paired-end mode. FASTQ files were generated using bcl2fastq-2.20. Adapter sequences and low-quality bases were trimmed from the raw reads using cutadapt ver v4.1 (Kechin et al, 2017). Trimmed reads were mapped to human reference genome sequences (hg38) using STAR ver 2.7.10a (Dobin et al, 2013) with GENCODE (release 32, GRCh38.p13) (Frankish et al, 2019) GTF file. The uniquely and properly mapped reads were used for further analysis. Raw read counts were calculated using htseq-count ver. 2.0.2 (Anders et al, 2015) with the GENCODE GTF file. Differential expression analysis was performed by DESeq2 v1.34.0 (Love et al, 2014) using the Wald test. The P-values were corrected for multiple testing using the Benjamini and Hochberg method and represented as the false discovery rate. Raw data from this study were submitted to the Gene Expression Omnibus (GEO) under accession number GSE263272.

scRNA-seq

Respiratory organoids were dissociated, and the cell concentration was adjusted to 1,000 cells/μl. The single-cell suspension, with a concentration of 1,200 cells/μl, was loaded onto a 10x Genomics Chromium Next GEM Single Cell G chip (10x Genomics) with enzyme mix, gel beads, and oils to generate gel beads-in-emulsion (Chromium Next GEM single cell 3′ kit v3.1) (10x Genomics). Subsequently, reverse transcription and cDNA amplification were conducted according to the manufacturer’s instructions using Verity thermal cycler (Thermo Fisher Scientific) for the Chromium Next GEM Single Cell kit. After adding index sequences to the library pool, the cDNA library was sequenced on the Nova-seq X platform (Illumina).

The 10x Genomics Cell Ranger pipeline (version 7.1.0) was used to perform sample demultiplexing, alignment to the GRCh38-2020-A human reference genome, barcode/unique molecular identifiers (UMI) processing, and gene counting for each cell. Gene count matrix data were processed as follows. Briefly, the low-quality cells were excluded based on the following criteria: (I) number of detected genes ≤ 200, (II) number of UMI molecules detected within a cell ≥ 100,000, and (III) percentage of mitochondrial genes expressed ≤ 10%. Filtered UMI feature-barcode matrices were processed with ICARUS v.2.0 (Jiang et al, 2023). Gene count matrices were normalized and scaled by 10,000. Dimensionality reduction with principal component analysis was performed using a set of 2,000 top variable genes (Seurat::FindVariableFeatures) (Hao et al, 2024). Clustering was performed with 50 principal components, a k-nearest neighbor value of 50, and the Louvain clustering algorithm. Cell type annotation was performed by scType (Ianevski et al, 2022). scRNA-seq expression datasets of bronchoalveolar lavage fluid of RSV-infected patient (Travaglini et al, 2020) were obtained from a public database. The accession numbers for scRNA-seq data obtained in this study (RSV+nirsevimab and RSV+palivizumab) are GSE263271. The accession numbers for scRNA-seq data (mock and RSV) are GSE260752.

Statistical analyses

Statistical significance was evaluated using an unpaired two-tailed t test, one-way analysis of variance (ANOVA) followed by Dunnet post hoc tests, or Wilcoxon rank-sum test with the Bonferroni correction. Statistical analyses were performed using GraphPad Prism 9. Data are representative of three independent experiments. Details are described in the figure legends.