The Critical Molecules Of Odontogenic Potential In Mouse Dental Mesenchyme

Many mammalian organ development, such as kidney, lung and teeth, depends on the multipl-interactions of mesenchyme and epithelium. Mouse molar development has been an excellent model for studying developmental process of organogenesis. Mammalian odontogenesis involves a series of sequential and reciprocal signal interactions between epithelium and the underlying mesenchyme. At the early stage of tooth development, growth factors functioning as signaling molecules that mediate signal interaction regulate gene expression and cellular function to define the odontogenic fate of both dental epithelium and mesenchyme.Tooth development relies on reciprocal tissue interactions between ectoderm-derived dental epithelium and cranial neural crest-derived mesenchyme. Based on this pricinciple, generation of an implantable tooth germ in vitro requires tow cell components, epithelial origin or mesenchymal origin and either of them must acquire odontogenic potential to initiate regenerative process. Stem cells are generally defined as cells that have the capacity to self-renew as well as to give rise to differentiated progeny. Tissue engineering is considered as one of the most powerful approaches to repair or replace an injured tissue or organ in the future. It was demonstrated recently that mouse stem cells, including ES cells, neural stem cells, and BMSCs, could be induced to reprogram into odontogenic fate to support tooth formation when proper odontogenic signals are provided. These studies support the idea that the odontogenic process can be initiated in stem cells with non-dental origin when proper odontogenic signals are provided. Dissection of molecular constitution of odontogenic potential would be a presquist for establishing approaches of stem cells based tooth regeneration and replacement therapy.Previous studies have shown that E10.5 second branchial arch epithelium (non-dental epithelium) could be induced by E13.5 malor mesenchyme that contains odontogenic potential to participate to form tooth structure. Since this recombinant approach requires two embryos at different developmental stages at the same time and the E10.5 second branchial arch epithelium is difficult to separate, the success ratio of tooth formation is low, presenting an obstacle for study. In this study, E13.5 and E14.5 dental mesenchyme were recombined with various non-dental epitheliums from the same embryo, including the head epithelium, mandibular epithelium, limb bud epithelium and back epithelium. We found that the recombinant of E14.5 dental mesenchyme and E14.5 non-dental mandibular epithelium exhibited the highest tooth formation efficiency at 61.9%. Further investigation revealed the developmental process in these regenerated teeth was similar with the normal one, including formation of ameloblast and secreting enamel. Our results indicated recombination of dental mesenchyme with E14.5 non-dental mandibular epithelium is a more efficient approach for making a tooth tissue recombinant to explore the molecular components of odontogenic properties. In addition, attempts have been made to identify alternative sources of human postnatal stem cells as the epithelial component for human whole tooth regeneration. Previous studies in our laboratory have proved that E13.5 dental mesenchyme was able to induce the formation of a bioengineered tooth crown when confronted with human keratinocytes. However, a low efficiency of sucees ratio for tooth formation is also account. In this study, human keratinocytes stem cells separated from pediatric foreskin, as another non-dental epithelium source from human, suspended keratinocytes stem cells recombined with dissociated E13.5 dental mesenchyme cells, ratio of human keratinocytes stem cells quantified in 10%, centrifuged and incubated for the formation of a firm cell pellet, followed by subrenal culture. This recombinant exhibited marvelous tooth formation efficiency at 92.6%. Its development process was consistent to the normal tooth germ. These results demonstrated that we established a set of effective system for research of inductive odontogenic potential.The odontogenic potential must consist of a unique combination of growth factors in different tissue layer at certain stages of tooth development. The expression profiles of numerous growth factors in different stages of tooth development have been well documented in mice. While currently unknown, it has become a central importance to reveal the constitution of odontogenic potential. In order to uncover the molecular component associated with the loss of odontogenic potential, we constructed a system to monitor the loss of odontogenic potential in mouse molar mesenchyme. Dental mesenchyme of E14.5 lower molar still live after long term (0 h,24 h, and 48 h) organ culture in vitro, but dental mesenchyme exhibited significant difference in odontogenic properties after cultured for long term. The capability of odontogenic potential in the E14.5 molar mesenchymal declined rapidly after 24h organ culture, furthermore, it totally lossed after 48h organ culture. The total RNA samples of dental mesenchyme which respectively harvested after 0h,24h and 48h organ culture were subjected to RNA sequencing analysis. The data generated from the RNA-seq were subjected to systematic bioinformatics analysis to screen genes implicated in odontogenic properties. Using GO category enrichment analysis to functional analysis the significant differentially genes screened in this study. The most significant enrichment of GO terms were oxygen binding, oxygen transport, oxygen transporter activity, organ development, binding, cytosolic part, multicellular organismal development, which contained 39 differentially expressed genes. According to the result of bioinformatics analysis, we suspected Fgf3 and Sfrp4 may play essential roles in the odontogenic potential of mouse molar mesenchyme.To further investigate the function of Fgf3 and Sfrp4 in odontogenic potential of mouse molar mesenchyme, we emploied the loss of function or gain of function analyisis to study the function of Fgf3 and Sfrp4 in the odontogenic potential. Recently, CRISPR/Cas9 system emerged as a powerful new tool in the genome editing. We constructed CRISPR/Cas9-mediated gene knockdown system in the dental mesenchyme. In the loss of function analysis, we employed this new gene knockdown system to investigate the role of Fgf3 and Sfrp4 in inductive odontogenic potential of molar mesenchyme, the Msxl and Pax9 as the positive control. First, a set of sgRNAs which target to Fgf3, Sfrp4, Msxl and Pax9 were screened according to the efficient on-target effects and inefficient off-target effects. Second, we constructed sgRNA-CRIPSR lentivirus vector and produce Fgf3, Sfrp4, Msxl and Pax9 knockdown lentivirus, respectively. Finally, the E13.5 molar mesenchyme cells were infected with the lentivirus and recombined with hKSC to analyse the odontogenic potential. As the result, the knockdown of Msxl and Pax9 in E13.5 molar mesenchyme cells loss capability of tooth formation. Knockdown of Fgf3 in mesenchyme didn’t affect tooth formation efficiency and knockdown of Sfrp4 in cultured 48h E14.5 mesenchyme which lost the odontogenic potential didn’t rescue the mesenchyme to formate the tooth structure. In the gain of function analysis, we employ beads which have the ability of soak the growth factor to express the FGF3 in the cultured 48h E14.5 mesenchyme, and recombined with E10.5 secondary branchial arch epithelium to analyse the odontogenic potential. As a result, the FGF3-soaked beads but not BSA-soaked beads rescue tooth formation in the recombinant. What’s more, the success ratio of tooth formation reaches to 43.8%. All the evidences indicated the Fgf3 have the ablity to rescued odontogenic potential in mouse dental mesenchyme.In conculsion, a highly efficient and convenient recombination system was set up for research of inductive odontogenic potential. We constructed a system of loss of odontogenic potential in mouse mesenchyme by organ culture system. The candidate genes for the odontogenic potential were screened from the RNA-seq and their function were investigated. Finally, our results indicated that Fgf3 was the critical molecules of odontogenic potential in mouse dental mesenchyme. Our results will have great implication in generation of bioengineered tooth and replacement therapy...