- Open Access
Characterization of an immortalized human small airway basal stem/progenitor cell line with airway region-specific differentiation capacity
Respiratory Researchvolume 20, Article number: 196 (2019)
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.
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.
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.
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.
The small airway epithelium (SAE), comprised of basal, intermediate, club, mucous and ciliated cells, plays a central role in the pathogenesis of chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF) and most lung cancers [1,2,3,4,5,6]. In humans, the basal cells (BC) function as the SAE stem/progenitor for the intermediate and differentiated club, mucous and ciliated cells [7,8,9,10,11,12]. Based on methods to sample SAE and to isolate BC from the SAE brushes obtained by fiberoptic bronchoscopy [9, 12,13,14], we have characterized SAE and SAE BC in normal non-smokers, asymptomatic smokers, and individuals with COPD using mRNA microarrays, RNAseq, single cell transcriptome, methylation, microRNA, metabolome and protein levels, characterizing the BC in health and dysregulation of the BC population associated with smoking and COPD [13,14,15,16,17,18,19,20].
While isolation of human SAE and SAE BC by bronchoscopy and brushing permits assessment of primary BC, the procedure is invasive, time consuming and expensive, and the primary BC can be cultured only for 3 to 4 passages before becoming senescent. In this context, if it is possible to immortalize normal human SAE BC that retain the capacity to differentiate to ciliated, secretory, and other differentiated cell types in vitro on air-liquid interface (ALI) culture, it would be very useful to the investigation of SAE biology in health and disease, and the assessment of pharmacologic agents targeted to modify dysregulated BC biology relevant to the pathogenesis of human lung disease. In the current study, we have generated hSABCi-NS1.1, an immortalized human small airway basal cell line from the brushed epithelium of a healthy non-smoker. hSABCi-NS1.1 cells can be passaged for at least 200 population doublings, have the capacity to differentiate into the major differentiated SAE club, mucous and ciliated cells, as well as rare SAE cell types, including surfactant protein positive cells and novel ionocytes, and have the capacity to recapitulate SAE disease-relevant biology when stressed with relevant signals.
Study population and biologic samples
The selected donor was a 50 yr. old African American male healthy non-smoker recruited under a protocol approved by the Weill Cornell Medical College Institutional Review Board. The subject was characterized as phenotypically normal on the basis of clinical history and physical examination, routine blood screening tests, urinalysis, chest X-ray, ECG and pulmonary function testing. The non-smoking status was confirmed by history, venous carboxyhemoglobin levels and urinalysis for levels of nicotine (< 2 ng/ml) and its derivative cotinine (< 5 ng/ml). Following written informed consent, bronchoscopy was used to collect the small airway epithelium (SAE) as previously described [13, 17, 20]. Part of the brushed SAE was used for genome-wide expression assessment by microarray (Affymetrix Human Genome U133 Plus 2.0 arrays, Santa Clara CA). Basal cells (BC) were purified from the remainder of the brushed small airway epithelium by trypsinization and selective culturing of BC on collagen-coated tissue culture flasks [9, 12]. On a morphologic level, these cells do not have motile cilia or mucous granules. The cells were expanded in vitro for one passage and stored in the liquid nitrogen for further use. A subset of published microarray dataset  from matched trachea, large (3rd -4th order bronchi) and small airway epithelium (10th to 12th order bronchi) of 9 healthy nonsmoker subjects was used as reference to confirm the expression pattern of donor SAE from current study. The gene expression pattern was compared by unsupervised-cluster analysis.
