Isolation, immunophenotypic characterization and pluripotency of dental pulp stem cells

Objective: The aim of this study has been the isolation and characterization of stem cells from dental pulp (DPSCs) in culture. Methods: The primary DPSCs cultures were obtained from human third molars. Immediately after extraction, the teeth were placed in Dulbecco’s modified Eagle medium (DMEM) culture medium supplemented with fetal bovine serum and antibiotics. In a laminar flow, the pulp was removed from the tooth, incubated with collagenase for 2 hours and placed on a culture plate. Phenotypic characterization and cell pluripotency was performed in the fifth passage (P5). To evaluate the expression of surface markers, the cells were incubated with antibodies against CD14, CD29, CD34, CD44, CD45, CD73, CD90, CD105 and HLA-DR antigens. For induction of cell differentiation in vitro, 104 cells/cm2 were plated in 12-well plates and cultivated in appropriate media for osteogenic, adipogenic and condrogencic differentiation after reaching at least 70% confluence. Results: The cells were positive above 95% for characteristic markers of the mesenchymal stem cells CD29, CD44, CD73 and CD90. In contrast, there was a low percentage of positivity (up to 1.1%) for the characteristic markers of hematopoietic cells such as CD14, CD34, CD45, CD184 and HLA-DR. Adipogenic differentiation was visualized by staining lipid vacuoles with Oil Red. Bone differentiation was visualized by staining calcium deposits with Alizarin Red. No chondrogenic differentiation was observed. Conclusions: The isolated cells were adherent to plastic, positive for the characteristic markers of mesenchymal stem cells and, therefore, an alternative source for tissue engineering studies