Study On The Potential Of Odontogenic Differentiation And Relevant Mechanism Of Ectomesenchymal Stem Cells Derived From Neural Crest

For a long time, people face embarass of tooth lossing due to caries, periodontosis, or trauma. Conventional treatments including tooth implantation have certain limitation and cannot completely restore the physiological and psychological function. The aim of tooth tissue engineering is to re-initiate the development of tooth for regenerating the tissues of tooth, such as dental periodontal ligament, cementum, dentin, even for whole tooth regeneration, possibilities for alternative, biology-based treatment strategy. The sources of seed cell are the most important among three factors of tissue engineering. The interaction betweencraniofacial neural crest-derived ectcomesenchymal stem cells（CNC-derived EMSCs） and ectoderm-derived epithlial cells have been occurring during the all stages of tooth development and companying with the regulation of signal molecule “cascade”. In classical tooth development theory, Neural crest cells delaminated through an epithelial-mesenchymal transition（EMT） from neural crest within midbrain and hindbrain region, migrated and formed the majority of the first branchial arch, which named ectomesenchymal stem cells（EMSCs）. The initiating singal molecules are delivered from dental lamina to EMSCs underlying lamina epithelium and induced the EMSCs to proliferation and condensation. Then, through the typical stages of dental lamina, bud, cap and bell stages, Sequentialy, the root of tooth began to develop and erupt after the dental crown completed. CNC-derived EMSCs form the majority of tooth except for enamel of dental crown, including pulp-dentin complex derived from odontoblast, and dental periodontal ligament and cementum derived from dental follicle progenitor cells（DFPCs）. So, CNC-derived EMSCs have bidirectional differentiation feature that the cells can differentiate to the odontoblast and DFPCs. CNC-derived EMSCs were selected as seed cell of tooth tissue engineering may have the capacity to form whole tooth tissues. Recent research results, however, demonstrated that the non-CNC-derived EMSCs also participate in the development of tooth. Thus, there are three difficult points need to be further studied:（1） Whether CNC-derived EMSCs can differentiate into odontoblast-like cell under odontogenic induction in vitro?（2） Whether there have differences of odontogenic differentiation between CNC-derived and non-CNC-derived EMSCs served as seed cells of tooth tissue engineering? and（3） the molecule mechanisms within CNC-derived EMSCs participated in epithelial-mesenchymal interaction at early stage of tooth development need to be further explored.Objective: This study aims to explore the differentiation difference between CNC-derived and Non-CNC-derived EMSCs from first branchial arch of embryo 11.5 days（E11.5） of SD rat under microenvironment of odontogenic induction in vitro and in vivo, and further to explore the molecule mechanism of odontogenic differentiation of CNC-derived EMSCs via gene microarray analysis and siRNA technique. We used the cell aggregation to simulate tooth development and expected to explore the differentiation model from the epithelial-mesenchymal interaction, which may provide promising data and strategy for tooth tissue engineering and tooth development theory.Methods:1. To determine the spatio-temporal location of P75-neurotrophin receptor（P75-NTR）,we used hematoxylin-eosin staining（HE） and immunohistochemistry（IHC） to observe the different stages of embryo of SD rat from E11.5 to E18.5d, and explore the spatio-temporal expression and biological function of P75-neurotrophin receptor compared with OCT4 and SOX2;2. To obtain the P75-positive ectomsenchymal stem cells(P₇₅⁺EMSCs), we used flow cytometry（FCM） to sort P₇₅⁺EMSCs. And then, they were cultured and characterised for stem cell makers.3. To study the capacity of odontogenic differentiation P₇₅⁺EMSCs and compare the differentence of odontogenic differentiation between P₇₅⁺ and P75-EMSCs, they were induced to differentiate into odontoblast-like cells using HAT-7 condition medium prepared from epithelial cell line of cervical loop of incisor of SD rat in vitro, and recombinating with cervical loop cell of the incisor of SD rat in vivo respectively. In vitro, P₇₅⁺ and P75-EMSCs were cultured with HAT-CM and collected at different time point of 0, 4, 8, 12 days to observe the appearance changes, cell cycle, proliferation, ultrastructure and preform real-time PCR and western blot to detect the expression changes of DSP and DMP1. The mineralized matrix nodules were detected by Alizarin Red S Staining. In vivo, recombination was preformed by injecting epithelial and EMSCs pellets into a drop of type-I collagen, and the recombinant were transplanted into renal capsule of adult SD