Establishment and characterization of novel epithelial-like cell lines derived from human periodontal ligament tissue in vitro

Abstract

In this study, novel human-derived epithelial-like cells (hEPLCs) lines were established from periodontal ligament (PDL) tissues, which were composed of a variety of cell types and exhibited complex cellular activities. To elucidate the putative features distinguishing these from epithelial rest of Malassez (ERM), we characterized hEPLCs based on cell lineage markers and tight junction protein expression. The aim of this study was, therefore, to establish and characterize hEPLCs lines from PDL tissues. The hEPLCs were isolated from PDL of third molar teeth. Cellular morphology and cell organelles were observed thoroughly. The characteristics of epithelial-endothelial-mesenchymal-like cells were compared in several markers by gene expression and immunofluorescence, to ERM and human umbilical-vein endothelial cells (HUVECs). The resistance between cellular junctions was assessed by transepithelial electron resistance, and inflammatory cytokines were detected by ELISA after infecting hEPLCs with periodontopathic bacteria. The hEPLCs developed into small epithelial-like cells in pavement appearance similar to ERM. However, gene expression patterns and immunofluorescence results were different from ERM and HUVECs, especially in tight junction markers (Claudin, ZO-1, and Occludins), and endothelial markers (vWF, CD34). The transepithelial electron resistance indicated higher resistance in hEPLCs, as compared to ERM. Periodontopathic bacteria were phagocytosed with upregulation of inflammatory cytokine secretion within 24 h. In conclusion, hEPLCs that were derived using the single cell isolation method formed tight multilayers colonies, as well as strongly expressed tight junction markers in gene expression and immunofluorescence. Novel hEPLCs lines exhibited differently from ERM, which might provide some specific functions such as metabolic exchange and defense mechanism against bacterial invasion in periodontal tissue.

Keywords: EMT; Epithelial rests of Malassez differentiation; Epithelial-like cells; Establishment cell lines; Periodontal ligament tissue.

Fig. 1

Morphologic characterizations of a human-derived epithelial-like cells (hEPLCs), b epithelial rests of Malassez (ERM), and c umbilical-vein endothelial cells (HUVECs) in phase-contrast microscope. hEPLCs and ERM appearances demonstrated highly orientated in pavement cellular arrangement with evenly small whereas HUVECs had a cobblestone appearance with large dark nuclei. Scale bar 50 µm

Fig. 2

Reverse transcriptase PCR (RT-PCR) were used to identify mRNA expression of TJ genes; Claudin-1, Claudin-2, Claudin-3, Zonula Occludens 1 (ZO-1), and Occludins (OCLN) among hEPLCs. HUVECs and ERM were used for comparison in lineage specifications. GAPDH was used as an endogenous control

Fig. 3

Immunofluorescence studies indicated the expression of epithelial cell markers; cytokeratin-14 (CK14), mesenchymal markers; mitochondria and vimentin in hEPLCs, but negatively expressed in amelogenin and ameloblastin. Scale bar 50 µm

Fig. 4

Immunofluorescence studies indicated the strong expression of the TJ proteins Claudin-1, Zonula Occludens 1 (ZO-1), and Occludins (OCLN) markers. The hEPLCs and ERM were negative for detection of endothelial cell marker; von Willebrand factor (vWF). Scale bar 50 µm

Fig. 5

Transepithelial electrical resistance was significantly higher, indicating that hEPLCs have higher resistance than epithelial rests of Malassez (ERM) at all FBS concentrations (a, *P < 0.05, n = 5). b Representative micrographs of transmission electron microscopy of hEPLCs organelles were depicted as follows; phagosomes (p) and lysosomes (l) were often observed in the cytoplasm. c Interdigitations with microvilli caused clusters to form within the intercellular space (asterisks). Fat droplets (f) and glycogen granules (g) were abundant. d Illustration demonstrates the long TJ (tj) contact between intercellular junctions. Tonofilaments (tf) were relatively well developed in the cytoplasms. Coated vesicles (arrowheads) were often observed. e Intermediate junctions (ij) and gap junctions (gj) are shown. Scale bar 500 nm

Fig. 6

Representative transmission electron microscopy micrographs showing bacterial phagocytosis at 2 h (a), 4 h (b), and 24 h (c). The concentration of inflammatory cytokines, including PGE2, IL-8, gradually increased in a time-dependent manner except in TNF-α (d) (*P < 0.05, n = 5). Scale bar 500 nm

Fig. 7

Distribution of the chromosome numbers in a representative hEPLCs line demonstrating a normal karyotype with diploid sets of chromosomes (2n = 46, XX)