Generation of a human airway epithelium derived basal cell line with multipotent differentiation capacity

Abstract

Background: As the multipotent progenitor population of the airway epithelium, human airway basal cells (BC) replenish the specialized differentiated cell populations of the mucociliated airway epithelium during physiological turnover and repair. Cultured primary BC divide a limited number of times before entering a state of replicative senescence, preventing the establishment of long-term replicating cultures of airway BC that maintain their original phenotype.

Methods: To generate an immortalized human airway BC cell line, primary human airway BC obtained by brushing the airway epithelium of healthy nonsmokers were infected with a retrovirus expressing human telomerase (hTERT). The resulting immortalized cell line was then characterized under non-differentiating and differentiating air-liquid interface (ALI) culture conditions using ELISA, TaqMan quantitative PCR, Western analysis, and immunofluorescent and immunohistochemical staining analysis for cell type specific markers. In addition, the ability of the cell line to respond to environmental stimuli under differentiating ALI culture was assessed.

Results: We successfully generated an immortalized human airway BC cell line termed BCi-NS1 via expression of hTERT. A single cell derived clone from the parental BCi-NS1 cells, BCi-NS1.1, retains characteristics of the original primary cells for over 40 passages and demonstrates a multipotent differentiation capacity into secretory (MUC5AC, MUC5B), goblet (TFF3), Clara (CC10) and ciliated (DNAI1, FOXJ1) cells on ALI culture. The cells can respond to external stimuli such as IL-13, resulting in alteration of the normal differentiation process.

Conclusion: Development of immortalized human airway BC that retain multipotent differentiation capacity over long-term culture should be useful in understanding the biology of BC, the response of BC to environmental stress, and as a target for assessment of pharmacologic agents.

Figure 1

Isolation and generation of the immortalized human airway basal cell line BCi-NS1. A. Morphology of the parental primary basal cells at passage 1. B. Morphology of the primary basal cells following senescence at passage 5 of culture. C. Morphology of immortalized basal cells (BCi-NS1) following passage 13 of culture. D. Telomerase activity of immortalized basal cells (TRAP assay). Lane 1 – Parental primary basal cells; lane 2 – immortalized basal cells BCi-NS1; lane 3 – positive control (Pos; telomerase positive HeLa cell extract); lane 4 – negative control (Neg; heat-inactivated telomerase positive HeLa cell extract). E. Characterization of immortalized basal cells by Western analysis of cell type specific markers. Lane 1 – Primary basal cells; lane 2 – BCi-NS1 immortalized basal cells; and lane 3 – bronchial brushing. For all cells, shown is the expression of basal cells markers (KRT5, TP63), secretory cell marker (MUC5AC), Clara cell marker (CC10) and ciliated cell marker (DNAI1). GAPDH was used as a loading control.

Figure 2

Isolation of BCi-NS1.1 clone of the BCi-NS1 immortalized human airway basal cell line. A. Morphology of BCi-NS1.1 clone at passage 18 and passage 44. B. Comparison of the parental and clonal immortalized basal cells by Western analysis of cell type specific markers. Lane 1 – Primary basal cells; lane 2 – BCi-NS1 parental immortalized basal cells, passage 24; and lane 3 – BCi-NS1.1 clonal immortalized basal cells, passage 3. For all cells, shown is the expression of the basal cell markers KRT5 and TP63. GAPDH was used as a loading control. C. Immunohistochemical characterization of cytopreps of BCi-NS1.1 cells (passage 22) with cell-type specific markers: KRT5 (basal cell); TP63 (basal cell); CD151 (basal cell); β-tubulin IV (ciliated cell); MUC5AC (secretory cell); TFF3 (goblet cell); CC10 (Clara cell); chromogranin A (neuroendocrine cell); N-cadherin (mesenchymal cell) and isotype control. Bar = 20 μm.

