Research On The Potential Characteristics Of Tooth Formation In Neural Crest Stem Cells

ObjectiveIn recent years, the development of the teeth, and stem cell theory of tooth regeneration technology become the research hot spot. Neural crest stem cells(NCSCs) are a multipotent embryonic cell population that contributes to the formation of various craniofacial structures including teeth, it is an important development in stem cell biology and regenerative medicine. Through the experiments on animals,The present study was aimed to observe the potential contribution of neural crest stem cells to tooth formation. The results will provide experimental data for the new renewable resources and the environment for tissue-engineered tooth body building.MethodsOne to three healthy adult ICR 8.5 days’ pregnant rats, 20 g~25 g of weight were chosen at the present study. The 4-6 somites were isolated from the pregnant rat embryos under the stereoscopic microscope.The neural crest stem cells were migrated from cranial neural tubes, then they were cultured and passaged. The neural crest stem cells were identified with the immunofluorescence.The results will provide theoretical basis for the potential contribution of neural crest stem cells for tooth formation.Results1. The morphological observation of the specimen in the experimental process:Under the stereoscopic microscope, the uterus and embryo of pregnant rat were observed in ED8.5. The 4-6 somites were successfully dissected and the cranial neural tube were isolated. It shows that the cranial neural tube is available.2. The observation of neural crest stem cells under the stereoscopic microscope:After the neural crest stem cells were cultured 2 days, the neural crest stem cells migrated from the edge of cranial neural tube, and grew aroud it. The cell is spindle shaped, medium nucleus and the cytoplasm is rich. We also observe it on 4 days, andon 2 days in the second generation. It shows that the culture and passage of the neural crest stem cells is available.3. Immunofluorescence test results: Identify the third generation neural crest stem cells with immunofluorescence. The results: Specific marker P75, HNK-1, AP2αpositive reaction; differentiated marker PAX6, SOX2 negative reaction. It shows the uniform and stable state of the neural crest stem cells.4. The result of preliminary experiment in vitro induced into tooth: In situ hybridization technique, immunofluorescence technique to detect the expression of gene PAX9, FGF10, BMP4. To locate the cells by inserting GFP into genes, it shows that Neural crest stem cells closely grow around epithelial tissue, its characteristic is similar to dental mesenchyme cells.Conclusion1. The isolation and culture with serum-free medium of neural crest stem cell has been established.2. The expression results of the neural crest stem cell in morphology and gene expression, and the detection of specific protein markers has been obtained stably.