Functional characterization of adult taste stem cells

 描述(申请人提供)：这项拟议的研究将研究成人味觉干细胞如何产生成熟的味觉细胞，并将开发一种培养系统来生长、扩增和分化味觉干细胞，以研究体外味觉再生过程。味觉干细胞的性质尚不清楚，但在舌后部的味觉干细胞表达Lgr5；在舌前部和舌后部的味觉干细胞表达Lgr6。为了扩大我们对这些细胞的初步了解，我们建议使用遗传谱系追踪策略并建立一种新的细胞培养方法来进一步鉴定Lgr5+和Lgr6+味觉细胞，并确定它们在体内和体外的再生能力。在这个项目中，我们将调查两个重叠的领域：在目标1中，我们 将在体内确定Lgr5+和Lgr6+细胞是否为味觉干/祖细胞。我们将使用现有的基因工程小鼠和各种标记诱导方案，详细描述Lgr5+和Lgr6+细胞的性质。Subaims将确定(Aim 1.1)强大的Lgr5+细胞是否是舌后的味觉干细胞，(Aim 1.2)Lgr5+细胞在通往成熟味蕾细胞的途中产生Shh+细胞，(Aim 1.3)单个味蕾是单克隆性的(来源于单个祖细胞)或寡克隆(源于少数祖细胞)，Lgr5+细胞是多能的(分化为不同的细胞类型)或单能的(发展为单一细胞类型)，以及(Aim 1.4)Lgr6+细胞代表舌前部和舌后部的成人味觉干/祖细胞。在目标2中，我们将测定Lgr5+和Lgr6+细胞的体外再生能力。单个Lgr5+肠道干细胞能够在体外构建隐窝绒毛结构。我们将通过FACS分选法从舌中分离出Lgr5+和Lgr6+干细胞，并将它们培养在特定的培养液中，在超低附着平板中培养，以检测它们的生长、扩增和长期传代(Aim 2.1)，以及Lgr5+和Lgr6+细胞的相关性。使用基于Matrigel的三维培养系统，我们将确定Lgr5+和Lgr6+细胞的再生能力，单个Lgr5+和Lgr6+细胞是多能还是单能，以及哪些培养成分影响生长、扩增和分化(目标2.2)。这些研究领域是朝着治疗味觉受损患者(如接受头颈部放射治疗或化疗的癌症患者)的再生方法迈出的重要的第一步，并可能导致开发一种用于筛选新型味觉调节剂的培养系统。

 DESCRIPTION (provided by applicant): This proposed research will examine how adult taste stem cells generate mature taste cells, and will develop a culture system to grow, expand, and differentiate taste stem cells to study taste regeneration processes in vitro. The nature of taste stem cells is unknown although those in the posterior tongue express Lgr5; those in both the anterior and posterior express Lgr6.To expand our preliminary understanding of these cells, we propose to use genetic lineage tracing strategies and to establish a novel cell culture approach to further characterize Lgr5+ and Lgr6+ taste cells and determine their regenerative capacity both in vivo and in vitro. In this project, we will investigate two overlapping areas: In Aim 1, we will determine whether Lgr5+ and Lgr6+ cells are taste stem/progenitor cells in vivo. We will characterize the nature of Lgr5+ and Lgr6+ cells in detail using existing genetically engineered mice and various marker induction regimens. Subaims will determine whether (Aim 1.1) the strong Lgr5+ cells are taste stem cells for posterior tongue, (Aim 1.2) Lgr5+ cells give rise to Shh+ cells en route to mature taste bud cells, (Aim 1.3) individual taste buds are monoclonal (derived from a single progenitor cell) or oligoclonal (derived from a few progenitor cells) and Lgr5+ cells are multipotent (differentiate into different cell types) or unipotent (develop into ony one cell type), and (Aim 1.4) Lgr6+ cells represent adult taste stem/progenitor cells in anterior and posterior tongue. In Aim 2, we will determine the regenerative capacity of Lgr5+ and Lgr6+ cells in vitro. Single Lgr5+ intestinal stem cells are able to build crypt-villus structures in vito. We will isolate Lgr5+ and Lgr6+ stem cells from tongue by FACS sorting and culture them in defined medium in ultra-low attachment plates to examine their growth, expansion, and long-term passages (Aim 2.1), as well as the relatedness of Lgr5+ and Lgr6+ cells. Using a Matrigel-based 3-D culture system, we will determine the regenerative capacity of Lgr5+ and Lgr6+ cells, whether single Lgr5+ and Lgr6+ cells are multipotent or unipotent, and which culture components affect growth, expansion, and differentiation (Aim 2.2). These areas of study are significant first steps toward a regenerative approach for treating patients who suffer from taste impairment, such as cancer patients receiving head and neck radiation therapy or chemotherapy, and may lead to the development of a culture system for screening novel taste modulators.