Characterization of an immortalized human small airway basal stem/progenitor cell line with airway region-specific differentiation capacity

Abstract

Background: The pathology of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and most lung cancers involves the small airway epithelium (SAE), the single continuous layer of cells lining the airways ≥ 6th generations. The basal cells (BC) are the stem/progenitor cells of the SAE, responsible for the differentiation into intermediate cells and ciliated, club and mucous cells. To facilitate the study of the biology of the human SAE in health and disease, we immortalized and characterized a normal human SAE basal cell line.

Methods: Small airway basal cells were purified from brushed SAE of a healthy nonsmoker donor with a characteristic normal SAE transcriptome. The BC were immortalized by retrovirus-mediated telomerase reverse transcriptase (TERT) transduction and single cell drug selection. The resulting cell line (hSABCi-NS1.1) was characterized by RNAseq, TaqMan PCR, protein immunofluorescence, differentiation capacity on an air-liquid interface (ALI) culture, transepithelial electrical resistance (TEER), airway region-associated features and response to genetic modification with SPDEF.

Results: The hSABCi-NS1.1 single-clone-derived cell line continued to proliferate for > 200 doubling levels and > 70 passages, continuing to maintain basal cell features (TP63⁺, KRT5⁺). When cultured on ALI, hSABCi-NS1.1 cells consistently formed tight junctions and differentiated into ciliated, club (SCGB1A1⁺), mucous (MUC5AC⁺, MUC5B⁺), neuroendocrine (CHGA⁺), ionocyte (FOXI1⁺) and surfactant protein positive cells (SFTPA⁺, SFTPB⁺, SFTPD⁺), observations confirmed by RNAseq and TaqMan PCR. Annotation enrichment analysis showed that "cilium" and "immunity" were enriched in functions of the top-1500 up-regulated genes. RNAseq reads alignment corroborated expression of CD4, CD74 and MHC-II. Compared to the large airway cell line BCi-NS1.1, differentiated of hSABCi-NS1.1 cells on ALI were enriched with small airway epithelial genes, including surfactant protein genes, LTF and small airway development relevant transcription factors NKX2-1, GATA6, SOX9, HOPX, ID2 and ETV5. Lentivirus-mediated expression of SPDEF in hSABCi-NS1.1 cells induced secretory cell metaplasia, accompanied with characteristic COPD-associated SAE secretory cell changes, including up-regulation of MSMB, CEACAM5 and down-regulation of LTF.

Conclusions: The immortalized hSABCi-NS1.1 cell line has diverse differentiation capacities and retains SAE features, which will be useful for understanding the biology of SAE, the pathogenesis of SAE-related diseases, and testing new pharmacologic agents.

Keywords: Basal cells; Immortalization; Single cell; Small airway; Telomerase.

Fig. 1

Typical small airway transcriptome features of the cell line donor’s small airway epithelium (SAE). Data shown is the unsupervised cluster analysis of microarray data from the cell line donor’s small airway epithelium with data from previously published-microarray datasets that include 9 matched-trachea, large airway and small airway epithelium samples. Genes differentially expressed between the paired trachea and SAE (fold changes > 2 fold, Benjamini-Hochberg corrected p < 0.05) were selected to generate the plot. Examples of SAE-enriched genes (GATA6, SOX9, LTF and SFTPB) are indicated. The donor’s SAE clusters with the reference SAE transcriptome, distinct from the large airway and trachea epithelium

Fig. 2

Isolation and growth of the hSABCi-NS1.1 immortalized small airway basal cells from a healthy nonsmoker subject. a Morphology of cells following drug selection, day 9. Left - parental cells without TERT, plus puromycin selection; right - hTERT infected cells with puromycin selection (mixed clones, hSABCi-NS1). Bar = 100 μm. b Morphology of the single cell-derived cell clone hSABCi-NS1.1 on day 6, passage 0. Single-cell clones were generated by culturing limited-numbers of cells of hSABCi-NS1 cell in 10 cm plate and tracked under the microscope from day 2. Bar = 100 μm. c Growth of hSABCi-NS1.1. Cell population doubling levels were quantified from passage 5. Each dot represents one passage in T25 flask. d Growth of hSABCi-NS1.1 after recovery from cryopreservation. Passage 46 of hSABCi-NS1.1 cells (red dots) which had been frozen in the liquid nitrogen for 131 days, was re-cultured to test the consistence of growth. The population doubling levels were re-counted from the day of re-culturing. Passage 1 parental pre-immortalized primary cells were cultured at the same time as control (blue triangles). e % live cells of the re-cultured cells. The % live cells of the re-cultured hSABCi-NS1.1 cells were counted during each passage. Only adherent cells were counted. Red – hSABCi-NS1.1, passage 46; blue – parental primary cells pre-TERT, passage 2. f Growth of the re-cultured hSABCi-NS1.1 cells at passage 52. Cell doubling time between day 2 and day 3 after seeding was 16 h. The rate of cell doubling was reduced after day 5 when the cells reached confluence

