Hard tissue regeneration using dental mesenchymal stem cells and embryonic stem cells that enabled us to trace neural crest cells and mesodermal cells

使用转基因小鼠，我们能够分别追踪NC细胞和中胚层细胞作为YFP^+细胞，我们建立了ES细胞系，以研究NC细胞或中胚层细胞是否由培养中的ESC产生。我们发现NC衍生的黑素细胞有效地从NC衍生的YFP^+细胞产生。NC衍生的YFP^+细胞也表达强烈的NC相关基因。我们还发现NC衍生的YFP^+细胞不仅分化为黑素细胞，还分化为神经元、脂肪细胞和成骨细胞。此外，为了检测成牙本质细胞在体内和体外的分化，我们通过将绿色荧光蛋白（GFP）插入Dspp基因的靶向载体构建了Dspp敲入ES（Dspp-KI ES）细胞系。然后，将Dspp-KI ES细胞注射到囊胚中，获得大量嵌合体小鼠。将培养6天的Dspp-KI ES细胞移植到肾被膜中，4周后在移植组织中检测到YFP^+细胞。我们进一步尝试通过NC在体外将这些ESC分化为成牙本质细胞。

Using transgenic mice that enabled us to trace NC cells and mesodermal cells as YFP^+ cells, respectively, we established ES cell lines to investigate whether NC cells or mesodemal cells were generated from ESCs in the culture. We found that NC-derived melanocytes were efficiently generated from NC-derived YFP^+ cells. The NC-derived YFP^+ cells also expressed strongly NC-associated genes. We also found that NC-derived YFP^+ cells differentiated into not only melanocytes, but also neurons, adipocytes, and osteoblasts. Furthermore, to detect the differentiation of odontoblasts in vivo and in vitro, we made Dspp Knock-In ES (Dspp-KI ES) cell lines by targeting vector that inserted GFP into Dspp gene. Then, Dspp-KI ES cells were injected into blastocysts, and large numbers of chimera mice were obtained. Dspp-KI ES-derived cells cultured for 6 days also were transplanted into kidney capsule, and 4 weeks after YFP^+ cells were detected in the transplanted tissues. We further try to differentiate these ESCs into odontoblasts in vitro through NC.