The involvement of histone methylation in osteoblastic differentiation of human periosteum‐derived cells cultured in vitro under hypoxic conditions

Abstract

Although oxygen concentrations affect the growth and function of mesenchymal stem cells (MSCs), the impact of hypoxia on osteoblastic differentiation is not understood. Likewise, the effect of hypoxia-induced epigenetic changes on osteoblastic differentiation of MSCs is unknown. The aim of this study was to examine the in vitro hypoxic response of human periosteum-derived cells (hPDCs). Hypoxia resulted in greater proliferation of hPDCs as compared with those cultured in normoxia. Further, hypoxic conditions yielded decreased expression of apoptosis- and senescence-associated genes by hPDCs. Osteoblast phenotypes of hPDCS were suppressed by hypoxia, as suggested by alkaline phosphatase activity, alizarin red-S-positive mineralization, and mRNA expression of osteoblast-related genes. Chromatin immunoprecipitation assays showed an increased presence of H3K27me3, trimethylation of lysine 27 on histone H3, on the promoter region of bone morphogenetic protein-2. In addition, mRNA expression of histone lysine demethylase 6B (KDM6B) by hPDCs was significantly decreased in hypoxic conditions. Our results suggest that an increased level of H3K27me3 on the promoter region of bone morphogenetic protein-2, in combination with downregulation of KDM6B activity, is involved in the suppression of osteogenic phenotypes of hPDCs cultured in hypoxic conditions. Although oxygen tension plays an important role in the viability and maintenance of MSCs in an undifferentiated state, the effect of hypoxia on osteoblastic differentiation of MSCs remains controversial. In addition, evidence regarding the importance of epigenetics in regulating MSCs has been limited. This study was to examine the role hypoxia on osteoblastic differentiation of hPDCs, and we examined whether histone methylation is involved in the observed effect of hypoxia on osteogenic differentiation of hPDCs.

The Role of Human Umbilical Vein Endothelial Cells in Osteogenic Differentiation of Dental Follicle-Derived Stem Cells in In Vitro Co-cultures

“…Flow cytometry (FACSCalibur, Becton Dickinson, CA, USA) was used to detect surface antigens on DFSCs (1 × 10 5 cells per marker). Specifically, cells were analyzed for the presence of mesenchyme markers (CD44, CD73, CD90, and CD105) and the absence of the hematopoietic markers CD34 and CD45, as described previously 25 . DFSCs that were at approximately 90% confluence were trypsinized, fixed in 3.7% formaldehyde solution, and incubated with fluorescein isothiocyanate (FITC)-conjugated primary antibodies (mouse anti-human) against CD34 (BD Pharmingen, CA, USA), CD44 (BD Pharmingen), CD45 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and CD90 (BD Pharmingen).…”

“…The activity of alkaline phosphatase (ALP), an early marker for osteoblast differentiation, and mineralized nodule formation (indicative of maturation of the osteoblast phenotype) were used as indicators of osteoblast differentiation. ALP activity, alizarin red S and von Kossa staining, and calcium content were examined using a previously published method 25 , 26 .…”

Development of bone regeneration strategies using human periosteum‐derived osteoblasts and oxygen‐releasing microparticles in mandibular osteomyelitis model of miniature pig

“…Patients provided informed consent prior to the collection of periosteal tissues, as required by the Ethics Committee at Gyeongsang National University Hospital. h PDCs were cultured using a previously described technique (Kim et al, ; Yoon et al, ). Periosteal tissues were cultured in 100‐mm culture dishes in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 μg/mL streptomycin, at 37 °C in 95% humidified air and 5% CO 2 .…”

Parthenolide Has Negative Effects on In Vitro Enhanced Osteogenic Phenotypes by Inflammatory Cytokine TNF-α via Inhibiting JNK Signaling

“…Our previous work demonstrated that these human periosteum-derived cells (hPDCs) were positive for markers present in MSCs (CD44, CD73, CD90, and CD105) and expressed the pluripotent transcriptional factors Oct4, Nanog, and Sox-2. Moreover, these hPDCs were successfully differentiated into mesenchymal lineages, particularly adipocytes, osteocytes, and chondrocytes; thus, they can be considered to be human periosteum-derived MSCs [17,18].…”