Establishment of immortalized clonal cells derived from periodontal ligament cells

Objectives: The periodontal ligament (PDL) is considered to contain heterogeneous cell populations that compose the connective tissues. However, it is difficult to identify what kinds of cells that make up those tissues, as well as whether they function alone or interact with other cell types, because the PDL cells used in previous studies were a mixture of heterogeneous populations. Further, it is difficult to isolate and expand single cell clones from PDL cells, as normal cells have a limited life span and are phenotypically unstable. The purpose of this study is to immortalize the PDL and to characterize the immortalized clonal cells. Methods: We extracted human periodontal ligament cells (HPL) were isolated from the human PDL tissues. The gene for hTERT was cloned into the pMSCVpuro retroviral vector from Clontechwith the EcoR± restriction enzyme. The cloned retrovirus vector was transfected into the RetroPack PT67, packaging Cell Line, where the vector was packaged into infectious, replication-incompetent retroviral particles. Telomerase activity was determined by polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP assay). Single cell clones were obtained using a limited dilution method. The detected cell clones by limited dilution were examined the alkaline phosphatase (ALP) activity. In addition, we confirmed the mRNA expression of osteogenic marker of detected clonal cells by RT-PCR methods. Results: Expression of the inserted gene and telomerase activity in each of the clones were confirmed. ALP activities showed distinctive differential patterns in each of single cell clones. There were two types for the mRNA expression of the osteogenic markers. A4 clone was detected the mRNA expression of osteogenic markers, despite C10 clone was not detected. Conclusion: Immortalization of HPL cells was successfully accomplished by transduction of hTERT gene and the osteogenic characteristics showed different patterns in single cell clones.