Generation of human dental epithelial lineage cells from embryonic stem cells

描述（申请人提供）：牙齿器官的形成是由外胚层上皮细胞和颅神经嵴分化的间充质细胞之间的信号相互作用而形成的。邻近牙上皮的牙间充质细胞分化成成牙本质细胞，形成牙本质。牙上皮细胞分化为成釉细胞，成釉细胞经过多个阶段分化，形成牙釉质。成人牙齿中不存在牙釉质上皮，目前也没有现成的牙上皮细胞来源。这一限制，以及缺乏对人类牙釉质形成机制的了解，影响了我们制定牙齿再生策略的能力。我们已经成功地显微解剖了人胎牙器官的成釉细胞，并开始用qPCR表征这些细胞对牙釉质基质蛋白的表达。在平行研究中，我们已经将人胚胎干细胞（hESCs）分化为上皮表型。我们假设牙齿上皮系细胞可以由多能性人胚胎干细胞产生。在这项应用中，我们将建立我们的初步研究，以确定hesc来源的上皮细胞分化为人类成釉细胞谱系细胞所需的基因和壁龛。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)鉴定人类成釉细胞谱系细胞与hesc来源的上皮细胞之间表达差异的基因。2)确定牙间质在诱导hesc源性上皮细胞向成釉细胞系转变中的作用。在目标1中，我们将使用人类全转录微阵列分析和qPCR来鉴定与hesc来源的上皮细胞相比，成釉细胞谱系细胞中显著上调的转录因子和细胞表面蛋白。在目标2中，我们将通过使用包含Pitx2阳性上皮细胞和牙间充质细胞的空间重构三维壁龛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代人类缺失的牙齿。在这项R03拨款中，我们将朝着这一目标迈进一步，重点研究人类牙齿上皮细胞谱系的工程，这是牙齿再生的关键挑战。此外，这些关于牙齿器官中上皮-间充质细胞相互作用的研究，可以作为一个模型系统，用于更广泛地了解其他上皮附属物的器官发生机制。

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.