Ontogeny and Biology of Human Small Airway Epithelial Club Cells

Abstract

Rationale: Little is known about human club cells, dome-shaped cells with dense cytoplasmic granules and microvilli that represent the major secretory cells of the human small airways (at least sixth-generation bronchi).

Objectives: To define the ontogeny and biology of the human small airway epithelium club cell.

Methods: The small airway epithelium was sampled from the normal human lung by bronchoscopy and brushing. Single-cell transcriptome analysis and air-liquid interface culture were used to assess club cell ontogeny and biology.

Measurements and main results: We identified the club cell population by unbiased clustering using single-cell transcriptome sequencing. Principal component gradient analysis uncovered an ontologic link between KRT5 (keratin 5)⁺ basal cells and SCGB1A1 (secretoglobin family 1A member 1)⁺ club cells, a hypothesis verified by demonstrating in vitro that a pure population of human KRT5⁺ SCGB1A1^- small airway epithelial basal cells differentiate into SCGB1A1⁺KRT5^- club cells on air-liquid interface culture. Using SCGB1A1 as the marker of club cells, the single-cell analysis identified novel roles for these cells in host defense, xenobiotic metabolism, antiprotease, physical barrier function, monogenic lung disorders, and receptors for human viruses.

Conclusions: These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.

Keywords: club cell; principal component gradient analysis; small airway epithelium.

Figure 1.

Source of club cells for analysis. SCGB1A1⁺ club cells were recovered from the normal human small airway epithelium (SAE) by bronchoscopy and brushing. (A and C) SCGB1A1⁺ immunohistochemistry, red stain; scale bars, 20 μm. (A) Source of club cells; SCGB1A1⁺ cells in the SAE in a section of normal lung. (C) Example of SCGB1A1⁺ cells recovered by brushing the SAE. B and D are the negative controls for A and C, respectively. (E and F) Transmission electron microscopy assessment of human SAE club cells recovered by brushing. Shown is a club cell with characteristic dense granules. (E) Whole club cell; scale bar, 2 μm. (F) Enlarged image of the granules; scale bar, 0.2 μm.

Figure 2.

Unsupervised clustering of the 157 single cells from human small airway. Small airway epithelium was recovered by bronchoscopy, treated with trypsin to generate single cells, and mechanically filtered to select 10- to 17-μm cells. A total of 157 cells pooled from three healthy individuals were sequenced. (A) Principal component analysis (PCA) of 157 human small airway single-cell transcriptomes using the genes in Figure E11 as the input dataset. Four different clusters were identified by the unsupervised clustering: cluster 1, red dots, 47 single cells; cluster 2, blue dots, 28 single cells; cluster 3, green dots, 67 single cells; cluster 4, purple dots, 15 single cells. (B) Expression of basal cell marker KRT5 in the small airway single cells in PCA plots. (C) Expression of club cell marker SCGB1A1 in the small airway single cells in PCA plots. In B and C, blue represents the relative expression of markers shown above the figures. (A–C, top row) y-axis is principal component 1 (PC1), and x-axis is PC2. (A–C, bottom row) y-axis is PC1, and x-axis is PC3. (D) Boxplots show the gene expression of basal cell marker KRT5 (left) and club cell marker SCGB1A1 (right) in the unsupervised clusters 1–4. The colors in the boxplot coordinated with the colors of clusters 1–4 in A. TPM = transcripts per kilobase million.

Figure 3.

Additional evidence of the close relationship between human small airway epithelium (SAE) basal and club cells using in vivo staining. (A and B, top) Immunofluorescence staining of the normal SAE using the club cell marker SCGB1A1 (red) and basal cell marker KRT5 (green). (A and B, bottom) Relative negative controls for the staining above. (A) Lung sections with SAE. (B) SAE recovered by brushing. Nuclei are stained with DAPI (blue). In addition to KRT5⁺SCGB1A1⁻ (basal cells) and SCGB1A1⁺KRT5⁻ (club cells), there are also KRT5⁺SCGB1A1⁺ putative “intermediate” cells in the lung sections and cells recovered by brushing. Scale bar, 20 μm. (C) Immunofluorescence staining quantification of the percentage of KRT5⁺SCGB1A1⁻ basal cells, KRT5⁺SCGB1A1⁺ putative intermediate cells, and SCGB1A1⁺ KRT5⁻ club cells in the cell populations recovered by brushing of the SAE. The data represent assessment of a total of 4,691 cells from nine healthy nonsmokers (mean ± SEM). P values are shown in the figure. n.s. = not significant.

Figure 4.

