Do airway epithelium air-liquid cultures represent the in vivo airway epithelium transcriptome?

Abstract

Human airway epithelial cells cultured in vitro at the air-liquid interface (ALI) form a pseudostratified epithelium that forms tight junctions and cilia, and produces mucin. These cells are widely used in models of differentiation, injury, and repair. To assess how closely the transcriptome of ALI epithelium matches that of in vivo airway epithelial cells, we used microarrays to compare the transcriptome of human large airway epithelial cells cultured at the ALI with the transcriptome of large airway epithelium obtained via bronchoscopy and brushing. Gene expression profiling showed that global gene expression correlated well between ALI cells and brushed cells, but with some differences. Gene expression patterns mirrored differences in proportions of cell types (ALIs have higher percentages of basal cells, whereas brushed cells have higher percentages of ciliated cells), that is, ALI cells expressed higher levels of basal cell-related genes, and brushed cells expressed higher levels of cilia-related genes. Pathway analysis showed that ALI cells had increased expression of cell cycle and proliferation genes, whereas brushed cells had increased expression of cytoskeletal organization and humoral immune response genes. Overall, ALI cells provide a good representation of the in vivo airway epithelial transcriptome, but for some biologic questions, the differences between in vitro and in vivo environments need to be considered.

Figure 1.

Correlation of overall gene expression of large airway epithelium air–liquid interface (ALI; n = 12) with brushed large airway epithelia of healthy nonsmokers (n = 12). Ordinate indicates relative gene expression in large airway epithelium ALI cultured cells. Abscissa indicates relative gene expression in brushed cells from large airway epithelia. Each is plotted on a logarithmic scale. Triangles represent each probe set that was present in at least 20% of samples. Gray rectangle indicates an up or down twofold change. Tight clustering indicates a good correlation between cultured cells and brushed cells (Spearman rank correlation coefficient, 0.89; P < 0.001). Overall, 81% of probe sets were similarly expressed within twofold.

Figure 2.

Correlation of expression values of ciliated, basal, and secretory cell-related genes in large airway epithelium ALI cultured cells and brushed large airway epithelia. (A, C, and E) Ordinate indicates relative gene expression in ALI cells, whereas abscissa indicates relative gene expression in brushed cells, plotted on a logarithmic scale. Triangles represent each gene present in at least 20% of samples. When multiple probe sets represent a gene, the probe set with the highest expression values was selected. Gray rectangle indicates an up or down twofold change. (B, D, and F) Ordinate indicates relative gene expression in ALI and brushed cells. Abscissa indicates examples of markers of each cell type. Dark gray bars, ALI cells; light gray bars, brushed cells from large airway epithelia. Each bar represents relative mean expression with standard error. P values are represented in brackets above bars. (A) Correlation of cilia-related genes (Spearman rank correlation coefficient, 0.85; P < 0.001). Overall, 67% of genes were similarly expressed within twofold. Of genes that were differentially expressed, 92% showed increased expression in brushed cells relative to ALI cultures. (B) Relative expression in examples of cilia-related genes (TEKT3, DNAH5, BBS4, and IFT40). Each was more highly expressed in brushed cells relative to ALI. (C) Correlation of basal cell–related genes (Spearman rank correlation coefficient, 0.64: P < 0.001). Overall, 42% of genes were similarly expressed within twofold. Of genes that were differentially expressed, 78% showed increased expression in ALI relative to brushed cells. (D) Relative expression of examples of basal cell markers (integrin A6, CD109, TP63, and keratin 5). Each was more highly expressed in ALI cells relative to brushed cells. (E) Correlation of secretory cell–related genes (Spearman rank correlation coefficient, 0.90; P < 0.02). Overall, 83% of genes were similarly expressed within twofold. (F) Relative expression levels of examples of secretory genes. TFF1, RAB27A, and MUC5B all showed similar expression in ALI cells relative to brushed cells, whereas MUC5AC was more highly expressed in brushed cells relative to ALI. Although the P value for MUC5B was significant, the fold change of −1.6 did not meet our criteria for differential expression of more than twofold. (B, D, and F) C, large airway epithelium ALI cultures; B, large airway epithelium brushed cells.

Figure 3.

Expression of the gene ontology categories cell cycle, cell proliferation, cytoskeleton organization, and humoral immune response genes in large airway epithelium ALI compared with brushed large airway epithelia. (A, C, E, and G) Ordinate indicates relative gene expression in ALI, whereas abscissa indicates relative gene expression in brushed cells, plotted on a logarithmic scale. Triangles represent each gene present in at least 20% of samples. When multiple probe sets represented a gene, the probe set with the highest expression values was used. Gray rectangle indicates twofold change. (B, D, F, and H) Ordinate indicates relative gene expression in ALI and brushed cells. Abscissa indicates examples of genes from each category. Dark gray bars, ALI; light gray bars, brushed cells. Each bar represents relative mean expression with standard error. P values are represented in brackets above bars. (A) Correlation of cell cycle genes (Spearman rank correlation coefficient, 0.86; P < 0.001). Overall, 72% of genes were similarly expressed. (B) Expression of cell cycle genes. Cyclin B2 (CCNB2) and anillin were more highly expressed in ALI relative to brushed cells, whereas tumor-suppressor genes ERF and ING1 were more highly expressed in brushed cells relative to ALI. (C) Correlation of cell proliferation genes. Overall, 66% of genes were similarly expressed within twofold (Spearman rank correlation coefficient, 0.83; P < 0.001). (D) Expression of cell proliferation genes. Amphiregulin (AREG), EMP1, fibroblast growth factor BP1 (FGFBP1), and PTH-like hormone (PTHLH) were more highly expressed in ALI relative to brushed large airway epithelial cells. (E) Correlation of cytoskeleton organization and biogenesis (Spearman rank correlation coefficient, 0.84; P < 0.001). Overall, 61% of genes were similarly expressed within twofold. (F) Expression of cytoskeleton organization genes. Tetkin 3 (TEKT3), dynein heavy chain 9 (DNAH9), and supervillin (SVIL) were more highly expressed in brushed cells relative to ALI. In addition to other keratins and tubulins within this category, keratin 6A was more highly expressed in ALI relative to brushed cells. (G) Correlation of humoral immune response genes (Spearman rank correlation coefficient, 0.84; P < 0.001). Overall, 56% of genes were similarly expressed within twofold. (H) Expression of humoral immune response genes. CD53, B-cell CLL/lymphoma 2 (BCL2), complement factor 5 (C5), and chemokine (C-C motif) ligand 14 (CCL14) were more highly expressed in brushed cells relative to ALI. (B, D, F, and H) C, large airway epithelium ALI cultures; B, large airway epithelium brushed cells.