Flow-Cytometric Analysis and Purification of Airway Epithelial-Cell Subsets

Abstract

The human airway epithelium is essential in homeostasis, and epithelial dysfunction contributes to chronic airway disease. Development of flow-cytometric methods to characterize subsets of airway epithelial cells will enable further dissection of airway epithelial biology. Leveraging single-cell RNA-sequencing data in combination with known cell type-specific markers, we developed panels of antibodies to characterize and isolate the major airway epithelial subsets (basal, ciliated, and secretory cells) from human bronchial epithelial-cell cultures. We also identified molecularly distinct subpopulations of secretory cells and demonstrated cell subset-specific expression of low-abundance transcripts and microRNAs that are challenging to analyze with current single-cell RNA-sequencing methods. These new tools will be valuable for analyzing and separating airway epithelial subsets and interrogating airway epithelial biology.

Keywords: airway epithelium; flow cytometry; single-cell RNA sequencing.

Figure 1.

Identification of cell-specific markers for flow cytometry. (A) Human bronchial epithelial cells (HBECs) grown in the absence (cyan histogram) or presence of IL-13 (magenta histogram) were stained using antibodies against cell subset–specific markers. Positive staining was determined by comparing the same sample stained with an appropriate isotype control (gray histogram). Histograms represent data from a single experiment with one donor; data from 10,000 cells were acquired. (B) Comparison of positive staining for cell subset–specific markers from unstimulated and IL-13–stimulated HBECs (staining of one technical replicate from n = 5 individuals; each donor is represented by a different color). Significance was evaluated using a Wilcoxon signed-rank test: *P < 0.05 and **P < 0.01. CDHR3 = cadherin-related family member 3; CEACAM = carcinoembryonic antigen-related cell-adhesion molecule 5; ITGA6 = integrin subunit α 6; NGFR = nerve growth factor receptor; ns = nonsignificant; TSPAN8 = tetraspanin-8; TUBA = acetylated α-tubulin.

Figure 2.

Characterization of airway epithelial-cell subsets and IL-13 stimulation using an analytical flow-cytometric panel. HBECs (n = 5 donors) were cultured with or without IL-13 and processed for multicolor flow cytometry. Data from 10,000 cells originating from a single replicate were acquired. (A–E) Gating strategy to identify airway epithelial subsets from unstimulated (IL-13⁻) and IL-13–stimulated (IL-13⁺) HBECs. (A) Doublets and (B) debris were removed, and resulting singlets were gated on (C) NGFR and TUBA. (D) Subsequently, TUBA⁻NGFR⁻ singlets were gated on ITGA6 and CEACAM6. (E) ITGA6⁻CEACAM6⁺ cells were then gated on MUC5AC and TSPAN8. The specific gate used in the subsequent analysis step is outlined in red. Positive antibody staining was determined using fluorescence-minus-one controls. Although antibodies for CDHR3 and CEACAM5 were also included in the panel, they were not used in our standard gating method because of substantial overlap with TUBA and CEACAM6 staining, respectively. (F) Quantification of four major epithelial subsets (B1+2; ciliated [Cil], TUBA⁺NGFR⁻; secretory [Sec], TUBA⁻NGFR⁻CEACAM6⁺ITGA6⁻; and Gob, TUBA⁻NGFR⁻CEACAM6⁺ITGA6⁻TSPAN8⁺MUC5AC⁺) from unstimulated (IL-13⁻) and IL-13–stimulated (IL-13⁺) HBECs derived from two individuals with no history of airway disease (NAD, pink) and three individuals with interstitial lung disease (ILD, cyan). The black bar represents the mean across HBECs, irrespective of disease history. (G–R) After downsampling, flow-cytometric results from 3,000 cells from each of three donors were combined and analyzed by t-distributed stochastic neighbor embedding. The t-distributed stochastic neighbor embedding plots are colored to show cells that stained for the ciliated cell markers (G) TUBA and (H) CDHR3 (red), the basal-cell markers (I) NGFR and (J) ITGA6 (blue), and the secretory cell markers (K) CEACAM6, (L) CEACAM5, (M) TSPAN8, and (N) MUC5AC (green). Cells that did not stain for the indicated marker are shown in gray. (O) FSC and (P) SSC are represented as a continuum from low (blue) to high (red). Cells came from unstimulated (⁻; cyan) or IL-13–stimulated (⁺; pink) (Q) cultures from three (R) individuals (R) (the R should be after individual rather than three; similarly (Q) could be moved after cultures 3 words earlier) (donors A, B, and C). **P < 0.01 for unstimulated versus IL-13–stimulated by Wilcoxon rank-sum test. B1 = TUBA⁻NGFR⁺; B2 = TUBA⁻NGFR⁻CEACAM6⁻ITGA6⁺; B1+2 = sum of B1 and B2; FSC = forward scatter; Gob = goblet; ILD = interstitial lung disease; NAD = no history of airway disease; ns = not significant; SSC = side scatter.

Figure 3.

Flow-cytometric sorting of HBEC subsets. Before processing for flow cytometry, six 12-mm Transwells each of HBECs (n = 3 donors) cultured in the absence or presence of IL-13 were stained with SiR-tubulin. SiR-tubulin–stained cells were trypsinized, stained with a cell-viability dye, and sorted by flow cytometric cell sorting. (A–E) Gating strategy for flow-cytometric cell sorting of unstimulated (IL-13⁻) and IL-13–stimulated (IL-13⁺) HBECs for a representative donor. After selection of (A) singlets and (B) live cells, cells were gated on (C) SiR-tubulin and NGFR staining. (D) SiR-tubulin⁻ cells were gated on NGFR and CEACAM6. (E) NGFR⁻CEACAM6⁺ cells were gated on CEACAM6 and TSPAN8. The specific gate/quadrant used in the subsequent analysis step is outlined in red. (F and G) To validate SiR-tubulin staining, SiR-tubulin⁺ cells were fixed in paraformaldehyde, immobilized to slides by cytospin, stained with TUBA (cyan), and counterstained with DAPI (magenta). Images show a representative cell from (F) unstimulated (IL-13⁻) and (G) IL-13–stimulated (IL-13⁺) cultures with TUBA-stained cilia (found in 58/60 cells examined). Scale bars, 10 μM. (H) qRT-PCR analysis of sorted cell subpopulations. Mean expression values calculated from triplicate experiments with different donors (n = 3) were normalized to the maximum expression of the gene in any cell type (0–1: white to red). CFTR = cystic fibrosis transmembrane conductance regulator; DCLK1 = doublecortin-like kinase 1; FOXJ1 = forkhead box J1; KRT5 = cytokeratin 5; miR = microRNA; MYB = MYB proto-oncogene, transcription factor; PRB1 = proline-rich protein BstNI subfamily 1; SCGB1A = secretoglobulin family 1A member; SPDEF = SAM pointed domain–containing ETS transcription factor; TSLP = thymic stromal lymphopoietin; TUBA1A = tubulin α 1A class 1.