Thanaphum Osathanon1, Philaiporn Vivatbutsiri1, Waleerat Sukarawan2, Wannakorn Sriarj3, Prasit Pavasant4, Sireerat Sooampon5. 1. Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Developing Research Unit in Genetic and Craniofacial Analyses of Craniofacial Structures, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. 2. Department of Pediatric Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Developing Research Unit in Tissue Engineering, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. 3. Department of Pediatric Dentistry, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Developing Research Unit in Cell Signaling and Protein Function, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. 4. Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. 5. Developing Research Unit in Cell Signaling and Protein Function, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand; Department of Pharmacology, Faculty of Dentistry, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: pl_sireerat@yahoo.com.
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 (CoCl2) could maintain human periodontal ligament (HPDL) cell stemness. METHODS: HPDL cells were treated with either 50 or 100 μM CoCl2. 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 CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 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 CoCl2 might inhibit osteogenic differentiation through an HIF-1α- dependent mechanism. CONCLUSION: This study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory.
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 (CoCl2) could maintain human periodontal ligament (HPDL) cell stemness. METHODS: HPDL cells were treated with either 50 or 100 μM CoCl2. 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 CoCl2 supplementation had no effect on cell proliferation. CoCl2 treatment increased the mRNA expression of the embryonic stem-cell markers REX1 and OCT4. Culturing HDPL cells in osteogenic medium containing CoCl2 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 CoCl2 might inhibit osteogenic differentiation through an HIF-1α- dependent mechanism. CONCLUSION: This study shows that CoCl2 treatment can induce stem-cell marker expression and inhibit the osteoblastic differentiation of HPDL cells. These findings suggest the potential application of CoCl2 for maintaining the stem-cell state in the laboratory.
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