Cell response analysis in SARS-CoV-2 infected bronchial organoids

Abstract

The development of an in vitro cell model that can be used to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is expected. Here we conducted infection experiments in bronchial organoids (BO) and an BO-derived air-liquid interface model (BO-ALI) using 8 SARS-CoV-2 variants. The infection efficiency in BO-ALI was more than 1,000 times higher than that in BO. Among the bronchial epithelial cells, we found that ciliated cells were infected with the virus, but basal cells were not. Ciliated cells died 7 days after the viral infection, but basal cells survived after the viral infection and differentiated into ciliated cells. Fibroblast growth factor 10 signaling was essential for this differentiation. These results indicate that BO and BO-ALI may be used not only to evaluate the cell response to SARS-CoV-2 and coronavirus disease 2019 (COVID-19) therapeutic agents, but also for airway regeneration studies.

Fig. 1. Generation of BO.

a Phase and HE staining images of human bronchial organoids (BO). b TEM images of BO. Larger images are shown in Supplementary Fig. 1. c Immunohistochemistry analysis of KRT5 (basal cell marker), acetylated α-tubulin (ciliated cell marker), MUC5AC (goblet cell marker), and CC10 (club cell marker) in BO. Panels a–c are representative of three independent experiments.

Fig. 2. SARS-CoV-2 infection experiments in BO.

a BO were infected with SARS-CoV-2 (1.3 × 10⁵ TCID50/well) in the presence or absence of 10 μM camostat and then cultured with differentiation medium for 5 days. b Immunohistochemistry analysis of SARS-CoV-2 Spike protein (SP) in the infected BO. b is representative of three independent experiments. c The amount of infectious virus in the supernatant was measured using the TCID50 assay. d Parametric Gene Set Enrichment Analysis (PGSEA) applied to GO biological process gene sets was performed in uninfected BO (mock), infected BO (control), and infected BO treated with 10 μM camostat (camostat). Three technical repeats were performed per sample for the RNA-seq analysis. e The amount of infectious virus in the supernatant of spherical and suspended BO was measured using the TCID50 assay. In panels c and e, statistical significance was evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests (*P < 0.05). In panels c and e, data represent mean ± SD from three independent experiments.

Fig. 3. SARS-CoV-2 infection experiments in BO-ALI.

a BO-ALI were infected with SARS-CoV-2 (1.3 × 10⁵ TCID50/well) and then cultured with differentiation medium for 2 days. b Immunofluorescence analysis of KRT5 (red) and acetylated α-tubulin (green) in uninfected BO-ALI. Nuclei were counterstained with DAPI (blue). c Immunofluorescence analysis of ACE2 (green) and acetylated α-tubulin (red) in uninfected BO-ALI. Nuclei were counterstained with DAPI (blue). d The amount of infectious virus in the supernatant of infected BO or BO-ALI was measured by the TCID50 assay. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05). Data represent the mean ± SD from three independent experiments. e Immunofluorescence analysis of SARS-CoV-2 Spike protein (SP) (green) and acetylated α-tubulin (red) in uninfected BO-ALI 2 days after the infection. Immunofluorescence analysis of SARS-CoV-2 SP (green) and KRT5 (red) in uninfected BO-ALI. Nuclei were counterstained with DAPI (blue) 2 days after the infection. f Immunofluorescence analysis of SARS-CoV-2 SP (green) and acetylated α-tubulin (red) in infected BO-ALI 7 days after the infection. Immunofluorescence analysis of SARS-CoV-2 SP (green) and KRT5 (red) in infected BO-ALI 7 days after the infection. Nuclei were counterstained with DAPI (blue). g Immunofluorescence analysis of KRT5 (red) and acetylated α-tubulin (green) in BO-ALI 15 days after the infection. Nuclei were counterstained with DAPI (blue). Panels b, c, e–g are representative of three independent experiments.

Fig. 4. Evaluation of COVID-19 therapeutic drugs using BO-ALI.

a BO-ALI were infected with SARS-CoV-2 (1.3 × 10⁵ TCID50/well) in the presence or absence of 10 μM camostat, 1 μM remdesivir, or 1 μM EIDD-2801 and then cultured with differentiation medium for 2 days. The amount of infectious virus in the supernatant was measured using the TCID50 assay. Statistical significance was evaluated using one-way ANOVA followed by Tukey’s post hoc test (*P < 0.05). b BO-ALI were infected with SARS-CoV-2 B, B.1, B.1.1.214, B.1.1.7, B.1.351, P.1, B.1.617.2, or B.1.1.529 (1.3 × 10⁵ TCID50/well) and then cultured with differentiation medium. The virus copy number in the supernatant was measured by qPCR 1, 2, or 3 days after the infection. Data represent the mean ± SD from three independent experiments.

Fig. 5. FGF10 is essential for the differentiation of basal cells.

a BO-ALI were infected with SARS-CoV-2 (1.3 × 10⁵ TCID50/well) and then cultured with a differentiation medium for 2 (a) or 15 (b) days. After the infection, BO-ALI were cultured with differentiation medium with or without FGF1, 2, or 10. The amount of infectious virus in the supernatant was measured by the TCID50 assay 2 days after the infection. Statistical significance was evaluated by one-way ANOVA followed by Dunnett’s post hoc test (*P < 0.05). Data represent the mean ± SD from three independent experiments. b Immunofluorescence analysis of acetylated α-tubulin (green) and KRT5 (red) 15 days after the infection. Nuclei were counterstained with DAPI (blue). Panel b is representative of three independent experiments.

Fig. 6. Infection experiments of BO-ALI generated from ten donors.

a Donor information of the NHBE cells used in this study. b, c BO-ALI were infected with SARS-CoV-2 (1.3 × 10⁵ TCID50/well) and then cultured with differentiation medium for 2 days. The viral RNA copy number in the cell-culture supernatant was measured by qPCR. Statistical significance was evaluated using an unpaired Student’s t test (**P < 0.01). Data represent the mean ± SD from three independent experiments.