The primary small airway BC were cultured in flasks coated with human type IV collagen (Sigma-Aldrich, St. Louis, MO) and supplied with Small Airway Epithelial Cell Growth Medium (SAGM; Lonza, Basel, Switzerland). Passage 1 small airway BC cells from the selected donor were immortalized using retrovirus-mediated expression of human telomerase reverse transcriptase (hTERT) with a puromycin resistance selection marker, as previously described . The parental immortalized small airway cell line was isolated using puromycin selection for 10 days. The surviving cell line was termed as human small airway basal cell immortalized-nonsmoker 1 (hSABCi-NS1). To isolate a single-cell clone, the parental hSABCi-NS1 cell was serially diluted into 10 cm dishes without collagen coating to facilitate cell clone formation . After seeding, the clones were marked and monitored daily to make sure each cell cluster was single-cell-derived. We observed that different cell clones demonstrated different biological features. After 12 days, relatively large clones were picked using sterile cloning cylinders and expanded in type IV collagen-coated flasks with SAGM. One single-cell-clone, termed hSABCi-NS1.1, was expanded and characterized. The morphology of the basal cells in culture over time was documented by an Olympus IX71 inverted microscope with an Olympus DP73 camera (New Jersey Scientific Inc., Somerset, NJ). The cell numbers of each passage were counted by a Countess automated cell counter (Thermo-Fisher, Waltham, MA). Since passage 5 of the hSABCi-NS1.1 culture, the cells were maintained in T25 flasks with type IV collagen. The cells were switched to PneumaCult Ex Plus medium (Stemcell Technologies, Cambridge, MA) from passage 40 onward. The population doubling level and cell doubling time were calculated according to ATCC Animal Cell Culture Guide (https://www.atcc.org/). Additional quality control was performed on passage 46 hSABCi-NS1.1 cells which had been stored in the liquid nitrogen for 131 days. As a control for comparison, a new vial of passage 2 primary parental cells was thawed and cultured under the same culture conditions (PneumaCult Ex plus medium; Stemcell Technologies).
Air-liquid Interface culture
The basal cell line was seeded at a density of 1.5 × 105 cells/100 μl/well onto a Transwell insert (0.4 μm size pore; 0.3 cm2 well; Corning, Corning, NY) coated with human type IV collagen (Sigma-Aldrich, St. Louis, MO) in PneumaCult Ex Plus medium (Stemcell Technologies). The lower chamber contained 0.5 ml PneumaCult Ex Plus medium. After 1 day, media in both upper and lower chambers were replaced with fresh Ex Plus medium. Two days post seeding, the media in the lower chamber was replaced with PneumaCult-ALI maintenance media (Stemcell Technologies). The media in the upper chamber was removed to expose the apical surface to air and establish the air-liquid interface (ALI; day of medium removal referred to as ALI day 0). The ALI cultures were then grown at 37 °C, 5% CO2, with fresh PneumaCult-ALI media changes every 2 to 3 days. The apical surface was washed with 1x PBS once a week to remove accumulated mucus. Transepithelial electrical resistance (TEER) was measured with a Millicell ERS-2 Voltohmmeter (Millipore, Burlington, MA). For each time point, 3 independent wells/group were measured.
Cells were assessed in chamber slides (Thermo-Fisher) or on ALI wells, fixed in 4% paraformaldehyde diluted in PBS from a 16% aqueous stock (Electron Microscopy Science, Hatfield, PA) for 20 min and washed with PBS. Standard immunofluorescence staining methods were used [22, 23]. The primary antibodies were rat anti-SCGB1A1 (MAB4218, R&D, Minneapolis, MN); mouse anti-TP63 (sc-8431, Santa Cruz Biotechnology, Dallas, TX), mouse anti-KRT14 (C8791, Sigma-Aldrich, St. Louis, MO), mouse anti-TJP1 (33-9100, Thermo-Fisher), mouse anti-CHGA (MA5-13096, Thermo-Fisher), mouse anti-MUC5AC (VP-M657, Vector Laboratories, Burlingame, CA); rabbit anti-KRT5 (PA5-32465, Thermo-Fisher,), rabbit anti-ARL13B (17711-1-AP, Proteintech, Rosemont, IL), rabbit anti-SFTPA (HPA.42638, Sigma-Aldrich), rabbit anti-SFTPB (LS-B8080-50, Lifespan Bioscience, Seattle, WA), rabbit anti-FOXI1 (HPA071469, Sigma-Aldrich, St. Louis, MO), rabbit anti-MUC5B (HPA008246, Sigma-Aldrich, St. Louis, MO) and rabbit anti-MSMB (HPA051257, Sigma-Aldrich). The specificity of the primary antibodies has been tested on commercial human tissues (US Biomax, Rockville, MD). Secondary antibodies were Alexa 555 or 488 or 647-labeled antibodies (Thermo-Fisher). The nucleus was stained by DAPI (Thermo-Fisher). Images were captures using an Olympus IX71 inverted microscope with an Olympus DP73 camera. Exposures in fluorescence channels were merged using ImageJ (imagej.nih.gov).
