Directional Differentiation Of Human Olfactory Mucosa Mesenchymal Stem Cells Into Vascular Endothelial Cells In Vitro

ObjectiveTo discuss the abillity of human olfactory mucosa mesenchymal stem cells?OM-MSCs?to differentiate into vascular endothelial cells?VECs?.The VECs were identified after differentiation.Methods1.Cultivation and identification of human OM-MSCs:The normal olfactory mucosa tissue were piked up by nasal endoscopy.OM-MSCs were used tissue adherent method to cultivate.The cell surface markers of cultured cells were detected by flow cytometry instrument.2.Inducing human OM-MSCs into VECs:P3 human OM-MSCs were cultured in flask with L-DMEM contained 2%FBS.When cells reached 90%fusion,cells were randomly divided into 2 groups:added inducing medium containd 50ng/ml VEGF in induction group while control group without VEGF.Then the cells were put in the incubator with 37?and 0.05%CO₂.3.Identify the VECs:?1?The morphological changes were observed under inverted phase contrast microscope.?2?Immunofluorescence staining:After inductiting 12 days,the CD31,CD34,vWF expression of cells were detected by immunofluorescence staining.?3?RT-qPCR:extract total RNA and test the expression level of Flt-1,vWF,Flk-1.?4?Cell phenotype:characterize the specific surface markers CD31and vWF of VECs.?5?Test of NO secretion level:use a NO test kit to identify OD value of culture medium 12d after induction and calculate the NO concentration.?6?Vascularization experiment in vitro:the cells of induced group were seeded onto semi-soild matrix medium and observed the abillity to form capillary-like net structure on the matrix.Results1.The cultured cells were adherent growth,and they were positive expression of CD73,CD90,CD105 and negative expression of CD34,CD45,CD11b,CD19 and HLA-DR.It conformed to the appraisal standard of mesenchymal stem cells.The cell purity was above 95%.2.Identification of VECs?1?Morphological observation:after induction,cell volume became smaller,cell morphology became short spindle,full and stereo.?2?Immunofluorescence:cells were positive expression of CD31,CD34,vWF.?3?RT-qPCR:after induction,the expression of Flt 1,vWF,Flk 1by cells was significantly increased,which had statistical significance.?4?Cell phenotype:The cells after induction were positive expression of CD31 and vWF and the proportion was about 30%.?5?NO production:the NO content in supernatant of induced group was higher than the control group,the difference was statistically significant.?6?Vascularization experiment in vitro:the cells of induced group formed tube-like structure and the tube-like structure connected with each other after seeded onto Matrigel.The cells of control group can not form tube-like structure.Conclusion1.Human olfactory mucosa tissue adherence method can isolate and amplify OM-MSCs in vitro.2.Vascular endothelial growth factor?VEGF?can induce the olfactory mucosa mesenchymal stem cells to differentiate into VECs.3.The VECs which came from OM-MSCs differentiation had the function of vascular endothelial cells and can form tube-like structure.