Immortalization of human periodontal ligament stem cells by transferring human telomerase reverse transcriptase gene

Li Gao, Fen Zhao, Wenxin Jiang, Chenguang Niu, Keyong Yuan, Wenzhen Lin, Xuchen Hu, Rui Ma, Zhengwei Huang

Abstract

Background: Human periodontal ligament stem cells (hPDLSCs) were considered to be potential seed cells for periodontal tissue engineering, however, it possesses a relatively short replicative lifespan and undergo senescence after several passages. So, this study aimed to establish a cell line of immortalized hPDLSCs to provide a consistent and homogeneous research cell model.
Methods: Primary human periodontal ligament cells (hPDLCs) were cultured and hPDLSCs were isolated using magnetic beads. The hPDLSCs were then immortalized by infection with a lentivirus vector carrying the hTERT gene. hTERT transcription was verified with RT-PCR. Characteristics of the immortalized cells, such as morphology, proliferation were evaluated. The expression of the odontoblastic/osteogenic markers dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), alkaline phosphatase (ALP) and osteomodulin (OMD) was analyzed with real time RT-PCR. Expression of p53 protein was verified with western blot to find whether the overexpression of hTERT could change the carcinogenicity of hPDLSCs.
Results: The results of the study showed that hPDLSCs were successfully isolated and hTERT was successfully integrated into the genomic DNA of hPDLSCs to form the immortalized human dental periodontal ligament stem cells (hPDLSCs-hTERT), furthermore, the ectogenic hTERT was successfully transcribed into mRNA. In long-term culture, hPDLSCs-hTERT bypassed senescence and continued to grow for more than 49 generations. Moreover, hPDLSCs-hTERT retained the phenotypic characteristics of the primary hPDLSCs. Furthermore, similar to hPDLSCs, hPDLSCs-hTERT expressed increased levels of ALP, DMP1, DSPP, and OMD in the odontoblastic/osteogenic induction medium (P<0.05). The expression levels of p53, which is an anti-oncogene, showed no significant difference between the hPDLSCs and hPDLSCs-hTERT.
Conclusions: We established a cell line of immortalized hPDLSCs, which remained in an undifferentiated state, exhibiting odontoblastic/osteogenic differentiation potential and low tumorigenicity.