Karyotype analysis of the immortalized hSABCi-NS1.1 cell at passage 50 was performed at the Molecular Cytogenetics-Core Facility at Memorial Sloan-Kettering Cancer Center using established protocols . At least 20 metaphase spreads were analyzed. All metaphases were fully karyotyped.
Real time PCR
cDNA was synthesized using TaqMan Reverse Transcriptase Reaction kit (Thermo-Fisher). All reactions were run on an Applied Biosystems Sequence Detection System 7500. Relative expression levels were determined using the △Ct method with 18S ribosomal RNA as the endogenous control. Premade TaqMan Gene Expression Assays (Thermo-Fisher) were used as listed in Additional file 1: Table S1.
Microarray and RNAseq
Trachea, large airway epithelium (LAE, 3rd -4th order bronchi) and small airway epithelium (SAE, 10th to 12th order bronchi) were collected from normal individuals via flexible bronchoscopy as described previously [13, 17, 20, 23]. Following RNA extraction and sample quality assessment, genome-wide expression assessment was performed using microarray (Human Genome U133 Plus 2.0 arrays, Affymetrix, Santa Clara, CA). The raw microarray data of the 9 matched trachea, large and small airway epithelium, and the SAE from 36 COPD smokers and 60 healthy nonsmokers have been published (GSE64614, GSE76327). The cluster analysis of cell line donor’s SAE with the 9 matched trachea, large and small airway epithelium was performed in Partek Genomics Suite (Partek, St. Louis, MO). Genes differentially expressed between the 9 paired trachea and SAE (fold changes > 2 fold, Benjamini-Hochberg corrected p < 0.05) were used as filter to generate heat map. For RNA-Seq (Illumina flow cells, San Diego, CA) , samples of passage 47 hSABCi-NS1.1 cell at basal stage (right before ALI culture) and ALI-d28 were used. The data are publicly available in the NCBI Gene Expression Omnibus (GEO accession number: GSE124265).
Lentivirus-mediated SPDEF expression
The methods for lentivirus production were as previously described [22, 23]. Lenti-SPDEF-IRES-GFP (expressing SAM Pointed Domain Containing ETS Transcription Factor) and lenti-IRES-GFP (control) plasmids were generated using the PLKO lentiviral vector backbone (Thermo Scientific). The replication deficient lentiviruses were generated in 293A cells using compatible packaging vectors. Both viruses were pseudo-typed with the VSV-G envelope and concentrated with PEG-it™ Virus Precipitation Solution (System Biosciences, Mountain View, CA) using the manufacturer’s protocol. For infection of the cells, recombinant lentiviruses were added to basal cells immediately prior to plating on Transwell inserts by combining lentiviruses with suspended basal cells at a multiplicity of infection sufficient to produce > 90% positive GFP staining. The cells and virus were deposited in the apical chamber of the transwell insert at a concentration of 1.5 × 105 cells/100 μl/well (designated ALI-day − 2; i.e., two days before removal of the apical medium). The following day, the infectious medium was removed, and the ALI culturing protocol continued as described above. The infection efficiency was confirmed by monitoring GFP positive staining.
The two-tailed Student’s t-test was used to compare gene expression in both in vivo and in vitro data. In all analyses, a p value less than 0.05 was deemed significant.
