Figure 3. Monolayers derived from lung organoids differentiate into proximal and distal airway components.

(A, B) Samples collected at various steps of lung organoid isolation and expansion in culture, and from the two types of monolayers prepared using the lung organoids were analyzed by bulk RNA seq and the datasets were compared for % cellular composition using the deconvolution method, CYBERSORTx. Schematic in (A) shows the workflow steps, and bar plots in (B) show the relative proportion of various lung cell types. (C, D) hiPSC-derived AT2 cells and alveolospheres (C) were plated as monolayers and analyzed by RNA seq. Bar plots in (D) show % cellular composition. (E, F) Submerged adult lung organoids (ALO) monolayers in transwells (E) or monolayers were grown as air-liquid interphase (ALI) models (F) were fixed and stained for the indicated markers and visualized by confocal immunofluorescence microscopy. The representative max projected z-stack images (left) and the corresponding orthogonal images (right) are displayed. Arrows in (E) indicate AT2 cells; arrowheads in (E) indicate club cells; asterisk in (F) indicates bundles of cilia standing perpendicular to the plane of the ALI monolayers; arrowheads in (F) indicate bundles of cilia running parallel to the plane of the ALI monolayers. Scale bar = 20 µm. (G) Monolayers of ALO1-3 were challenged with SARS-CoV-2 for indicated time points prior to fixation and staining for KRT5, SARS-COV2 viral nucleocapsid protein and DAPI and visualized by confocal microscopy. A montage of representative images are shown, displaying reticulovesicular network patterns and various cytopathic effects. Scale bar = 15 µm. (H) Monolayers of ALO, hiPSC-derived AT2 cells, and other alternative models (see Figure 3—figure supplements 1–2) were infected or not with SARS-CoV-2 and analyzed for infectivity by qPCR (targeted amplification of viral envelope, E gene). See also Figure 3—figure supplement 3B, C for comparison of the degree of peak viral amplification across various models. (I) ALO monolayers pretreated for 4 hr with either vehicle (DMSO) control or EIDD-parent (NHC) or its metabolite EIDD-2801/MK-4482 were infected with SARS-CoV-2 and assessed at 48 hpi for infectivity as in (H). Line graphs display the relative expression of E gene. Error bars display SEM. p value **<0.01; ***<0.001.

Figure 3—figure supplement 1. Monolayers derived from adult lung organoids (ALO) can form an epithelial barrier.

(A–G) Two different types of 2D polarized monolayers are prepared using adult lung organoids. Schematics in (A) and (E) show growth as submerged or air-liquid interphase (ALI) models, respectively. Panel (B) shows bar graphs with transepithelial electrical resistance (TEER) across submerged monolayers grown in transwells. Panel (C) shows bar graphs for relative fluorescence unit (RFU) of the FITC-labeled dextran flux from the apical to basolateral chambers of a submerged monolayer. (D) Brightfield images show representative fields of submerged monolayers grown on transwells. Scale bar = 100 µm. Arrows indicate self-organized vacuolar regions were seen. (F) Bar graphs with TEER across ALO-derived monolayers grown as ALI models. (G) Brightfield images show representative fields of ALI monolayers at two different time points during culture. Scale bar = 100 µm. (H, I) Submerged monolayers of ALO were fixed with methanol (H) or paraformaldehyde (I) prior to co-staining with DAPI (blue; nuclei) and either occludin (green [H] or phalloidin [red; I]). Scale bar = 20 µm. (J) ALO monolayers were grown as ALI models were fixed and co-stained for SFTPC (red), Ac-Tub (green), and DAPI (blue; nuclei) and visualized by confocal immunofluorescence microscopy. Scale bar = 20 µm. (K, L) Schematic in (K) shows the study design for challenging submerged monolayers with 500 ng/ml LPS, followed by TEER measurement. Bar graphs in (L) display the % change in TEER observed with or without LPS treatment normalized to the baseline TEER. p-values were analyzed by one-way ANOVA. Error bars denote SEM; n = 3–6 datasets. **p< 0.01.

Figure 3—figure supplement 2. Alternative models of lung epithelial cells used in this work for modeling SARS-CoV-2 infection and/or as a control for gene expression studies.

(A–D) Monolayers of primary airway epithelial cells (small airway epi; A B; bronchial epi; C, D) were visualized by bright field microscopy (A, C) or by fixing, staining, and visualizing by confocal microscopy (B, D). Representative images in (B) and (D) are presented as maximum projected z-stacks on the left and as an orthogonal view on the right. (E–G) hiPSC-derived AT2 cells, prepared using the i-HAEpC2 cell kit, were grown in monolayers on transwell inserts to form a polarized. Brightfield images are shown in (F). Monolayers were fixed and stained for several markers and analyzed by confocal microscopy. Representative images are shown in (G). Scale bar = 20 µm.

Figure 3—figure supplement 3. Proof of SARS-CoV-2 infectivity.

(A) Monolayers of ALO1-3 were challenged with SARS-CoV-2 for indicated time points prior to fixation and staining for KRT5 (red) and viral nucleocapsid protein (green) and DAPI (blue; nuclei) and visualized by confocal microscopy. Representative images are shown, displaying various cytopathic effects. Scale bar = 15 µm. (B) Monolayers of adult lung organoids (ALO) (either transwell submerged models or air-liquid interphase [ALI], left) and monolayers of hiPSC-derived AT2 cells (right) were infected or not with SARS-CoV-2 and analyzed for viral envelope gene (E gene). Bar graphs display the relative expression of E gene in infected ALO monolayers, indicative of viral infection. (C) Line graphs show the change in E gene expression in infected monolayers over 24 hr period (from 48 hpi to 72 hpi) where values at 72 hpi are normalized to that at 48 hpi. Data is presented as SEM of three independent repeats.