Basal cells as stem cells of the mouse trachea and human airway epithelium

Abstract

The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreER(T2) transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay.

Fig. 1.

Lineage tracing of tracheal BCs. (A) KRT5-CreER^T2 transgene construct. (B) Timeline for steady-state experiments with KRT5-CreER^T2;Rosa26R-LacZ adult mice. (C–E) Paraffin sections of X-gal–stained (blue) trachea. Anti-acetylated tubulin (brown, cilia) (C) 6 days, (D) 3 weeks, and (E) 12 weeks after final Tmx injection. (F) Bar graph showing percentage of labeled cells scored as basal, Clara, or ciliated after Tmx exposure and chase. Data shown are mean ± SEM. (G) Timeline for postnatal growth experiments with Rosa26R-eYFP reporter. (H and I) Cryosections stained for GFP (green, lineage label), Scgb1a1 (red, Clara cells), and T1α (blue, BCs). (H) P3 weeks. (I) P15 weeks. [Scale bar in C (for C–E), 20 μm; in H (for H and I), 25 μm.]

Fig. 2.

Characterization of mouse tracheal epithelial populations. Tracheal epithelial cells were sorted on the basis of labeling with GSI-A3B isolectin and expression of KRT5-GFP. (A) KRT5-GFP tracheal epithelium not labeled with GSI-A3B. (B) Transgenic cells labeled with GSI-A3B. p63+ cells make up 90.5% of cells in P4, 40.4% of cells in P6, and 1.1% of cells in P5. Averaged over 7 experiments, P4 contained 6% of total sorted cells, P5 67%, and P6 12%. (C) Heatmap showing the 100 most differentially expressed genes by Affymetrix microarray of P4, P5, and P6. (D) RT-PCR confirmed the upregulation of 11 genes in KRT5-GFP^hi BCs vs. KRT5-GFP⁻ cells.

Fig. 3.

A novel sphere-forming assay for BC behavior. (A) Single viable KRT5-GFP⁺ BCs (P6) or KRT5-GFP⁻ (P5) tracheal epithelial cells were seeded. (B) Tracheospheres formed within 9 days of seeding. (C) Approximately 3% of KRT5-GFP⁺ BCs form tracheospheres, whereas ≈0.5% of KRT5-GFP⁻ cells form spheres. (D and E) 9 days. Tracheospheres stained for (D) p63 (red) and DAPI (blue, nuclei). (E) Basal KRT14 (red) and luminal KRT8 (green). (F and G) Twenty-day differentiated tracheospheres. (F) Hematoxylin and eosin stain. Arrows indicate ciliated cells. (G) Acetylated tubulin (red, cilia) and NGFR (green, BCs). (Scale bars, 1 mm in B; 50 μm in D–G.)

Fig. 4.

NGFR⁺ BCs of mouse and human airways. (A) NGFR (green) is confined to p63⁺ (red) BCs in the mouse trachea. (B) FACS using an anti-NGFR antibody to isolate BCs from primary mouse tracheal epithelium. p63+ cells make up >86% of cells in P6. (C) NGFR expression is confined to the BCs of normal human bronchus. (D) FACS using anti-NGFR and anti-ITGA6 antibodies to enrich for BCs from primary human bronchial epithelial cultures. p63+ cells make up 96% of cells in P5, 83% of cells in P4, and only 15% of cells in P6. (E–G) Human spheres at 18 days of culture. (E) Light micrograph. (F) Hematoxylin and eosin–stained section. (G) Basal KRT14 (red) and luminal KRT8 (green). The KRT14⁺ basal layer appeared somewhat discontinuous. (H and I) Human spheres at 25 days stained for (H) p63 (green, BCs) and (I) acetylated tubulin (red, cilia). (Scale bars, 50 μm in A and C; 25 μm in E–I.)