Generation of hSABCi-NS1.1
Based on our previous published sub-dataset , small airway epithelium has a different gene expression pattern than matched-tracheal and large airway epithelium from healthy nonsmokers (Fig. 1). For example, expression of SFTPB (surfactant protein), LTF (secretory cell gene) and small airway development-associated transcription factors GATA6 and SOX9 [24,25,26,27] are enriched in the small airway epithelium (Fig. 1). To ensure that the small airway epithelium recovered from the donor had typical SAE transcriptome, unsupervised clustering was carried out on the SAE transcriptome of the donor to compare with the previous small, large and trachea epithelium dataset. As expected, the microarray data of the donor clustered with the SAE samples when differential expression gene list of trachea vs small was assessed.
After retro-hTERT genetic modification, the SABC were resistant to puromycin selection (Fig. 2a). The resulting cell population was a mixed cell population termed as hSABCi-NS1. A single cell clone was isolated from hSABCi-NS1 (termed as hSABCi-NS1.1) (Fig. 2b). The heterogeneous morphology is likely because these cells were at different phases of the cell . The hSABCi-NS1.1 clone survived and was expanded for more than 1 year in vitro (Fig. 2c). The cells entered consistent growth after 200 days. The population doubling levels between passage 5 and passage 55 were ~ 200 (Fig. 2c). As additional quality control, one vial of passage 46 hSABCi-NS1.1 cells, frozen in liquid nitrogen for > 100 days, was re-cultured and compared with a vial of passage 1 parental primary cells under the same culture conditions at the same time. Even after > 110 additional population doubling levels, the re-cultured passage 46 hSABCi-NS1.1 cells had no signs of replicative senescence (Fig. 2d, Additional file 1: Figure S1). The percentage of live cells over each passage in the re-cultured cells was always > 80% (Fig. 2e). The doubling time of hSABCi-NS1.1 cells during exponential growth of passage 52 was approximately 16 h (Fig. 2f). Based on morphology, the cells remain healthy in the late passages.
Karyotyping analysis of 23 hSABCi-NS1.1 cells from passage 50 cell showed that all of the cells hade an abnormal male karyotype with trisomy 8 in all cells analyzed. Additionally, 22 (96%) cells contained abnormalities of chromosome 19 (resulting in net gain of 19q and/or 19p) and 15 (65%) cells contained trisomy 20.
Basal/stem cell markers
Consistent with the source of the hSABCi-NS1.1 cell line, immunostaining showed that the parental cells prior to immortalization expressed KRT5 (Additional file 1: Figure S2). hSABCi-NS1.1 cells were positive for basal cells markers, TP63 and KRT5, at passage 6 (Fig. 3a). The positive staining was maintained at passage 49 (Fig. 3b). TaqMan PCR confirmed that hSABCi-NS1.1 (passage 8 to passage 54) had similar mRNA expression levels of TP63 compared with primary small airway basal cells (Fig. 3c).
To assess whether the hSABCi-NS1.1 cells still retain the potential to form tight junctions, the cells were cultured on ALI. Measurement of trans-epithelial electrical resistance showed that the resistances were consistently > 200 Ohms x cm2 after ALI-day 14 (Fig. 4a). Immunofluorescence staining revealed that there was sharp and continued tight junction protein 1 (TJP1) staining between the cells on the apical side (Fig. 4b).
To assess the differentiation capacity of hSABCi-NS1.1 cells on ALI, the ALI cells were stained with markers for SAE-associated lineages (Fig. 5a-e), including ciliated (ARL13B+ motile cilia), club (SCGB1A1+ organelles), mucous (MUC5AC+ or MUC5B+ organelles), ionocytes (FOXI1+ nuclei), neuroendocrine (CHGA+ organelles), and less-defined alveolar-like cells (SFTPA+ or SFTPB+ organelles). ARL13B staining showed a characteristic apical collection of curvilinear structures while SCGB1A1, MUC5AC, MUC5B, SFTPA, SFTPB, and CHGA staining occurred in collections of intracellular puncta typical of secretory vesicles. The transcription factor, FOXI1, co-localized with nuclei consistent with its role in regulation of gene expression. Ciliated cell and club cell staining were non-overlapping while MUC5AC and MUC5B were partially overlapping as expected.
