Cobalt chloride supplementation induces stem-cell marker expression and inhibits osteoblastic differentiation in human periodontal ligament cells

Abstract Objective Low oxygen tension is one of the crucial factors of the stem-cell niche. However, the long-term hypoxic culture of stem cells is difficult and requires special equipment. In this study, we investigated whether mimicking hypoxia using cobalt chloride (CoCl 2 ) could maintain human periodontal ligament (HPDL) cell stemness. Methods HPDL cells were treated with either 50 or 100 μM CoCl 2 . Cell proliferation was determined by an MTT assay. The mRNA expression of stem-cell marker and osteogenic associated genes were analyzed by RT-PCR and Real-time PCR. Osteogenic differentiation was determined by assaying alkaline phosphatase activity and in vitro mineralization. Results The results showed that the CoCl 2 supplementation had no effect on cell proliferation. CoCl 2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4 . Culturing HDPL cells in osteogenic medium containing CoCl 2 resulted in a decrease in alkaline phosphatase activity, down-regulation of osteogenic associated gene expression, and suppression of mineralization. The use of Apigenin, an HIF-1α inhibitor, indicated that CoCl 2 might inhibit osteogenic differentiation through an HIF-1α- dependent mechanism. Conclusion This study shows that CoCl 2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl 2 for maintaining the stem-cell state in the laboratory.