Abstract: Objective: To examine the effect of hypoxia on human periodontal ligament stem cells (hPDLSCs). Methods: HPDLSCs were collected and validated. Cells were cultured in normoxia (20% O₂) or hypoxia (5%O₂) for 5 days. MTT was applied to detect the cell proliferation ability in each day. OCT4, SOX2, and NANOG genes were analyzed by RT-PCR in cells culture in hypoxia at day 1 and day 5. Ki67 staining and crystal violet staining assay were used to detect the self-renewal ability of hPDLSCs. Osteogenic and adipogenic differentiation abilities were investigated after 21-day-induction. Results: The results from MTT assay showed that the proliferation ability of hPDLSCs treated with hypoxia was higher (P<0.05). Hypoxia treatment enhanced the related gene expression at both day 1 and day 5 (P<0.05). SOX2 was enhanced strongly at day 5 (P<0.01). Crystal violet staining assay indicated that colony-forming efficiency was increased in hypoxia group (P<0.05). In addition, hypoxia also enhanced the osteogenic differentiation capacity of hPDLSCs. Osteogenic gene RUNX2 was significantly increased (P<0.05). However, although hypoxia increased the expression of adipogenic gene PPAR γ2 and adipogenic differentiation, the results was not statistically significant. Conclusion: Hypoxic preconditioning of hPDLSCs can increase cell proliferation and expression of stemness-related genes. In addition, hypoxia environment can also improve the osteogenesis differentiation of hPDLSCs, but has little effect on adipogenic differentiation.

Key words: Hypoxia, Human periodontal ligament stem cells, Proliferation, Differentiation