RNAseq was used to confirm the differentiation capacities of hSABCi-NS1.1 on ALI. Compared to the undifferentiated basal cell stage, ALI-day 28 hSABCi-NS1.1 had down-regulation of basal cell genes (KRT5, KRT14, TP63, KRT15), with up-regulation of genes typical of ciliated cells (FOXJ1, DNAI1, SPAG6, TEKT1), secretory cells (SCGB1A1, SCGB3A1, MUC5B, LYZ), alveolar-like cells (SFTPA1, SFTPA2, SFTPB and SFTPD), neuroendocrine cells (CHGB and SCG5), ionocyte cells (FOXI1, ATP6V1B1 and CFTR; Table 1). All of these markers have been detected in small airway epithelium observed by brushing during fiberoptic bronchoscopy using RNAseq . By functional term enrichment analysis, “cilium” and “immunity” were among the top 3 categories in the top 1500 up-regulated genes (Table 2), while “ribosomal protein” was the top 1 functional category associated with the top 1500 down-regulated genes (Table 2), consistent with the fact that basal cells contain abundant ribosomes . In addition, many Notch pathway genes showed more than 1.5-fold changes during differentiation, e.g., NOTCH1 (− 2.1-fold), NOTCH2 (2.7-fold), NOTCH4 (1.5-fold), JAG2 (− 2.9-fold), HES1 (− 6.4-fold), HES2 (− 8.6-fold), HES4 (− 1.6-fold), HES6 (3.0-fold), HEY1 (1.8-fold) and HEYL (2.9-fold). The data is consistent with the knowledge that the dynamic changes of Notch pathway are involved in the commitment of airway epithelial lineages.
Interestingly, differentiated hSABCi-NS1.1 cells expressed inflammatory cell genes CD4 (lymphocyte cell marker) and MHC-II genes (CD74, HLA-DRA; dendritic cell markers; Additional file 1: Table S2). For additional confirmation, RNAseq reads were aligned with exons of selected mRNAs. The alignment clearly demonstrated the expression of immunity genes CD4, CD74 and HLA-DRA (Fig. 6a, b and c). Since hSABCi-NS1.1 was single-cell-derived, contamination with inflammatory cells can be excluded. The findings have corroborated our previous observation from single-cell RNAseq data that some airway epithelial cells were positive for inflammatory cell genes, including CD4, CD74 and HLA-DRA .
Regional identity in the airways
To assess whether differentiation of airway cell lines have regionally enriched features, the transcriptome of hSBACi-NS1.1 cells was compared with our previously-reported large airway cell line BCi-NS1.1  using the same culture condition in basal and ALI culture. TaqMan PCR analysis demonstrated that hSABCi-NS1.1 cells had lower expression levels of basal, club, and ciliated cell genes, while significantly higher expression levels of small airway-associated genes, including SFTPA1, SFTPB, SFTPD and LTF (Fig. 7a). Consistent with this data, there was a trend of lower expression of SOX2 (proximal airway transcription factor) and significantly higher expression levels of small airway development transcription factors (NXK2–1, GATA6, SOX9, HOPX, ID2, ETV5) [24,25,26,27] (Fig. 7b).
Recapitulation of SAE-related disease
To test potential applications of the cell line in the studies of SAE diseases, hSABCi-NS1.1 cells were genetically modified by lentivirus to express SPDEF, a key transcription factor controlling secretory cell differentiation . As shown in Fig. 8a, SAE from COPD smokers demonstrated dysregulation of multiple secretory cell genes, including SPDEF. Based on GFP distribution, the infection efficiency of lentivirus in hSABCi-NS1.1 cells was high and maintained during ALI culture (not shown). As a control, SPDEF overexpression in hSABCi-NS1.1 cells on ALI induced remarkable up-regulation of MUC5AC staining (Fig. 8b). TaqMan PCR data demonstrated that upregulation of SPDEF led to increased-expression of MUC5AC, MUC2, TFF3, CEACAM5, MSMB and decreased-expression of SCGB3A1 and LTF. However, no consistent changes in SCGB1A1 or MUC5B were found (Fig. 8c), supporting our previous proposal that a transcription factor network might be involved . Immunostaining confirmed the striking up-regulation of MSMB (Fig. 8d), which has not been identified as a SPDEF-regulated gene in previous studies .