Figure 3

Secretion of VEGFA by immortalized BCi-NS1.1 cells. A. VEGFA levels assessed by ELISA in growth media from primary airway basal cell cultures (n = 4) and immortalized BCi-NS1.1 cell cultures (n = 4). Secreted VEGFA was normalized to cell number and calculated as pg/cell/ml. Data shown is the average ± the standard error of n = 4 independent cultures of primary BC and n = 4 independent passages of BC-NS1.1 cells from passage 10 to 14. Statistics were calculated by a 2-tailed Student’s t test. B. Endothelial cells support the growth of primary airway basal cells and immortalized BCi-NS1.1 cells in the absence of growth factors. Human primary BC and immortalized BCi-NS1.1 cells were co-cultured with Akt-activated human umbilical vein endothelial cells (HUVEC-Akt) in cytokine- and serum-free conditions. At the desired time points, cells were harvested and the GFP-labeled HUVEC-Akt cells was determined as the GFP⁺VE-cadherin⁺ population by flow cytometric analysis, and the GFP^-VE-cadherin^- population quantified as expanded basal cells. Data shown is the average ± the standard error of n = 3 independent cultures of primary BC and n = 3 independent passages of BC-NS1.1 cells from passage 5 to 26 compared directly in tandem for each independent experiment. Statistics were calculated by a 2-tailed Student’s t test.

Figure 4

Tight junction formation of immortalized BCi-NS1.1 cells during air-liquid interface culture. A. Transepithelial electric resistance (TER) of BCi-NS1.1 cells cultured on air-liquid interface (ALI). Resistance (Ohms x cm²) was measured at every media change. Data shown is the average TER ± the standard error of n = 8 independent experiments from passage 6–30 cells between day 10 to 40 of ALI culture. B. Expression of tight junction-related genes of BCi-NS1.1 cells. At ALI day 0 and day 28, TaqMan quantitative PCR analysis was used to assess expression of the tight junction related genes claudin 3 (CLDN3) and claudin 8 (CLDN8). Data shown is the average ± the standard error of n = 10 independent experiments for BC-NS1.1 cells from passage 6 to 43. Statistics were calculated by a 2-tailed Student’s t test.

Figure 5

Differentiation capacity of immortalized BCi-NS1.1 cells over long-term culture. A. Morphology of ALI day 28 of BCi-NS1.1 cells, passage 30 demonstrating a multilayer ciliated epithelium, hematoxylin and eosin staining B. Immunofluorescent staining of ALI day 28 of BCi-NS1.1 cells, passage 30, for KRT5 (basal cell, red), β-tubulin IV (ciliated cell, green) and nuclei (blue). A, B. Bar = 20 μm. C. Quantification of differentiation of ALI day 28 cross-sections of BCi-NS1.1 cells. The number of positive ciliated and secretory cells was scored for BCi-NS1.1 cells for n = 10 independent experiments from cells between passage 6 to 43. The data is presented for each cell type as percentage relative to the total number of cells. In addition, the average ± the standard error of n = 10 independent experiments is shown. D. Comparison of the differentiation capacity of BCi-NS1 cells to that of primary airway BC. Primary BC were cultured under identical differentiation-inducing conditions on ALI to that of BCi-NS1.1 cells and quantified in an identical manner. The number of positive ciliated and secretory cells was scored for primary BC and presented for each cell type as percentage relative to the total number of cells. The average ± the standard error of n = 5 independent experiments is shown and compared to the average data for BCi-NS1.1 described in panel C. Statistics were calculated by a 2-tailed Student’s t test.

