Establishment and characterization of a telomerase immortalized human gingival epithelial cell line

Abstract

Background and Objective Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase-immortalized human gingival epithelial cell line and compare its in vitro behavior to that of human GECs. Material and Methods Human primary gingival epithelial cells were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, Telomerase Immortalized Gingival Keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors (TLRs) and invasion by P. gingivalis. Results TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for TLRs 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild type Porphyromonas gingivalis and an invasion defective ΔserB mutant. Conclusion Results confirm bmi1/hTERT-immortalization of primary gingival epithelial cells generated a robust cell-line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells.

Porphyromonas gingivalis-Induced Reactive Oxygen Species Activate JAK2 and Regulate Production of Inflammatory Cytokines through c-Jun

“…Wild-type (WT) P. gingivalis ATCC 33277 and the isogenic ⌬fimA mutant (33) were cultured anaerobically in Trypticase soy broth supplemented with yeast extract (1 mg ml Ϫ1 ), hemin (5 g ml Ϫ1 ), and menadione (1 g ml Ϫ1 ) at 37°C. Telomerase-immortalized gingival epithelial cells (TIGKs) (41) were maintained in supplemented keratinocyte-SFM (Invitrogen) (19). TIGKs were challenged with P. gingivalis at a multiplicity of infection (MOI) of 10.…”

Suppression of T-Cell Chemokines by Porphyromonas gingivalis

“…Both species were grown anaerobically at 37°C. Human telomerase immortalized gingival keratinocytes (TIGKs) (33), derived from the gingival epithelium, were routinely cultured at 37°C and 5% CO 2 in keratinocyte serum-free medium (Invitrogen) supplemented with 0.4 mM calcium chloride, 25 g/ml bovine pituitary extract (BPE), and 0.2 ng/ml epidermal growth factor (EGF) (TIGKM). Human neutrophils and peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors using plasma-Percoll gradients as previously described (34) and approved by the Institutional Review Board of the University of Louisville.…”

Noncanonical Activation of β-Catenin by Porphyromonas gingivalis

“…All bacteria were cultured anaerobically at 37°C. Telomerase immortalized gingival epithelial keratinocytes (TIGKs) derived from a primary gingival epithelial cell line were maintained in DermaLife keratinocyte medium with supplements (Lifeline Cell Technology, Carlsbad, CA) as described previously (36). Cells between passages 10 and 20 were cultured to 80% confluence and infected with P. gingivalis under tissue culture conditions (5% CO 2 , 37°C).…”