BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity

Abstract

Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.

Keywords: air-liquid interface; airway model; lung; mucociliary differentiation; primary ciliary dyskinesia.

Fig. 1.

BMI-1 maintains healthy cell morphology in 2D culture. The morphology of NHBE cells at passage 1 (A) and passage 3 (B) was observed under light microscopy and compared with NHBE BMI-1 cells after passages 11 (C), 17 (D), and 25 (E). White arrows highlight elongated cells and black arrows highlight squamous cells. Scale bars are 100 µm.

Fig. 2.

Karyotype analysis of NHBE-BMI-1 cells. Karyotype of passage 23 NHBE-BMI-1 cells was undertaken by The Doctors Laboratory, London.

Fig. 3.

Elevated p16^Ink4a precedes senescence and BMI-1 functions by inhibiting p16^Ink4a and retains a normal cell doubling time. Western blot was used to assess levels of BMI-1 and p16^Ink4A in serially passaged NHBE cells and BMI-1-transduced cells (A), and cell counting was used to determine the replication kinetics of NHBE and NHBE BMI-1 cells at varying passages (B). Growth curves are presented as percent of mean of day 1 cell count. Data are means ± SE. For each data point n = 3 biological replicates.

Fig. 4.

BMI-1 cells retain their mucociliary differentiation capacity. Extensively passaged BMI-1-transduced cells (passage 15) were differentiated on ALI and cilia beat frequency of NHBE (A) and CFBE (B) cells was determined using ciliaFA plugin (25) for ImageJ. Data are means ± SE; n = 4 independent ALI cultures, 5 fields videoed per culture. Immunostaining of NHBE-BMI-1 cells was used to show tight junction formation (occludin; C), mucin production (MUC5AC and MUC5B; D and E, respectively), the presence of basal cells (p63+; F), widespread BMI-1 expression (BMI-1; G), and extensive ciliation (acetylated α-tubulin; H). TEM was used to determine cilia ultrastructure (I). Images are representative of 4 independent ALI cultures per marker. Scale bars for C–H are 50 µm and 100 nm for I.

Fig. 5.

BMI-1 cells form ALI cultures suitable for Ussing chamber studies. Representative Ussing chamber traces and changes in short-circuit current (I_sc) in response to administration of amiloride (apical), forskolin (apical and basolateral), and GlyH-101 (apical) in primary and BMI-1-transduced NHBE (A and B) and CFBE (C and D) cells are shown. Data are means ± SE; n = at least 3 independent ALI cultures (see Table 4 for exact values).

Fig. 6.

DNAH5 knockdown recapitulates PCD phenotype. A: qRT-PCR was used to assess DNAH5 mRNA expression in NHBE-BMI-1 cells and NHBE BMI-1-transduced with lentivirus expressing either a scrambled or DNAH5-targeting shRNA and grown in submerged 2D culture. **P < 0.01; 1-way ANOVA with Bonferroni’s posttest used to assess significance. Data are means ± SE. B: immunostaining for DNAH5 and acetylated α-tubulin was used to assess the presence or absence of DNAH5 in the ciliary axoneme of shRNA-transduced and untransduced NHBE BMI-1 cells differentiated at ALI. Presence of GFP fluorescence denotes cells transduced with the GFP-shRNA construct and so expressing the shRNA. Scale bars are 20 µm. Images are representative of 4 independent ALI cultures per condition.