Functional Study Of CD24 Positive Dental Mesenchymal Cells In Late Embryonic Developmental Stage

Tooth regeneration is one of the ultimate goals in oral research.How to obtain seed cells with the ability of inducing tooth formation is the key to successfully construct regenerative teeth.Previous studies have reported a variety of seed cells involved in tooth regeneration.However,researchers have not been able to construct regenerative teeth based solely on seed cells obtained from adults without the help of some cells or tissues derived from tooth embryos.Therefore,further analysis of more comprehensive characteristics of tooth germ cells that can issue instructions or have the potential to induce tooth formation during development will provide the solid theoretical and practical knowledge for defining the characteristics of seed cells required for tooth regeneration.In mice,tooth development comprises lamina stage,bud stage,cap stage,and bell stage.In a series of spatiotemporal events in tooth development regulated by signaling pathway such as BMPs,FGF,Wnts and SHH,epithelial and mesenchymal cells interact and induce the tooth structure.Previously,our research group analyzed the mesenchymal cells in bud and bell-like stages that have the potential to induce odontogenesis during the mouse tooth germ development via single-cell RNA-seq.Two highly active Gene regulatory networks(GRNs)were found to be regulated by transcription factors Barx1 and Foxf1,respectively.The expression of Barx1 and Foxf1 during the important stages of tooth germ development were examined.Furthermore,we discovered a cell surface marker CD24 was highly co-expressed with Barx1.Dental mesenchymal cells strongly expressing CD24mainly distributed in the upper part of the dental papilla during late development(bell stage).Whether this group of cells is the most core and most effective group of instructing cells in the mesenchymal of tooth germ,which has the potential to induce tooth formation,remains to be systematically studied.In this study,the distribution of CD24 protein in bell stage molars(Embryonic day 16.5,E16.5)was confirmed by immunohistochemistry,similar to the expression position of Cd24a.Furthermore,flow cytometry was used to detect the proportion of CD24 positive cells in mesenchymal cells of E16.5 molar germ.It was found that there were three cell populations,CD24⁺⁺,CD24⁺,and CD24^-cells,as 40%,36%,and 3.1%,respectively.We also observed these three cell populations in mesenchymal cells in Postnatal 1(PN1)molar DM as 7.4%,20%and 17.8%,respectively.To further clarify the function of these three cell subsets with CD24marker in tooth development,we compared the expression level of odontogenic genes in CD24⁺⁺cell group and CD24⁺cell group.Through q PCR detection,we observed high expression levels of dental genes such as Barx1,Bmp4,Lef1,Pax9,and Wif1 in CD24⁺⁺cells.On the other hand,according to the expression distribution of CD24,flow sorting method was used to obtain CD24⁺⁺and CD24⁺cells from the mesenchymal of E16.5 molar germ.The upper part of Dental papilla(DP1)and the lower part of Dental papilla(DP2)were obtained by section method.The odontogenic potentials of the cell subsets were verified by recombination with the odontogenic or non-odontogenic epithelium.We found that the rate of tooth formation of CD24⁺⁺and CD24⁺cells recombined with E16.5 odontogenic epithelium was 79.41%(n=27/34)and 23.08%(n=6/26),respectively.The odontogenesis rate was 16.7%(n=1/6)and 0%(n=0/5)in the recombination with non-odontogenic E10.5 second branchial epithelial.Our results indicates that CD24⁺⁺cell group has a significantly higher ability to induce odontogenesis compared to CD24⁺cell group.DP1 group(64.52%,n=20/31)corresponding to the distribution of CD24⁺⁺cells also showed a higher rate of tooth formation in the recombination experiment compared to DP2 group(23.81%,n=5/21)corresponding to the distribution of CD24⁺.It also showed a high rate of odontogenesis,that is,a significant potentiality of induced odontogenesis.We further cultured CD24⁺⁺and CD24-cells from PN1 stage into the hollow cavity of porcine Tooth dentin matrix(TDM)with specific acellular matrix,and found that the teeth of the former formed functional tissue module(pulp and dentin complex)showed a significant advantage in differentiation potential.In addition,micro-CT and immunohistochemical methods were used to confirm that the CD24⁺⁺group could form relatively larger teeth with better shape.In conclusion,we found that the cell population marked by CD24 existed in the dentogenic mesenchymal cells of late E16.5 tooth embryo development,which not only had a significant potential to induce epithelial tissue co-odontogenesis,but also had the potential to differentiate into dentine pulp complex.The results of this study confirmed that the dental germ mesenchymal cell subpopulation marked by CD24 is the core cell population that issues the instruction of tooth formation in the late dental germ mesenchymal and has the potential to determine the fate of tooth orientation differentiation.This is the first high-resolution mapping and functional interpretation of the cell subsets during molar embryo development,providing a scientific basis for defining the characteristics of mesenchymal seed cells during tooth regeneration.