A comparative study of in vitro air-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution

Abstract

Background: The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air-liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized.

Methods: BCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens.

Results: We confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus.

Conclusions: Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

Keywords: Airway epithelium; Air–liquid interface epithelial culture; Innate immunity; Respiratory infection; Single cell transcriptomics.

Fig. 1

Analysis of epithelial morphology and cell types in differentiated BCi-NS1.1- and primary-derived HAE cultures. a Experimental design. HAE cultures were seeded from either primary cells or the hTERT-expressing airway basal cell line BCi-NS1.1. Cells were differentiated for three weeks at air–liquid interface to generate three replicates of BCi-NS1.1-HAE cultures (derived from one donor), and three HAE cultures from three separate primary donors. b Hematoxylin & Eosin (H&E) and Periodic Acid-Schiff (PAS)-Alcian blue staining of formalin-fixed paraffin-embedded transections of HAE cultures. All cross-sectional images are oriented with the basolateral surface of the culture at the bottom of the image and the apical surface at the top of the image. Scale bars = 150 µm c. Cell type marker immunofluorescence of transections of BCi-NS1.1- or primary-derived HAE cultures. DAPI (nuclei) stained in blue. Cytokeratin 5 (KRT5, basal cells), villin-1 (ciliated cells), CC10 (encoded by SCGB1A1, secretory cells) and MUC5B (secretory cells) labeled in green. MUC5AC (secretory cells) labeled in red. Scale bars = 150 µm. d–g Quantification for each cell type marker, KRT5 (c), villin-1 (d), CC10 (e), and mucins (f) (sum of MUC5B and MUC5AC), represented as a percentage of the total phalloidin (actin)-labeled area of the HAE cultures. h Quantification of cells using Imaris software labeled positive for MUC5B, MUC5AC, or both, represented as a percentage of total MUC5B and/or MUC5AC positive labeling

Fig. 2

Cell annotation and pseudotime analyses of scRNA-Seq data from BCi-NS1.1 and primary–derived HAE cultures. a UMAP (gene expression data for n = 12,801 single cells), colored by cell population assignment. b Cell frequencies by biological replicate. Cell populations with significantly different frequencies (BCi-NS1.1 versus primary HAE cultures) are noted in population label key (*P < 0.05, **P < 0.01, ***P < 0.001). c A dot plot summarizing the expression level (z-scaled, dot color) and percentage of cells expressing the indicated gene (dot size) of signature marker genes for each cell population per biological replicate. d Pseudotime trajectory lineage spanning basal-to-secretory cells (both HAE precursor groups, integrated). Proliferating cells and low frequency cell types (grey) were excluded from trajectory analysis. e Cell distributions for each precursor cell group along the basal-to-secretory trajectory (Kolmogorov–Smirnov Goodness of Fit Test, p = 2.2e−16). f Cell pseudotime values by population and scRNA-Seq replicate. Statisical testing for differential median pseudotimes for basal, intermediate and secretory I cells all reached the minimal p value of 0.1 via a Wilcoxon Rank Sum Test with 3 replicates per group (‡)

Fig. 3

Gene expression analyses of BCi-NS1.1 and primary–derived HAE cultures. a Heatmap of Spearman correlation coefficients for pseudobulk transcriptional profiles aggregated by cell population (color) and precursor cell group (shape) by scRNA-Seq replicate. Row and column clustering were determined by Ward’s linkage method and plotted as a marginal dendrogram. b. PCA of pseudobulk transcriptional profiles displaying principal components 1 through 4 for the top 3000 most variable genes across plotted samples. The percentage of total variance described by each component is indicated in axis titles. c–e Differential gene expression analyses contrasting BCi-NS1.1 and primary –derived HAE cultures by cell population for basal, suprabasal, intermediate, secretory I, and secretory II populations. c Volcano plots indicating differentially expressed genes (DEGs) in pseudobulk profile contrasts; significance defined as adjusted p-value (Benjamini-Hochberg) < 0.05, log2FC > 1 or < − 1, and per-cell population expression in > 10% of cells. TERT and ISGs IFIT1, ISG15, and MX1 are labeled when significantly differentially expressed. The number of significant DEGs for each cell population contrast are indicated. d. Intersection of DEG lists by cell population. A core set of 30 DEGs across all contrasts is highlighted in gold. e The top 30 gene sets enriched in any tested cell population (ranked by FDR, ordered by directional p-value; C2 canonical pathways collection). Dark grey indicates sets that were not significantly enriched. A positive directional p-value indicates enrichment in BCi-NS1.1 relative to primary-derived HAE cultures and a negative value represents enrichment in primary-derived HAE cultures relative to BCi-NS1.1. Gene sets related to interferon signaling are highlighted in red

Fig. 4

Infection of BCi-NS1.1- and primary-derived HAE cultures with IAV and S. aureus. a, c, e RNA levels of MX1, IFN-β, IFIT1 and ISG15 were measured using RT-qPCR at steady-state (a), after IAV infection (c) and after IFN-β treatment (e). b, d, f Representative western blots for MX1 and phosphorylated STAT1 (p-STAT1), along with GAPDH controls for mock treated (b), IAV-infected (d) and IFN-β-treated (f) cultures, and quantification of western blot band intensity from three individual blots for each condition. Blots are cropped to the relevant size for the indicated protein. g S. aureus colony-forming units (CFU) after 18 h of infection h IAV plaque-forming units (PFU) after 24 h of infection i. IAV-infected cultures were fixed and labelled with an anti-NP antibody. Infection was quantified as total NP positive area. j. LDH release was quantified after IAV or S. aureus infection and plotted as a fold change of LDH release over mock infected cultures. k Top-down images of representative IAV-infected HAE cultures, showing DAPI (blue) and influenza virus-NP (green). Scale bars = 1 mm. l. Top-down images of representative S. aureus and IAV-infected HAE cultures, showing Phalloidin (red) and DAPI (blue). Scale bars = 1 mm

Fig. 5

Expression and function of CFTR channels in HAE cultures. a Cell type marker immunofluorescence of transections of BCi-NS1.1- or primary-derived HAE cultures. DAPI (nuclei) stained in blue. CFTR labeled in green. Scale bars = 200 µm. b Diagrammatic representation of CFTR functional assay. TEER of CFTR channels (purple) was measured at baseline, then activators of CFTR channels or controls were added and TEER was measured at 10–60 min post-treatment. c TEER measurement of HAE cultures treated with media control, vehicle control (DMSO) or CFTR channel activator (forskolin and IBMX)