Abstract
As a key factor for cell pluripotent and self-renewing phenotypes, SOX2 has attracted scientists’ attention gradually in recent years. However, its exact effects in dental pulp stem cells (DPSCs) are still unclear. In this study, we mainly investigated whether SOX2 could affect some biological functions of DPSCs. DPSCs were isolated from the dental pulp of human impacted third molar. SOX2 overexpressing DPSCs (DPSCs-SOX2) were established through retroviral infection. The effect of SOX2 on cell proliferation, migration and adhesion ability was evaluated with CCK-8, trans-well system and fibronectin-induced cell attachment experiment respectively. Whole genome expression of DPSCs-SOX2 was analyzed with RNA microarray. Furthermore, a rescue experiment was performed with SOX2-siRNA in DPSC-SOX2 to confirm the effect of SOX2 overexpression in DPSCs. We found that SOX2 overexpression could result in the enhancement of cell proliferation, migration, and adhesion in DPSCs obviously. RNA microarray analysis indicated that some key genes in the signal pathways associated with cell cycle, migration and adhesion were upregulated in different degree, and the results were further confirmed with qPCR and western-blot. Finally, DPSC-SOX2 transfected with SOX2-siRNA showed a decrease of cell proliferation, migration and adhesion ability, which further confirmed the biological effect of SOX2 in human DPSCs. This study indicated that SOX2 could improve the cell proliferation, migration and adhesion ability of DPSCs through regulating gene expression about cell cycle, migration and adhesion, and provided a novel strategy to develop seed cells with strong proliferation, migration and adhesion ability for tissue engineering.
Highlights
In the field of regenerative medicine, stem cells have been investigated for years, for their ability to repair injured tissue and restore organ function partially
The results indicated that the dental pulp stem cells (DPSCs) used in our study were positive for CD29 (90.1%), CD44 (95.8%), CD73 (90.2%), CD90 (91.4%), CD105 (92.1%), and CD166 (93.6%), and nearly negative for CD14 (0.09%), CD34 (0.26%) and CD45 (0.31%), which were usually regarded as the specific markers of hematopoietic cells (Fig 1A)
In the negative control group, the DPSCs cultured with normal medium were used for each stain, and those cells could not be stained with alkaline phosphatase (AP) and Oil red O kit (Fig 1B1 and 1B2)
Summary
Isolation and Characterization of Human DPSCs. Normal human impacted third molar was collected from patient after giving informed consent. The participant has provided the written informed consent to participate in this study. DPSCs were isolated using the method described as the reference [32]. After cleaning the tooth surface and cutting around the cementoenamel junction, the dental pulp was isolated and further digested in a solution of 3 mg/mL collagenase type I for 1 hour at 37°C. Cell suspensions of dental pulp were seeded into 6-well plate and cultured with Alpha-Minimum Essential Medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 0.1g/mL streptomycin (Hyclone) at 37°C under 5% CO2 condition. DPSCs were differentiated into adipocytes and osteocytes as previously described [33, 34]
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