Ordering of expression of small airway epithelium single cells using a semisupervised gradient analysis. Principal component (PC) analysis was performed on the 114 single cells from unsupervised clusters 1 and 3. Principal component analysis plots are colored by (A) KRT5 expression, (B) SCGB1A1 expression, and (C) pseudotime gradient values. The red arrows shown in A and B were fit using linear regression; specifically, each marker gene's expression was regressed on the first four PCs. The black arrow in C shows the direction of the pseudotime gradient, which was constructed by averaging the slopes of the red lines shown in A and B and assigning direction arbitrarily. The gradient analysis demonstrates that the KRT5⁺ and SCGB1A1⁺ cell types are related but cannot distinguish direction (i.e., KRT5⁺→SCGB1A1⁺ or SCGB1A1⁺→KRT5⁺). (D and E) Examples of genes significantly correlated with the gradient values. The blue curves show three-parameter sigmoid functions fit to each gene. (D) SCGB1A1⁺. (E) KRT5⁺. TPM = transcripts per kilobase million.

Figure 5.

In vitro demonstration that basal cells (BCs) are the progenitor cells for club cells in the human small airway epithelium (SAE). Highly purified BCs isolated from the SAE of healthy individuals (see Figures E3B–E3F) were placed on air–liquid interface (ALI) on type IV collagen-coated transwells and cultured for 28 days. The BCs initiating the cultures were 99.0 ± 1.1% KRT5⁺ and 0% SCGB1A1⁺ (see Figures E4A and E4C). (A and B) TaqMan PCR assessment of SAE BC differentiation in ALI cultures over 28 days. BC and club cell characteristic genes were based on the single-cell transcriptome analysis of signature genes that characterize cluster 1 and 3 cells (see Table E2). (A) BC signature genes: KRT5, CCND2, FAT2, and PDGFA. (B) Club cell signature genes: SCGB1A1, PIGR, MUC5B, and XBP1. (C) Immunofluorescence staining of BC marker KRT5 (green) and club cell marker SCGB1A1 (red) in sections of the ALI culture at Days 0, 7, 14, and 28. Over time, the KRT5⁺ cells disappear, and the SCGB1A1⁺ cells appear. Nuclei are stained with DAPI. Scale bar, 20 μm. (D) Immunofluorescence top staining of additional club cell marker MUC5B in the SAE derived from BC at Days 7, 14, and 28 in ALI culture. Top row, Day 7; middle row, Day 14; bottom row, Day 28. Left column, SCGB1A1 (green); middle column, MUC5B (red); right column, overlapping of SCGB1A1 and MUC5B. The appearance over time of the MUC5B⁺ cells tracks with SCGB1A1⁺ cells. Nuclei are stained with DAPI (blue). Scale bar, 20 μm. (E) Similar to D, but with SCGB1A1 and SLPI. The relative negative control subjects for C–E are shown in Figures E5A–E5C. (F–M) Transmission electron microscopic assessment of the airway epithelium in ALI culture at Day 28. The Day 28 cultures included cells with the characteristic features of club cells, including microvilli (F–H and L), dense cytoplasmic granules (F–K), and abundant smooth endoplasmic reticulum (K and M). Scale bar, 1 μm. SLPI = secretory leukocyte peptidase inhibitor.

Figure 6.

Analysis of small airway epithelium single-cell transcriptomes of healthy individuals. Venn diagram of the single cells based on the expression of cell markers of the major cell populations in human small airway epithelium. Shown are the numbers of single cells expressing the markers SCGB1A1 (club cells), KRT5 (basal cells), DNAI1 (ciliated cells), and MUC5AC (mucin-secreting cells). The numbers of “pure” and various combinations of MUC5AC⁺, SCGB1A1⁺, KRT5⁺, and DNAI1⁺ cells are indicated.

Figure 7.

Cell type–specific genes that characterize the differentiation program from basal cells (BC) to club cells. Comparison of gene expression of pure club cells versus pure BC to identify club cell and BC “signature” genes. (A) Volcano plot showing differential gene expression between the 37 KRT5⁻SCGB1A1⁺ club cells and 29 KRT5⁺SCGB1A1⁻ BC. Genes with significantly higher expression in BC are indicated in red, and genes significantly higher in club cells are indicated in blue. See Table E12 for a list of significant genes. (B and C) BC, red; intermediate cells, purple; club cells, blue. (B) Box plot examples of BC signature genes in SCGB1A1⁻KRT5⁺ BC, KRT5⁺SCGB1A1⁺ intermediate cells, and KRT5⁻SCGB1A1⁺ club cells. (C) Box plot examples of club cell signature genes in SCGB1A1⁻KRT5⁺ BC, KRT5⁺SCGB1A1⁺ intermediate cells, and KRT5⁻SCGB1A1⁺ club cells.