Apical-Out Human Airway Organoids Modeling SARS-CoV-2 Infection

Abstract

The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases.

Keywords: Omicron variant; SARS-CoV-2; epithelial polarity; human airway organoids.

Figure 1

Generation of apical-out airway organoids. (a) A schematic graph outlines the expansion of lung organoids (LOs) in expansion (Exp) medium and differentiation of apical-in and apical-out airway organoids (AwOs) in proximal differentiation (PD) medium; (b) Bright-field images of the organoids during the expansion or differentiation culture. Scale bar = 200 μm; (c) Confocal images of basolateral marker pan-Keratin (green) in lung organoids and airway organoids. Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar = 10 μm.

Figure 2

Characterization of airway organoids. (a) Confocal images of ciliated cell markers (ACCTUB, green; FOXJ1, red) and basal cell markers (P63, red; CK5, green) in apical-in (top) and apical-out (bottom) airway organoids (AwOs). Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar = 10 μm. (b) Non-differentiated lung organoids (LOs) and differentiated airway organoids (AwOs) were assessed through qPCR analysis to detect the expression level of marker genes specific for basal (P63, CK5), ciliated (FOXJ1, SNTN), goblet (MUC5AC) and club (CC10) cells. Data represent means ± SD of a representative experiment, n = 4. Ordinary one-way ANOVA with Dunnett’s multiple comparison test comparing airway organoids to the lung organoids. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (c,d) Non-differentiated lung organoids (LOs) and differentiated airway organoids (AwOs) were assessed through flow cytometry to examine the abundance of CK5+ basal cells, ACCTUB+ ciliated cells, MUC5AC+ goblet cells and CC10+ club cells. Representative histograms are shown in (c): red, cells stained with target antibodies; blue, cells stained with isotype controls. Data shown in (d) represent means ± SD of a representative experiment in one organoid line, n = 3. Ordinary one-way ANOVA with Dunnett’s multiple comparison test comparing airway organoids to the lung organoids.

Figure 3

Integrin β1 mediates epithelial polarity of the airway organoids. Matrigel-embedded apical-in airway organoids were treated with function-blocking anti-integrin β1 antibody or control IgG. Confocal images of ciliated cell marker ACCTUB (green) and basal cell marker P63 (red) in control and treated organoids (top). Confocal images of the basolateral marker pan-Keratin (green) in control and treated organoids (bottom). Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar = 10 μm.

Figure 4

SARS-CoV-2 variants infection in apical-out airway organoids. (a) At the indicated hours post-infection (hpi) with SARS-CoV-2 wildtype (WT), Omicron variants B.1.1.529 and BA.5 (1 MOI), culture media were harvested from the infected apical-out airway organoid (AwO) and applied to viral load detection (left) and viral titration (right). Data represent means ± SD of a representative experiment, n = 3. Multiple unpaired t-test with multiple comparisons using the Holm–Sidak method was carried out. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (b) At 24 hpi, SARS-CoV-2 WT-, B.1.1.529- and BA.5 (1 MOI)-infected apical-out airway organoids were harvested and assessed using flow cytometry to quantify dsRNA+-virus-infected cells. Data on the left represent means ± SD of a representative experiment, n = 3. Ordinary one-way ANOVA with Tukey’s multiple comparison test comparing every other group. Representative histograms are shown in the right: red, infected cells stained with target antibodies; blue, mock-infected cells stained with target antibodies. (c,d) Confocal images of ciliated cell marker ACCTUB (red) and SARS-CoV-2 nucleoprotein (NP, green) in infected apical-out airway organoids at (c) 24 hpi or (d) 72 hpi. Nuclei and actin filaments were counterstained with DAPI (blue) and Phalloidin-647 (white), respectively. Scale bar = 20 μm.