Waleerat Sukarawan

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

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.

Effect of Jagged-1 and Dll-1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth

OBJECTIVE The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. DESIGN Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. RESULTS The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. CONCLUSION The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model.

Basic Fibroblast Growth Factor Regulates REX1 Expression Via IL‐6 In Stem Cells Isolated From Human Exfoliated Deciduous Teeth

Basic fibroblast growth factor (bFGF) regulates pluripotent marker expression and cellular differentiation in various cell types. However, the mechanism by which bFGF regulates REX1 expression in stem cells, isolated from human exfoliated deciduous teeth (SHEDs) remains unclear. The aim of the present study was to investigate the regulation of REX1 expression by bFGF in SHEDs. SHEDs were isolated and characterized. Their mRNA and protein expression levels were determined using real‐time polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. In some experiments, chemical inhibitors were added to the culture medium to impede specific signaling pathways. Cells isolated from human exfoliated deciduous tooth dental pulp tissue expressed mesenchymal stem cell surface markers (CD44, CD73, CD90, and CD105). These cells differentiated into osteogenic and adipogenic lineages, when appropriately induced. Treating SHEDs with bFGF induced REX1 mRNA expression and this effect was attenuated by pretreatment with FGFR or Akt inhibitors. Cycloheximide pretreatment also inhibited the bFGF‐induced REX1 expression, implying the involvement of intermediate molecule(s). Further, the addition of an IL‐6 neutralizing antibody attenuated the bFGF‐induced REX1 expression by SHEDs. In conclusion, bFGF enhanced REX1 expression by SHEDs via the FGFR and Akt signaling pathways. Moreover, IL‐6 participated in the bFGF‐induced REX1 expression in SHEDs. J. Cell. Biochem. 118: 1480–1488, 2017.

Effect of lithium chloride on cell proliferation and osteogenic differentiation in stem cells from human exfoliated deciduous teeth

Lithium Chloride (LiCl) has been used as a canonical Wnt pathway activator due to its ability to inhibit a glycogen synthase kinase-3. The aim of the present study was to investigate the effect of LiCl on cell proliferation and osteogenic differentiation in stem cells isolated from human exfoliated deciduous teeth (SHEDs). SHEDs were isolated and cultured in media supplemented with LiCl at 5, 10, or 20mM. The results demonstrated that LiCl significantly decreased SHEDs colony forming unit ability in a dose dependent manner. LiCl significantly enhanced the percentage of cells in the sub G0 phase, accompanied by a reduction of the percentage of cells in the G1 phase at day 3 and 7 after treatment. Further, LiCl markedly decreased OSX and DMP1 mRNA expression after treating SHEDs in an osteogenic induction medium for 7 days. In addition, no significant difference in alkaline phosphatase enzymatic activity or mineral deposition was found. Together, these results imply that LiCl influences SHEDs behavior.

Influence of Jagged1 on apoptosis-related gene expression: a microarray database analysis

Abstract Notch signaling regulates various cell behaviors including cell death and survival. The present study aimed to investigate the influence of Notch ligand, Jagged1, on the expression of apoptosis related genes using the microarray database analysis. In this regard, microarray dataset from Gene Expression Omnibus (GEO) repository was searched with keywords. The included GEO series were downloaded and analyzed using the Connection Up- and Down-Regulation Expression Analysis of Microarrays Extension program. The predicted protein–protein interaction was determined using GenesFANs program. The results demonstrated that Jagged1 enhanced pro-apoptosis gene expression in human lung and breast cancer cell line while promoted the expression of anti-apoptosis gene in human lymphatic epithelial cells. In human umbilical endothelial vein cells, Jagged1 promoted both pro- and anti-apoptosis gene expressions. No significant change in apoptotic related gene was noted in Jagged1 overexpressing human endometrial stromal cells. However, Jagged1 down-regulated both pro- and anti-apoptosis gene in human lung and breast cancer cell line as well as human umbilical endothelial vein cells. In conclusion, Jagged1 regulated both pro- and anti-apoptosis gene expressions, depending on cell types. However, the influence of Jagged1 on cell apoptosis or survival is needed further confirmation experiments.