Figure 6

Comparison of the differentiation capacity of immortalized BCi-NS1 cells to that of primary basal cells. Primary airway BC and immortalized BCi-NS1 cells were cultured under differentiation inducing conditions on air-liquid interface (ALI). A, B. mRNA transcripts; A. Primary BC. B. BCi-NS1.1 cells. At ALI day 0 and day 28, TaqMan quantitative PCR analysis was used to assess cell type specific mRNA markers: KRT5 (basal cell); TP63 (basal cell); MUC5AC (secretory cell); MUC5B (secretory cell); TFF3 (goblet cell); CC10 (Clara cell); DNAI1 (ciliated cell) and FOXJ1 (ciliated cell). Data shown is the average ± the standard error of n = 5 independent experiments for primary BC and n = 10 independent experiments for BC-NS1.1 cells. Statistics were calculated by a 2-tailed Student’s t test. Asterisk (*) indicates not detected. For B, the data represents an average ± the standard error for BCi-NS1.1 cells from passage 6 to 43. C. Characterization of differentiation by Western analysis of cell type specific markers. Lane 1 – Primary BC, ALI day 0; lane 2 – Primary BC, ALI day 28; lane 3 – BCi-NS1.1, ALI day 0; and lane 4 – BCi-NS1.1, ALI day 28. Shown is the expression of a basal cell marker (TP63); Clara cell marker (CC10); and ciliated cell markers (DNAI1 and FOXJ1). GAPDH was used as a loading control. D,E,F. Immunofluorescent staining of ALI day 28 primary BC and BCi-NS1.1 cells for D. CC10 (Clara cell, red), E. MUC5AC (secretory cell, green) or F. TFF3 (goblet cell, red) and nuclei (blue). Bar = 20 μm. For C-F all data shown is representative of n = 3 independent experiments.

Figure 7

IL-13 modulation of differentiation of BCi-NS1.1 immortalized airway basal cells. BCi-NS1.1 cells were cultured under differentiation inducing conditions on air-liquid interface (ALI) in the absence and presence of IL-13. A. Transepithelial electric resistance (TER) of the airway epithelia in the absence and presence of IL-13. The resistance (Ohms x cm²) was measured at ALI day 28. Data is presented as the percentage resistance relative to untreated cells and plotted as the average ± the standard error of n = 5 independent experiments (BCi-NS1.1, passage 6–15). B. Morphology of untreated and IL-13 treated cells. Alcian blue staining of ALI day 28 sections of BCi-NS1.1 cells untreated or treated with IL-13. Bar = 20 μm. C. Quantification of differentiation. Shown is Alcian blue staining of ALI day 28 sections of BCi-NS1.1 cells untreated or treated with IL-13. The number of positive ciliated and secretory cells scored. Data is presented for each cell type as percentage relative to the total number of cells. Data shown is the average ± the standard error of n = 5 independent experiments (BCi-NS1.1, passage 6–15). All statistics were calculated by a 2-tailed Student’s t test.

Figure 8

Quantification of IL-13 modulation of the differentiation of BCi-NS1.1 immortalized airway basal cells. BCi-NS1.1 cells were cultured on air-liquid interface (ALI) in the absence and presence of IL-13. A. TaqMan analysis of cell type specific mRNA markers at ALI day 28, including basal cell markers (KRT5, TP63); secretory cell markers (MUC5AC, MUC5B); goblet cell marker (TFF3); Clara cell marker (CC10); and ciliated cell markers (DNAI1, FOXJ1). Data shown is the average ± the standard error of n = 5 independent experiments (BCi-NS1.1, passage 6–15). Statistics were calculated by 2-tailed Student’s t test. U = untreated; IL-13 = treated with IL-13. B. Western analysis of untreated and IL-13 treated cells of cell type specific markers at ALI day 28. Lane 1 – untreated, lane 2 – IL-13 treated. Shown is the expression of a basal cell marker (TP63); Clara cell marker (CC10) and ciliated cell markers (DNAI1, FOXJ1). GAPDH was used as a loading control. C. Immunofluorescent staining of ALI day 28 membranes for untreated and IL-13 treated cells for MUC5AC (secretory cell, green) and nuclei (blue). Bar = 20 μm. For B and C, all data shown is representative of n = 3 independent experiments.

Figure 9

Wound healing capacity of immortalized BCi-NS1.1 cells. BCi-NS1.1 cells were cultured on air-liquid interface (ALI) culture and the ability of the cells to repair injury to the epithelium assessed at ALI day 0, 7, 14, 21 and 28. Images were obtained at the time of injury (t = 0) and 20 hr later (t = 20) once repair had taken place. All data shown is representative of n = 5 independent experiments using BCi-NS1.1 cells between passage 7 to 44.