rat. Samples were harvested 4 weeks after subrenal capsule culture, then, processed for immunohistochemical staining（IHC）.4. To ascertain the molecular mechanism of the difference of odontogenic differentiation between P₇₅⁺ and P75-EMSCs, microarray analysis and KEGG was performed to evaluated the gene changes and metabolic pathway between P₇₅⁺ and P75-EMSCs. Smad4 specific small interfering RNA and activator kartogenin treatment were performed to evaluated the effects of Smad4 protein of TGF-βpathway to odontogenic differentiation of P₇₅⁺ and P75-EMSCs.Results:1. P75-NTR served as the marker of neural crest cells continuously expressed in the dental mesenchymal cells at all development stages of tooth and is able to be considered the reliable marker tracing the migration and differentiation of EMSCs. Meanwhile, P75-NTR play a role in participation in epithelial-mesenchymal interactions; Expressions of OCT4 and SOX2 were comparatively weaker at the early stage（dental lamina） than later stage（ bell stage）, which indicated they participated in epithelial-mesenchymal interactions with P75-NTR.2. The sorted P₇₅⁺EMSCs showed a high percentage（24.5%） in mesenchymal cells isolated from the first branchial arch of embryo of E11.5 SD rat and exhibited more uniformed shape than those of pre-sorting, atypical spindle shaped fibroblast morphology, in which there have different cell groups living together. CCK8 detection between the P₇₅⁺EMSCs and P75-EMSCs showed that the sorted P₇₅⁺EMSCs have stronger proliferation ability than that of P75-EMSCs. The sorted P₇₅⁺EMSCs not only positively expressed the mesenchymal stem cell marker Stro-1 and vimentin, but also the lineage-specific markers of CNC cells P75 NTR,Ap2α and HNK-1. The results showed that the P₇₅⁺EMSCs are the CNC-derived ectomesenchymal stem cells that have the double features with mesenchymal and neural crest cells. Expression of Oct-4 suggested that the sorted P₇₅⁺EMSCs have certain features of early embryonic stemness.3. In vitroinduction under HAT-7 condition medium, the changes of cell morphology, proliferation, cell cycle, and ultrastructure demonstrated that P₇₅⁺EMSCs differentiated into odontoblast-like cells which possessed secretion function. The expressions of gene and protein, Dspp（DSP） and DMP1, the marker of odontoblast, of P₇₅⁺EMSCs were significantly higher than P75-EMSCs after induction. Meanwhile, Mineralized nodules of P₇₅⁺EMSCs were remarkable more than P75-EMSCs. In vivo,P₇₅⁺EMSCs hierarchically closely encircled with the island-like structures of dental epithelial cells in collagen gel. The results of IHC showed that DSP and DMP1 proteins were detected only in P₇₅⁺EMSCs closely surrounding epithelial-mesenchymal interface. In contrast, DSP or DMP1 were not detected in P75-EMSCs between epithelial-mesenchymal interface. Via in vitro and in vivo induction model, the results of odontogenic induction demonstrated that the odontogenic potential of P₇₅⁺EMSCs were significantly higher than that of P75-EMSCs.4. To understand the molecular mechanisms underlying the more active odontogenic differentiation ability of P₇₅⁺ EMSCs compared to P75-EMSCs, we performed genome-wide gene expression profiling of P₇₅⁺ EMSCs and P75-EMSCs. Compared to the P75-EMSCs, there were 128 genes upregulated and 15 genes downregulated. KEGG pathway mapping of difference expression genes showed that TGF-βsignaling pathway might be involved in the difference of odontogenic differentiation ability between P₇₅⁺EMSCs and P75-EMSCs. Specially, as a key coactivator, Smad4 was confirmed downregulated in P75-EMSCs cells by Real-time PCR and Western blot analyses. Cells treated with HAT-CM for induction of odontogenic differentiation, P₇₅⁺EMSCs showed a higher level of DSP and DMP1 expression than P75-EMSCs. However, inhibition of Smad4 expression by si RNA resulted in a significantly decreased DSP and DMP1 expression in P₇₅⁺EMSCs. In P75-EMSCs cells, cells treated with SMAD4 activator Kartogenin significantly increased DSP and DMP1 expression. The results showed that the molecular mechanisms underlying the more active odontogenic differentiation ability of P₇₅⁺ EMSCs than P75-EMSCs may be regulated by TGF-β pathway in which SMAD4 served as a key mediator.Conclusions: Based on these findings, we can conclude that the sorted P₇₅⁺EMSCs derived from first branchial arch of E11.5d SD rat embryo, which were characterised as define CNC-derived ectomesenchymal stem cells, have more active potential of odontogenic differentiation compared with Non- CNC-derived ectomesenchymal stem cells under odontogenic induction in vitro and in vivo model. Smad4 might play a critical role in determination of the odonto-differentiation potential of CNC-derived EMSCs. P₇₅⁺EMSCs might act as a novel and potent stem cell resource that could be used for cell and gene therapy for tooth repair and regeneration.