Novel immortalized human vocal fold epithelial cell line: In vitro tool for mucosal biology

Abstract

Study of vocal fold (VF) mucosal biology requires essential human vocal fold epithelial cell (hVFE) lines for use in appropriate model systems. We steadily transfected a retroviral construct containing human telomerase reverse transcriptase (hTERT) into primary normal hVFE to establish a continuously replicating hVFE cell line. Immortalized hVFE across passages have cobblestone morphology, express epithelial markers cytokeratin 4, 13 and 14, induced hTERT gene and protein expression, have similar RNAseq profiling, and can continuously grow for more than 8 months. DNA fingerprinting and karyotype analysis demonstrated that immortalized hVFE were consistent with the presence of a single cell line. Validation of the hVFE, in a three-dimensional in vitro VF mucosal construct revealed a multilayered epithelial structure with VF epithelial cell markers. Wound scratch assay revealed higher migration capability of the immortalized hVFE on the surface of collagen-fibronectin and collagen gel containing human vocal fold fibroblasts (hVFF). Collectively, our report demonstrates the first immortalized hVFE from true VFs providing a novel and invaluable tool for the study of epithelial cell-fibroblast interactions that dictate disease and health of this specialized tissue.

Keywords: human vocal fold epithelial cells; immortalization; telomerase; three-dimensional VF mucosal model.

FIGURE 1

Morphology of primary hVFE from VF tissue samples, and their purification process under phase microscope. At Day 2, hVFE started growth from the edge of tissue, at Day 8 hVFF (white arrows) started mixed growth with hVFE (black arrows), at Day14 all hVFF and hVFE mixed growth together. After passing (P1, P2, and P3) with 0.05% trypsin, the mixed hVFF became less and less, hVFE population reached over 95% and demonstrated the typical “cobblestone” morphology. Scale bar: 100 µm

FIGURE 2

Identification of hVFE from hVFF. Immunocytochemistry revealed epithelial cell markers, cytokeratin 4 (CK4), cytokeratin 13 (CK13), and cytokeratin 14 (CK14), were positive in hVFE (A‐C) and fibronectin (FN) and collagen I (Col1) were negative in hVFE (D and E). hVFF showed positive staining for FN (I) and Col1 (J), negative for CK4 (F), CK13 (G), and CK14 (H). Nuclei were counted‐stained with DAPI. Scale bar: 50 µm

FIGURE 3

Process of hVFE immortalization. Primary hVFE (A) were transduced by retrovirus containing hTERT gene. Because large amounts of virus were applied, a significant number of cells underwent apoptosis (B). After 2‐week G418 treatment (C), only a few cells survived with the transfection efficiency being less than 3%. After 5‐week continued culture (D), cells that survived formed colonies. E‐H shows control hVFE transfected by empty vector without hTERT gene. After 2‐week G418 treatment (G) and 5‐week additional culture (H), there were no living cells

FIGURE 4

Immortalization of hVFE was identified and confirmed by standard PCR, PCR ELISA, and immunocytochemistry. A, PCR was performed following RNA extraction from immortalized hVFE at four different passages using the primers specific to hTERT gene and product was resolved on 1.5% agarose gel for confirming hTERT gene in immortalized hVFE. Strong hTERT gene expression (501bp band) was observed in immortalized hVFE at passages 1, 7, 14, and 21 (P1, P7, P14, and P21). B, telomerase activity of immortalized hVFE was measured by PCR ELISA. Similar level of telomerase activity was observed in immortalized hVFE at passages 3, 7, 14s and 21 (P3, P7, P14, and P21). C, cellular location of induced hTERT was identified by immunocytochemistry. Immortalized cells were labeled by epithelial cell marker, cytokeratin 14 (green), and hTERT (red) and imaged by confocal microscope demonstrating hTERT was positive in the nucleus of the epithelial cells. Scale bar: 50 µm

FIGURE 5

Characterization of immortalized hVFE. Immortalized (A) and primary cultured hVFE (B) showed similar cobblestone morphology feature of epithelial cells; Immortalized cells were positively labeled by hTERT (red, D), comparing to primary hVFE (hTERT negative, G). DAPI (blue) was cell nuclei‐counter stained (C, F), merged images of DAPI and hTERT in immortalized (E) and primary hVFE (H). Stratified epithelial cell markers, cytokeratin 4 (CK4), 13 (CK13), and 14 (CK14), were positively expressed in immortalized hVFE (I, J, K) and primary hVFE (L, M, N). DAPI (blue) was cell nuclei‐counter stained. Scale bar: 50 µm; (O) Cell proliferation rates of immortalized and primary hVFE were measured by MTT assay. During 7‐day period, two primary hVFE cells at passages 2‐3 (♦ and ■) showed rather flat proliferation curves, while immortalized hVFE at passage 3 (▲) showed significant higher cell numbers at Day4 and Day7 compared with primary cells (P = .0009 and P = .0002, respectively)

FIGURE 6

Cytogenetic analysis of immortalized hVFE cell line (passage 9) show an abnormal female karyotype with trisomy 7, trisomy 20, loss of one copy of chromosome 11, and gain of two marker chromosomes of unknown origin

FIGURE 7

Characterization of immortalized hVFE in 3D VF mucosa construct. A, Schematic of VF mucosa construct by co‐culturing of immortalized hVFE and primary hVFF. B, H&E staining shows morphology of the VF mucosa construct developed with immortalized hVFE. C, Anti‐E‐cadherin staining shows E‐cadherin expressed in all layers of immortalized hVFE. D, Anti‐Laminin alfa 5 staining demonstrates lack of basement membrane found in developed VF mucosa construct. E‐G, IHC staining for stratified epithelial markers, CK4 (E), CK13 (F), and CK14 (G). CK4 and CK14 were expressed in all layers of hVFE, CK13 expressed in super‐basal layers of epithelium. DAPI (blue) was cell nuclei‐count stain. Scale bar: 200 µm

FIGURE 8

Cell migration evaluation of immortalized hVFE from the surface of collagen gel, collagen gel with primary hVFF, and collagen‐fibronectin coated plate. A, Immortalized hVFE were grown on the surface of the collage gel (4 mg/mL collagen I, Col‐Gel), collagen gel containing hVFF (Col‐Gel with hVFF), and collagen‐fibronectin coated plate (30 µg/mL collagen I and 10 µg/mL fibronectin, Col‐FN) until confluent. A cell‐free area was introduced with a pipette tip, and migration was evaluated after 24 hours. Representative examples of cells at Day 0, 1, 3, 5, and 7 from four independent experiments are shown. Scale bar: 100 µm. B, Quantification of the results of four separated scratch wound assays using ImageJ. On Col‐FN surface, cells began to infiltrate the wound area by Day 1 and completely covered the wound by Day 5, compared to cells on Col‐Gel that had 20%‐30% of the area covered over the same 7‐day period. On collagen gel with fibroblasts, by Day 3 epithelial cell migration covered the 40% of the wound and by Day 5‐7 covered 60% of the wound. Asterisks (*) indicate a significant difference (P < .05) in scratch wound healing compared to the different substrate at same time point