The Role Of SFRP2 In Neural Differentiation From Human Dental Pulp Stem Cells

A variety of optic nerve diseases could cause progressive and irreversible damage to the optic nerve of human,even resulting in loss of vision.The optic nerve cells are terminally differentiated cells with poor ability of regenerating,so it’s difficult for the injured retinal nerve to be repaired or regenerated by themselves.That is to say,barely no method is applied clinically to cure the diseases caused by the injury of optic nerve.Recently,the application of stem cells in the treatment of retinal diseases shows great prospect.Dental pulp stem cells(DPSCs),derived from the neural crest ectoderm,which is same as optic nerve,is easier to be obtianed than neural stem cells.Thus,DPSCs can be used as seed cells to repair and reconstruct nerve tissue defect.The studies show that the Notch and Wnt/β-catenin signaling pathways are essential for the development of retinal nerve in the vertebrate animals.Notch gene is widely expressed in a variety of species,which is highly conservative in evolution.Notch mainly mediates an inhibitory signal during cell differentiation.The Wnt/β-catenin signaling pathway runs through the whole process of retina and optic nerve development,indicating that it plays an important role in neural development.Secreted frizzled related protein 2(SFRP2)is an inhibitor of Wnt signaling,meanwhile,it also participates in other signaling pathways,such as Notch and BMP signaling.When being combined with metal protease ADAM10,SFRP2 will inhibit the Notch signaling pathway.Other studies have shown that SFRP2 is highly expressed during the process of neurogenesis.We hypothesized that SFPR2 can promote the differentiation of dental pulp stem cells into neural like cells by inhibiting Notch and Wnt signaling pathways.In this experiment,with the help of a variety of cell biology and molecular biology methods,we invesitigated the function of SFRP2 in the differentiation of human dental pulp stem cells(h DPSCs)into neuron like cells.We transfected h DPSCs with adenovirus carrying SFRP2 gene in vitro,and induced neural differentiation by being co-cultured with retinal pigment epithelial(RPE)cells.Our study will provide an evidence for the clinical application prospect of optic nerve repairing.Method:The pulp tissue was isolated from the intact extracted third molars,and the primary culturing of h DPSCs was processed by the enzyme digestion method.The growth curve of the 3rd generation h DPSCs was drawn by MTT.The multilineage differentiation potential was detected by oil red O and alizarin red S staining after adipogenic and osteogenic induction.The multiplicity of infection(MOI)of adenovirus was screened by flow cytometry and cytotoxicity assay(MTT).A MOI of 800 particles / cell was used to transfect SFRP2 into h DPSCs,and RT-PCR was applied to detect the expression of SFRP2.Afterwards,the cells were transfected with Ad-SFRP2 and / or co-cultured with RPE cells to induce neural differentiation.The expression of nerual differentiation related genes,such as GFAP,Nestin,MAP2,HES1,β-3tublin and ATOH7,was detected by q PCR.Result:Under the microscope,the 3rd generation of h DPSCs cultured in vitro were adherent to the dish.The morphology of the cells was long spindle shaped,with round nucleus in the center of the cells.After the cells being induced in adipogenic and osteogenic conditioned media respectively,oil red O staining on day 35 and alizarin red S staining on day 21 showed the lipid droplets and calcified nodules,and toluidine blue staining showed that cells had a tendency to form colonies.The growth curve showed that on the fourth day,cells began to be in the logarithmic phase of growth,and on the ninth day,the cells entered the plateau stage.The signal of green fluorescent protein could be observed by fluorescent microscope after h DPSCs had been transfected with Ad-EGFP.The expression of SFRP2 was detected by RT-PCR at 72 h after transfection.The result of co-culture with RPE cells and transfection with Ad-SFRP2 showed that in the earlier period of culture,the expression of GFAP in the co-culture group is more obviously increased than that in the transfection group;on the contrary,in the later period,the expression of GFAP in the transfection group is more obviously increased;the expression of Nestin in the co-culture and transfection group showed an increased tendency in the earlier period and a decreased tendency in the later period;the expression of β-3tublin was continuously down-regulated in the co-culture and transfection group;the expression of MAP2 in the co-culture group was higher in later period;HES1 in the co-culture group had a higher expression in earlier period and lower expression in later period;on the contrary,the expression of HES1 in the transfection group showed an decreased tendency;the expression of ATOH7 showed no statistical significance.Conclusion:SFRP2 can positively regulate the neural differentiation of dental pulp stem cells,however,the specific mechanism still needs to be elucidated.