Odontogenic Potential Study Of In Vitro Cultured Murine Neural Crest Stem Cells

The embryonic mesenchyme of the mouse tooth bud after budding has the potential of dental implantation and can be re-formed with non-odontogenic epithelium.Therefore,it is often used to induce epithelial cells with stem cell characteristics to participate in the construction of regenerated teeth.It is known that the dental mesenchymal cells are developed from the neural crest cells,which migrate to the first branchial arch during the early embryo development.We have established a platform for differentiation of human induced pluripotent stem cell(iPS)into neural crest like cells.However,the exact way to induce iPS-derived neural crest like cells to become the odontogenic dental mesenchymal cells is still unknown.Based on the similarity of tooth development between human and mouse,we investigated the in vitro developed characteristics of the mouse neural crest cells and tooth formation potential of murine neural crest stem cells.At first,the mouse neural crest cells were isolated from embryonic mice of 8.5 days and cultured in vitro with the appropriate culture environment.The obtained neural crest cells were then evaluated by the specific gene expression and the surface marker molecular analyses.Meanwhile,the neural crest stem cells were induced by mineralized osteogenesis to exam their osteogenic ability,providing the basis and guarantee for the differentiation ability of the neural crest stem cells.The CCK8 experiment was used to further evaluate the proliferation ability of the third generation neural crest stem cells.Through the recombination between the cultured neural crest cells and dental epithelium at embryonic day 14.5(E14.5),the recombinant germs were then transplanted into the kidney capsule to explore whether the neural crest stem cells with dental potential.In this study,the neural tube isolated from rat embryo was transferred to cell culture dish,and the neural stem cells migrated from the neural tube showed an shuttle morphology.Immunofluorescence showed these cells were positive for E-Caherin,Pax3,P75 and HNK1,and negative for Pax6 in passage 0(P0).Whereas the cells in passage 3(P3)showed positive for P75,HNK1 and Ap2α,and negative for Pax6.P0 cells and P3 cells both express the markers of neural crest,and did not express the marker of neural stem cell.It is suggested that the mouse neural crest stem cells can be cultured in serum-free conditioned medium to maintain the characteristics of mouse neural crest stem cells in vitro.The results showed that although the neural crest gene was missing in the process of passaging,the genotype of the neural crest was still stronger than that of the neural crest stem cells.Although expression of the source gene is low but the expression level is relatively low.For neural stem cell genes in late passaged neural crest cells were expressed.The results showed that with the increase of the number of days,the neural crest stem cells showed a gradual increase in the form of CCK8.Indicating that neural crest stem cells can be stable in the culture medium we provide,to maintain cell proliferation.The neural crest stem cells osteogenic induction differentiation in vitro,through alizarin red dye staining,orange deposition.The expression of mineralized neural crest stem cells in the four groups was significantly higher than that in the control group(p<0.05).The expression of Ocn,Bmp3 and Bmp2 was much higher than that of the other three groups.The results showed that neural crest stem cells can differentiate into bones.The neural crest stem cells and tooth germ epithelium reorganization,transplantation and other experiments,by HE staining we can observed the presence of P3 and P5 in the presence of tooth structure,which proved that neural crest stem cells have dental potential.As described in the review,through the culture,identification and detection of neural creal stem cells in vitro,showed that neural crest stem cells had the potential of dental potential.This result provides a good basis for finding ways to obtain neural stem cells in vitro to induce access to dental mesenchymal cells,and then provide an important reference for the differentiation of neural crest cells derived from human iPS into adipogenic mesenchymal cells,thus promoting the differentiation of stem cells into adipogenic cells in vitro.