Generation of human dental epithelial lineage cells from embryonic stem cells

描述(申请人提供)：牙齿器官是由外胚层来源的上皮细胞和颅顶分化的间充质细胞之间顺序的相互信号相互作用形成的。与牙齿上皮相邻的牙齿间充质细胞分化为成牙本质细胞，形成牙本质。牙齿上皮细胞分化为成釉细胞，成釉细胞经历多个分化阶段，以协调釉质的形成。釉质上皮在成人牙齿中不存在，目前也没有现成的牙上皮细胞来源。这一局限，加上对引导人牙釉质形成的机制缺乏了解，影响了我们制定牙齿再生策略的能力。我们已经成功地从人胎牙器官中分离出成釉细胞，并开始用定量聚合酶链式反应鉴定这些细胞表达釉质基质蛋白。在平行研究中，我们将人类胚胎干细胞(HESCs)分化为上皮表型。我们假设牙齿上皮细胞系可以从多能的人类胚胎干细胞中产生。在这项应用中，我们将在我们的初步研究的基础上，确定hESC来源的上皮细胞分化为人类成釉细胞系所需的基因和生态位。这些研究将使我们更接近牙齿再生的最终目标。我们的具体目标如下：1)确定人类成釉细胞系细胞与人胚胎干细胞来源的上皮细胞相比差异表达的基因。2)探讨牙本质间充质在诱导人胚胎干细胞来源的上皮细胞向成釉细胞定向分化中的作用。在目标1中，我们将使用人全转录微阵列分析和qPCR来鉴定成釉细胞系细胞中显著上调的转录因子和细胞表面蛋白，与hESC来源的上皮细胞相比。在目标2中，我们将利用包含Pitx2阳性上皮细胞和牙间充质细胞的空间重组三维生态位来研究牙源性相互信号相互作用在促进上皮细胞分化中的作用。我们的最终目标是开发基于细胞的策略来替换人类缺失的牙齿。在这笔R03拨款中，我们将向这一目标更近一步，专注于工程人类牙齿上皮细胞，这是牙齿再生中的一个关键挑战。此外，这些关于牙齿器官中细胞间充质-上皮相互作用的研究，可以作为一个模型系统，以更广泛地了解其他上皮性附件组织发生的机制。 与公共健康相关：对生物工程牙齿的需求正在增加。然而，设计人类牙齿上皮细胞是解决这一需求的关键障碍。我们在这个方案中的目标是将人类胚胎干细胞分化为牙齿上皮细胞。我们假设，如果提供适当的合适的壁龛，人类牙齿上皮细胞可以从多潜能的人类胚胎干细胞中产生。

DESCRIPTION (provided by applicant): Tooth organs form through the sequential reciprocal signaling interaction between ectodermally-derived epithelial cells and cranial neurocrest differentiated mesenchymal cells. Dental mesenchymal cells adjacent to the dental epithelium, differentiate into odontoblasts, which form dentin. The dental epithelial cells differentiate into ameloblasts that go through multiple stages of differentiation to orchestrate enamel formation. The enamel epithelium does not exist in adult teeth, and currently there are not readily available sources of dental epithelial cells. This limitation, along with a lack of knowledge on mechanisms guiding the formation of human enamel, impacts our capacity to develop strategies for tooth regeneration. We have successfully microdissected ameloblasts from human fetal tooth organs and have begun to characterize these cells for expression of enamel matrix proteins by qPCR. In parallel studies we have differentiated human embryonic stem cells (hESCs) into epithelial phenotype. We hypothesize that dental epithelial lineage cells can be generated from pluripotent human embryonic stem cells. In this application, we will build on our preliminary research to identify genes and niches required for differentiation of hESC-derived epithelial cells into human ameloblast lineage cells. These studies will take us closer to our ultimate goal of tooth regeneration. Our specific aims are follows: 1) To identify genes that differentially expressed by human ameloblast lineage cells as compared to hESC-derived epithelial cells. 2) To determine the role of the dental mesenchyme in inducing hESC-derived epithelial cells commitment to ameloblast lineage cells. In aim 1, we will use Human Whole Transcript Microarray analysis and qPCR to identify transcription factors and cell surface proteins that are significantly up-regulated in ameloblast lineage cells as compared to hESC-derived epithelial cells. In aim 2, we will investigate odontogenic reciprocal signaling interactions in promoting epithelial cell differentiation by using spatially reconstituted three- dimensional niches containing Pitx2 positive epithelial cells and dental mesenchymal cells. Our ultimate goal is to develop cell-based strategies to replace missing teeth in humans. In this R03 grant, we will move a step closer to this goal, focusing on engineering human dental epithelial lineage cells, which is a critical challenge in tooth regeneration. In addition, these studies of epithelial- mesenchymal interactions of cells in tooth organ, can be used as a model system for a broader understanding of the mechanisms involved in the organgenesis of other epithelial appendages. PUBLIC HEALTH RELEVANCE: Demand for bioengineered teeth is increasing. However, engineering human dental epithelial cells is a critical hurdle in addressing this demand. Our goal in this proposal is to differentiate human embryonic stem cells into dental epithelial cells. We hypothesize that human dental epithelial cells can be generated from pluripotent human embryonic stem cells if the appropriate niches are provided.