Osteo-/odontogenic differentiation of BMP2 and VEGF gene-co-transfected human stem cells from apical papilla
Stem cells from apical papilla (SCAP) possess clear osteo-/odontogenic differentiation capabilities, and are regarded as the major cellular source for root dentin development. Bone morphogenetic protein 2 (BMP2) and vascular endothelial growth factor (VEGF) serve pivotal roles in the modulation of tooth development and dentin formation. However, the synergistic effects of BMP2 and VEGF on osteo-/odontogenic differentiation of SCAP remain unclear. The current study aimed to investigate the proliferative and osteo-/odontogenic differentiating capabilities of BMP2 and VEGF gene-co-transfected SCAP (SCAP-BMP2-VEGF) in vitro. The basic characteristics of the isolated SCAP were identified by the induction of multipotent differentiation and by flow cytometry. Lentiviral vector-mediated gene transfection was conducted with SCAP in order to construct blank vector-transfected SCAP (SCAP-green fluorescent protein), BMP2 gene-transfected SCAP (SCAP-BMP2), VEGF gene-transfected SCAP (SCAP-VEGF) and SCAP-BMP2-VEGF. The Cell Counting Kit 8 assay was used to analyze the proliferative capacities of the four groups of cells. The expression of osteo-/odontogenic genes and proteins in the cells were evaluated by reverse transcription-quantitative polymerase chain reaction and western blotting. The mineralized nodules formed by the four group cells were visualized by alkaline phosphatase (ALP) staining. Among the four groups of cells, SCAP-VEGF was demonstrated to exhibit increased proliferation, and SCAP-BMP2-VEGF exhibited reduced proliferation during eight days observation. SCAP-BMP2-VEGF exhibited significantly increased expression levels of ALP, osteocalcin, dentin sialophosphoprotein, dentin matrix acidic phosphoprotein gene 1 and dentin sialoprotein than the other three groups at the majority of the time points. Furthermore, the SCAP-BMP2-VEGF group exhibited a significantly greater number of ALP-positive mineralized nodules than the other groups following 16 days culture in vitro. In conclusion, lentiviral vector-mediated BMP2 and VEGF gene co-transfection significantly activated the osteo-/odontogenic differentiation of human SCAP.Human SCAP were isolated from an extracted immature mandibular third molar of a male patient aged 18 years old by a method used in a previous study (11). All protocols were reviewed and approved by the Ethics Committee of Guanghua School and Hospital of Stomatology, Sun Yat-sen University (Guangzhou, China). The osteogenic and adipogenic differentiation capacities of SCAP were identified by Alizarin Red staining and Oil Red O staining (Cyagen Biosciences, Inc., Guangzhou, China), respectively (11). The typical phenotypes, including STRO-1/Alexa Fluor 647-Allophycocyanin (BioLegend, Inc., San Diego, CA, USA), CD146/Phycoerythrin (BD Pharmingen, San Diego, CA, USA), CD24/Fluorescein Isothiocyanate (FITC) (BD Pharmingen) and CD45/FITC (BD Pharmingen), using the 2nd passage of SCAP were assessed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).The kidney-shaped papillae, which had a dense and pink appearance, were separated from the third molars (Fig. 1A). The isolated primary SCAP presented with a short spindle-like appearance and formed classic cell colonies subsequent to eight days culture (Fig. 1B). Alizarin Red staining illustrated that SCAP formed abundant mineralized nodules 32 days subsequent to osteogenic induction (Fig. 1C), and Oil Red O staining indicated that SCAP formed lipid droplets 16 days following adipogenic induction (Fig. 1D). The freshly isolated SCAP exhibited typical forward scatter/side scatter characteristics (Fig. 1E), similar to those reported previously (4,7). The presence of the phenotypic markers, including STRO-1, CD146 and CD24, were observed to be 23.1%, 89.0% and 22.2%, respectively in the isolated cells (Fig. 1F–H).