Induction Of Bone Marrow Stromal Cells Into Neuron-Like Cells By Vascular Endothelial Growth Factor

Background and objectiveStudies have demonstrated that bone marrow stromal cells (BMSCs) undergo neuronal differentiation under certain in vitro conditions.However, very few inducers of BMSCs neural differentiation have been used in clinical application.The effects and mechanism of vascular endothelial growth factor (VEGF) on neural differentiation of BMSCs in vitro remain poorly understood.In this experiment,we aimed at investigating the effect of VEGF on inducing BMSCs into neuron-like cells in vitro, and at determining the best VEGF concentration for experimental induction.Methods(1)BMSCs were harvested from 6～8-weeks-old male and female adult Sprague Dawley rats. The stem cell marker CD90 and the hematopoietic cell marker CD45 were detected by flow cytometry to identify the third passage of BMSCs.(2)After 3 passages, the BMSCs were pre-induced with 10ng/mL basic fibroblast growth factor(b-FGF) for 24 hours,followed by randomly being assigned to 5,10,and 20ng/mL VEGF-treated groups,as well as the control groups,in which the BMSCs were not treated with VEGF.The morphological changes in BMSCs prior to and following VEGF induction was observed under a phase-contrast microscope (×200).(3)Expression of NSE following induction was determined by immunocytochemistry after 3 and 10 days after induction.The total number of NSE-positive cells was quantified from 20 randomly selected visual fields (×200) per coverslip.Results(1)After 72 hours in culture, some adherent BMSCs were observed with varying appearances:primarily round cells accompanied by triangle and spindle-shaped cell bodies with short processes.At 7–10 days in primary culture,the majority of spindle-shaped cells formed colonies.After 2–3 passages,the BMSCs were relatively homogeneous in appearance,and the majority of cells were spindle-shaped and displayed a whirlpool pattern;some cells were large and flat.(2)Flow cytometry demonstrated that BMSCs from the third passage were positive for the stem cell marker CD90 (97.5%) and negative for the hematopoietic cell marker CD45.(3)Shrunken,round cells,with a strong refraction and thin bipolar or multipolar primary and secondary branches were observed 3 days after induction with 5,10,and 20ng/mL VEGF. However, these changes were not observed in the control group.(4)At 10 days after induction, the number of NSE-positive cells was greatest in the 10 ng/mL VEGF-treated group(P< 0.05).The number of NSE-positive cells was least in the control group at 3 and 10 days post-induction(P<0.05).Moreover, the number of NSE-positive cells was greater at 10 days compared with 3 days after induction(P<0.05).Conclusion(1)rat BMSCs were capable of differentiating into neuronal-like cells.(2)In this experiment,VEGF played an important role on inducing BMSCs into neuron-like cells in vitro and compared with 5 and 20ng/mL,10ng/mL was the most appropriate VEGF concentration.(3)VEGF could be utilized to determine neuronal differentiation of BMSCs in vitro and repair central neural damage and neural degeneration.