De novo generation of mammalian tooth from stem cells

描述（由申请人提供）：大约20%的美国人口有先天性缺牙。牙齿脱落的机会也随着人的年龄沿着增加。在这种情况下，自然再生或体外生成的可植入牙齿将是完美的替代品。利用具有自我更新能力的干细胞进行组织工程是修复或替代受损组织或器官的最有力和最有前途的方法之一。由定向分化的干细胞组装功能器官将具有重要的临床应用。我们的长期努力是使人类牙齿再生成为现实。我们设想了两种方法来再生人类牙齿在未来：（1）确定可以启动牙齿发育程序的关键因素，并应用这些因素来重建牙齿;（2）从干细胞中体外生成可植入牙齿。在本申请中，我们将使用啮齿动物作为模型系统，测试从骨髓基质细胞（BMSCs）生成可植入牙齿器官的可能性。提出了三个具体目标。目的1研究大鼠骨髓间充质干细胞的成牙潜能。我们将首先证实我们的初步结果，即骨髓间充质干细胞可以分化成成牙本质细胞时，牙源性微环境中遇到。BMSCs是否能以与牙间充质相似的方式对牙源性信号作出反应，在诱导基因表达和牙芽形成方面，将通过组织重组和蛋白浸泡的珠粒植入来测试。此外，如果BMP 4可以赋予BMSCs成牙能力也将被测试。目的2将确定BMSCs是否可以通过牙源性微环境或潜在的牙源性信号分子诱导或“调节”，并获得牙源性潜能，当与胚胎上皮重组时能够诱导牙齿形成，或成为牙源性主管细胞，能够响应牙源性信号以支持牙齿形成。体外组织重组、器官培养、原位杂交和肾被膜移植将被用来解决这些问题。最后，在目标3中，我们将测试体外组装的牙胚是否可植入，并可通过子宫外手术在口腔中发育成功能性牙齿。从这些研究中获得的信息可以在不久的将来转化为人类的应用。

DESCRIPTION (provided by applicant): About 20% of U.S. population have congenitally missing teeth. Chance of tooth loss also increases along with a person's age. A naturally regenerated or in vitro generated implantable tooth would be a perfect replacement in such situations. Tissue engineering using the self-renewing stem cells is becoming one of the most powerfull and promising approaches to repair or replace an injured tissue or organ. Assembly of a functional organ from directed differentiating stem cells will have significant clinical application. Our long-term effort is to make human tooth regeneration become a reality. We envision two approaches to regenerate the human tooth in the future: (1) identifying key factor(s) that can initiate tooth developmental program, and applying the factor(s) to allow the rebuilding of teeth; and (2) generating implantable teeth in vitro from stem cells. In this application, we will use rodents as a model system test the possibility of generating an implantable tooth organ from bone marrow stromal cells (BMSCs). Three specific aims are proposed. Aim 1 is proposed to examine the properties of the potential odontogenic competence in the rat BMSCs. We will first confirm our preliminary results that BMSCs could differentiate into odontoblasts when an odontogenic microenvironment was encountered. Whether BMSCs can respond to odontogenic signals in a similar way as dental mesenchyme, in terms of induction of gene expression and tooth bud formation, will be tested by tissue recombination and protein-soaked bead implantation. Additionally, if BMP4 can confer BMSCs odontogenic competence will also be tested. Aim 2 will determine if BMSCs can be induced or "conditioned" by an odontogenic microenvironment or potential odontogenic signaling molecules, and to acquire odontogenic potential, capable of inducing tooth formation when recombined with an embryonic epithelium, or to become odontogenic competent, capable of responding to odontogenic signals to support tooth formation. In vitro tissue recombination, organ culture, in situ hybridization, and kidney capsule grafting will be employed to address these questions. Lastly in Aim 3, we will test if in vitro assembled tooth germ is implantable and can develop into a functional tooth in the oral cavity by exo uteru surgery. Information obtained from these studies can be translated into application in humans in the near future.