Fig. 3

Maintenance of basal cell features in the immortalized hSABCi-NS1.1 cells. a Basal cell markers staining at passage 6. Left, TP63 (green); middle, KRT5 (red); right, merged image. Bar = 50 μm. b Basal cell marker staining at passage 49. Left, TP63 (red); middle, KRT5 (green); right, merged image. Bar = 20 μm. Note that some nuclei appear negative for TP63 because of image contrast. c Expression of TP63 mRNA. Gene expression was assessed by TaqMan PCR. Each circle is one passage of hSABCi-NS1.1 cells. Primary small airway basal cells from 3 healthy nonsmokers at passage 2 were used as controls (blue triangle on right)

Fig. 4

Cell junction formation of the immortalized hSABCi-NS1.1 cells on air-liquid interface (ALI) culture. a Trans-epithelial electrical resistance (TEER). Each color represents one independent experiment (n = 7 in total, passage 47 to 56 cells). Each dot represents average TEER of 3 ALI wells between day 0 and day 35 of ALI culture. b Junction protein staining of hSABCi-NS1.1 on ALI culture. Shown is tight junction protein 1 (TJP1) staining (green) of passage 47 cells on ALI-day 35. Bar = 20 μm. This composite image was created from a stack of individual images acquired at different depths in the monolayer (z-stack covering 40 μm). As a result, the merged image includes cell junction staining at the apical surface overlaid on the image of the epithelial cell nuclei. The difference in depth of the two structures accounts for difference in alignment of nuclei and apical tight junctions

Fig. 5

Differentiation capacity of immortalized hSABCi-NS1.1 cells on ALI culture. Passage 47 to passage 56 of hSABCi-NS1.1 cells were assessed on ALI day 14 to 35 for airway epithelial cell lineage markers. a Ciliated and club cells of passage 49 cells, ALI-day 21. Red-ARL13B; Green-SCGB1A1. Inset, higher magnification, showing cilia and SCGB1A1⁺ granules. Bar = 20 μm. b Mucous cells in passage 49 cells, ALI-day 16. Red-MUC5AC; green-MUC5B; yellow-co-localization (arrowheads). Bar = 20 μm. c Surfactant protein expressing cells, passage 49, ALI-day 35. Left, red-SFTPA; right, red-SFTPB. In both fields, nuclei were stained with DAPI (blue). d Ionocyte, passage 47, ALI-day 28. Red-FOXI1. Nuclei were stained with DAPI (blue). e Neuroendocrine cells, passage 49 cells, ALI-day 35. Red-CHGA. Nuclei were stained with DAPI (blue). Bar = 10 μm

Fig. 6

Example of non-inflammatory cell immunity genes in hSABCi-NS1.1 on ALI. The reads from the RNAseq data were aligned to the introns and exons of reference mRNAs of the selected genes. Both basal and ALI-d28 data were plotted. a CD4 alignment; b CD74 alignment; c HLA-DRA alignment

Fig. 7

Expression of small airway development-relevant transcription factors in immortalized hSABCi-NS1.1 cells. Gene expression in hSABCi-NS1.1 cells (from small airway) was compared with gene expression in BCi-NS1.1 cells (from large airway). Both cell lines (passage 52–55) were cultured on ALI using identical culture conditions for comparison. Large and small airway lineage markers and transcription factors relevant to the development of large airway (SOX2) and small airway (NKX2–1, GATA6, SOX9, HOPX, ID2 and ETV5) were assessed by TaqMan PCR. Data shown is the mean fold-changes ± standard error of n = 3 independent side-by-side comparison experiments. *, p < 0.05; **, p < 0.01; #, consistent changes in all 3 independent experiments, but not significant because of high variability. a Airway cell lineage marker genes. b Transcription factors relevant to the development of large airway and small airway epithelium

Fig. 8

Consequences of overexpression of SPDEF on the differentiation of immortalized hSABCi-NS1.1 cells on ALI, as a model of secretory cell hyperplasia in COPD. Based on the knowledge that SPDEF, a transcription factor that induced secretory cell differentiation is up-regulated in the SAE of COPD smokers. hSABCi-NS1.1 cells were genetically modified by lentivirus expressing SPDEF and cultured on ALI to day 14 to test whether the hSABCi-NS1.1 cells can be used as a model of secretory cell hyperplasia. a Secretory cell gene expression in the SAE of 36 COPD smokers compared to 60 healthy nonsmokers in vivo. Five up-regulated and 4 down-regulated secretory cell genes were used as examples. Data shown is relative fold-change of microarray-based gene expression ± standard error. b Staining of a known SPDEF-regulated gene in ALI. MUC5AC was used as an example. Shown are passage 47 cells on ALI-day 21. Red-MUC5AC; blue-nucleus. Top panel, lenti-GFP infected cells, as control; bottom panel, lenti-SPDEF-infected cells. Bar = 50 μm. c TaqMan PCR assessment of lentivirus-SPDEF-induced gene expression changes in hSABCi-NS1.1 cells. The same genes as in panel A were assessed. Data shown is the mean fold-change ± standard error of n = 3 independent experiments using passage 52 to passage 55 cells. d Staining of MSMB as an example of a SPDEF-regulated secretory gene in ALI. Shown are passage 47 cells on ALI-day 21. Red-MSMB; blue-nucleus. Top panel, lenti-GFP infected cells, as control; bottom panel, lenti-SPDEF-infected cells. For a, c *, p < 0.05; **, p < 0.01; ***, p < 0.001; #, consistent changes in all 3 independent experiments, but not significant because of variability