We have generated and characterized a single-cell-derived normal human small airway basal cell line (hSABCi-NS1.1) with diverse differentiation capacities and regional features. This human SAE cell line can achieve high cell doubling levels and passage numbers. The hSABCi-NS1.1 cell line provided a novel tool to study SAE biology and pathophysiology in an airway region relevant manner. The availability of hSABCi-NS1.1 cells will permit studies of diseases for which the SAE plays a central role. It also can be used to assess the effect of specific mediators on the SAE. As an example, we found transcription factor SPDEF can induce human COPD-relevant changes in hSABCi-NS1.1 cells. Also relevant to small airway biology, hSABCi-NS1.1 can differentiate to ionocytes, a recently identified airway cell type [30, 31], surfactant protein positive cells [13, 17, 20, 32] and neuroendocrine cells . Basal cells are likely the common progenitor for all these cell types, and hSABCi-NS1.1 cells will be a convenient tool to study these cell populations.
The hSABCi-NS1.1 cell line demonstrates regional (small airway epithelium) identity. Along the proximal to distal axis, the airway epithelium has different morphological features [4, 7, 20, 24,25,26,27]. As examples, compared to the tracheal epithelium, small airway epithelium has a thinner epithelial layer, is composed of basal, secretory and ciliated cells in different ratios, and has surfactant protein positive cells [13, 14, 32]. These airway regional features are apparent in the airway epithelium transcriptome, including SAE-specific surfactant protein genes and SAE enrichment of distal airway patterning-associated transcription factors NKX2–1, GATA6, SOX9 and ETV5 [24,25,26,27].
The regional identity is likely directly involved in the pathogenesis of SAE-related diseases. For example, SAE express surfactant protein genes (SFTPB, SFTPA2), genetic variation of which are associated with COPD and IPF [33, 34]. SAE-enriched transcription factor, NKX2–1, is relevant to lung adenocarcinoma , a cancer type that is derived from the small airway epithelium. The expression of MUC5B, an IPF risk gene , is enriched in SAE . GATA6 is involved in regulating MUC5AC gene expression  and can regulate an IPF-associated mesenchymal phenotype . LTF plays important anti-infection roles in CF .
In vitro differentiation models of airway epithelial cells have been widely used in studies for airway development, carcinogenesis, environmental insults and infection by pathogens [41, 42]. Importantly, since the SAE plays a central role in many chronic lung disorders, hSABCi-NS1.1 cells should be a valuable tool for drug screening. In this context, hSABCi-NS1.1 cells are available for other investigators by contacting the senior author.
Availability of data and materials
The data are publically available in the NCBI Gene Expression Omnibus (GEO accession number: GSE124265).
Chronic obstructive pulmonary disease
Idiopathic pulmonary fibrosis
Small airway basal cell
Small airway epithelium
Small airway epithelial cell growth medium
Transepithelial electrical resistance
Telomerase reverse transcriptase
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We thank N. Mohamed for help in preparing this manuscript.
Role of funder
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
These studies were supported, in part, by HL107882, HL107882-S1, HL113443 and HL118541; and Boehringer Ingelheim Pharmaceuticals Inc. Research reported in this publication was supported by NIH and the Family Smoking Prevention and Tobacco Control Act. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Food and Drug Administration.
Ethics approval and consent to participate
Weill Cornell Medicine (WCM) Institutional Review Board approved the protocols. Research subjects were evaluated and consented at the Weill Cornell Medical College Clinical Translational and Science Center and the Department of Genetic Medicine Clinical Research Facility.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Table S1. List of primers for TaqMan assays. Table S2. List of immunity-related genes among the top 1500 up-regulated genes expressed by hSABCi-NS1.1 on air-liquid interface. Figure S1. Morphology of hSABCi-NS1.1 at late passage. Figure S2. Basal cell marker KRT5 staining in the parental cell prior to immortalization. (PDF 641 kb)