Immortalized periodontal ligament mesenchymal cells retain undifferentiation, proliferation and osteogenic...

Periodontal ligament-derived mesenchymal stem cells (PDLCs) are accessible and multipotent, with potential in regenerative therapies. However, their limited replicative capacity restricts long-term in vitro studies and hinders deeper understanding of mineralization mechanisms. To overcome this, we established an immortalized PDLC line (iPDLCs) by introducing the human telomerase reverse transcriptase (hTERT) gene via lentiviral transduction. We compared iPDLCs to primary PDLCs in terms of phenotype, proliferation, and osteogenic differentiation. Surface markers (CD105, CD166, CD34) were assessed by flow cytometry, while gene expression of hTERT, OCT4, NANOG, MAPK14, YAP1, CREB1, RUNX2, SP7, and ALPL was analyzed by RT-qPCR. ALPL activity and Alizarin Red staining were used to evaluate mineralization. iPDLCs showed a 2,300-fold increase in hTERT expression and maintained mesenchymal features, with high CD105/CD166 and low CD34 expression, as well as elevated OCT4 and NANOG levels. Upon osteogenic induction, iPDLCs upregulated proliferation-related genes (MAPK14, YAP1, CREB1) and showed early and enhanced expression of osteogenic markers (RUNX2, SP7, ALPL), particularly on day 7 (p ≤ 0.05 vs. PDLCs). Strong correlations were found between YAP1, RUNX2, and SP7 expression on days 7 and 10. Despite this early gene activation in iPDLCs, mineral deposition was greater in PDLCs at day 28 (p ≤ 0.05), confirmed by ALPL activity. These findings suggest that iPDLCs retain their undifferentiated state, proliferation capacity, and mineralization potential, while offering a reliable and long-lasting model for studying the molecular mechanisms involved in osteogenic differentiation of PDLCs.