Development of technical bases for biotooth formation by using iPS cells

诱导性多能干细胞（Induced pluripotent stem cells，iPS）是由体细胞通过同时导入多种因子而产生的，并分化为具有广泛增殖能力的3种胚层细胞。最近，许多研究人员报道，iPS细胞在适当条件下可以分化为不同的细胞类型，如神经元、心肌细胞和肾谱系细胞。因此，iPS细胞已成为牙齿再生的潜在细胞来源。本研究的目的是通过诱导的iPS细胞诱导的牙胚细胞与小鼠牙胚细胞重组形成生物牙。首先，我们建立了iPS向间充质干细胞（MSC）的诱导方法。分化后的细胞表达MSC标志物STRO-1，表达率达90%以上，同时也表达其它MSC标志物。此外，我们建立了一种培养方案，以诱导iPS细胞分化为神经嵴样细胞（NCLC），这是一种牙齿间充质细胞的前体细胞。经诱导后，大部分细胞表达神经嵴细胞标志物。裸鼠皮下移植后未形成畸胎瘤，提示其临床应用安全。此外，在NCLC和小鼠牙上皮的重组培养中，NCLC表现出涉及牙齿间充质细胞的基因表达模式。部分NCLC也表达牙本质涎蛋白。小鼠牙上皮条件培养液进一步促进NCLC向成牙本质细胞分化。这些结果表明，iPS细胞是牙齿再生和牙齿发育研究的有用细胞来源。

Induced pluripotent stem(iPS) cells are generated from somatic cells by the simultaneous introduction of several factors ; and they differentiate into the3embryonic germlayers with an extensive proliferative capacity. Recently, many researchers have reported that iPS cells can differentiate into different cell types, such as neurons, cardiac myocytes, and renal lineage cells, under appropriate conditions. Therefore, iPS cells have emerged as potential cell sources for tooth regeneration. In this study, we aimed to form biotooth by recombination of iPS cell-induced dental germ cells with mouse dental germ cells. First, we established the induction methods of iPS into mesenchymal stem cell(MSC). The differentiated cells expressed STRO-1, MSC marker, more than90%, as well as other MSC markers. Further, we established a culture protocol to induce the differentiation of iPS cells into neural crest like cells(NCLC), which is a precursor cell of dental mesenchymal cells. After the induction, the most of cells expressed neural crest cells markers. The cells did not form teratomas after transplantation into nude mice subcutaneously, suggesting the safety for clinical applications. Moreover, in recombination cultures of NCLC and mousedental epithelium, NCLC exhibited a gene expression pattern involving dental mesenchymal cells. Some NCLC also expressed dentin sialoprotein. Conditioned medium of mouse dental epithelium cultures further enhanced the differentiation of NCLC into odontoblasts. These results suggest that iPS cells are useful cell sources for tooth regeneration and tooth development studies.