De novo generation of mammalian tooth from stem cells

描述(申请人提供)：大约20%的美国人有先天缺失的牙齿。牙齿脱落的几率也会随着年龄的增长而增加。在这种情况下，自然再生或体外生成的种植牙将是完美的替代品。使用自我更新干细胞的组织工程正在成为修复或替换受损组织或器官的最强大和最有前途的方法之一。由定向分化干细胞组装成功能器官将具有重要的临床应用价值。我们的长期努力是让人类牙齿再生成为现实。我们设想未来有两种方法可以再生人类牙齿：(1)确定启动牙齿发育程序的关键因素(S)，并应用该因素(S)允许牙齿重建；(2)从干细胞体外生成种植牙。在这项应用中，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检测大鼠骨髓间充质干细胞潜在成牙能力的特性。我们将首先确认我们的初步结果，即当遇到牙源性微环境时，BMSCs可以分化为成牙本质细胞。在诱导基因表达和牙芽形成方面，BMSCs对牙源性信号的反应方式是否与牙间充质相似，将通过组织重组和蛋白质浸泡微珠植入来测试。此外，如果BMP4可以赋予BMSCs成牙能力，也将进行测试。目的2将确定骨髓间充质干细胞能否被牙源性微环境或潜在的牙源性信号分子诱导或调节，并获得成牙潜能，当与胚胎上皮重组时能够诱导牙齿形成，或者成为成牙能力，能够对牙源性信号做出反应，以支持牙齿形成。将通过体外组织重组、器官培养、原位杂交和肾包膜移植来解决这些问题。最后，在目标3中，我们将测试体外组装的牙胚是否可以植入，并通过宫外手术在口腔内发育成功能牙齿。从这些研究中获得的信息可以在不久的将来转化为对人类的应用。

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.