Novel human bronchial epithelial cell lines for cystic fibrosis research

Abstract

Immortalization of human bronchial epithelial (hBE) cells often entails loss of differentiation. Bmi-1 is a protooncogene that maintains stem cells, and its expression creates cell lines that recapitulate normal cell structure and function. We introduced Bmi-1 and the catalytic subunit of telomerase (hTERT) into three non-cystic fibrosis (CF) and three DeltaF508 homozygous CF primary bronchial cell preparations. This treatment extended cell life span, although not as profoundly as viral oncogenes, and at passages 14 and 15, the new cell lines had a diploid karyotype. Ussing chamber analysis revealed variable transepithelial resistances, ranging from 200 to 1,200 Omega.cm(2). In the non-CF cell lines, short-circuit currents were stimulated by forskolin and inhibited by CFTR(inh)-172 at levels mostly comparable to early passage primary cells. CF cell lines exhibited no forskolin-stimulated current and minimal CFTR(inh)-172 response. Amiloride-inhibitable and UTP-stimulated currents were present, but at lower and higher amplitudes than in primary cells, respectively. The cells exhibited a pseudostratified morphology, with prominent apical membrane polarization, few apoptotic bodies, numerous mucous secretory cells, and occasional ciliated cells. CF and non-CF cell lines produced similar levels of IL-8 at baseline and equally increased IL-8 secretion in response to IL-1beta, TNF-alpha, and the Toll-like receptor 2 agonist Pam3Cys. Although they have lower growth potential and more fastidious growth requirements than viral oncogene transformed cells, Bmi-1/hTERT airway epithelial cell lines will be useful for several avenues of investigation and will help fill gaps currently hindering CF research and therapeutic development.

Fig. 1.

Decreased Bmi-1 and elevated p16^Ink4a protein content precedes senescence in primary human bronchial epithelial (hBE) cells. A: growth curve from a representative hBE preparation. B: corresponding Western blots for Bmi-1, p16^Ink4a, and actin. The actin blot represents reprobing of the p16^Ink4a blot. Similar results were obtained in 2 additional specimens.

Fig. 2.

Bmi-1 plus human telomerase reverse transcriptase (hTERT) extends the growth capacity of hBE cells. Growth curves of UNCN1T, UNCN2T, UNCN3T, UNCCF1T, UNCCF2T, and UNCCF3T cell lines on plastic were recorded in sham-infected cells (•) or cells infected with green fluorescent protein (GFP) lentivirus (⧫) or the combination of Bmi-1 and hTERT lentivirus (▪) as described in materials and methods. The addition of Bmi-1 plus hTERT enabled at least 38 population doublings, whereas all controls senesced before a maximum of 25 doublings. All Bmi-1 plus hTERT cell lines have grown for at least 30 passages.

Fig. 3.

Ussing chamber studies of parental primary cells and each of the novel cell lines. Representative Ussing chamber traces and changes in short-circuit current (I_sc) are shown in response to amiloride, forskolin, CFTR(inh)-172, and UTP in passage 2 (P2) primary cells of all 6 parental cell lines and their Bmi-1 plus hTERT growth-extended derivatives at P14–P16. Each data point represents the mean ± SD of between 3 and 14 individual 12-mm Snapwell inserts per cell type, cultured for 18–24 days. CF, cystic fibrosis.

Fig. 4.

CFTR protein detection in Bmi-1 plus hTERT cell lines. Immunoprecipitation (IP)-Western blot analysis was performed using lysates of 12 × 12-mm Millicell CM inserts per lane, beads coupled to monoclonal antibody (MAb) 596, and probing of the Western blot with MAb 596. The strongest putative CFTR band C signals originated from UNCN1T and UNCN3T cells, the same 2 cell lines with the greatest forskolin-stimulated and CFTR(inh)-172-inhibitable currents in Ussing chambers. We assume that the bands marked with asterisks are nonspecific and are due to the necessarily high levels of input protein.

Fig. 5.

Bmi-1 plus hTERT cell lines at P14–P16 exhibit a pseudostratified morphology. Representative photomicrographs are shown of histology sections from day 28 air-liquid interface (ALI) cell cultures of all 6 novel Bmi-1/hTERT growth-enhanced hBE cell lines. Note the sparse ciliated cells and abundant mucous secretory cells, routine formalin-fixed paraffin sections, hematoxylin and eosin (H&E) and alcian blue-periodic acid-Schiff (ABPAS) stain (original magnification, ×400). Sections are representative of at least 3 individual cultures for each cell line.

Fig. 6.

No systematic differences in IL-8 production between non-CF and CF cell lines. IL-8 content in apical washings and basolateral conditioned media was measured 24 h after IL-1β, TNF-α, or Pam3Cys (Toll-like receptor 2 ligand) challenge of well-differentiated ALI cultures of all 6 Bmi-1/hTERT novel hBE cell lines. Results are expressed as the total amount of IL-8 secreted into the apical and basolateral culture compartments over a 24-h period and are means ± SE of quadruplicate wells.