Functional characterization of adult taste stem cells

 描述（由申请人提供）：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，但舌后部的味觉干细胞表达Lgr 5，舌前部和后部的味觉干细胞均表达Lgr 6。为了进一步了解味觉干细胞，我们拟采用遗传谱系追踪的方法，建立一种新的细胞培养方法，进一步鉴定Lgr 5+和Lgr 6+味觉细胞的特性，并测定其在体内和体外的再生能力。在这个项目中，我们将研究两个重叠的领域：在目标1中，我们 将确定Lgr 5+和Lgr 6+细胞是否是体内味觉干/祖细胞。我们将使用现有的基因工程小鼠和各种标记物诱导方案详细描述Lgr 5+和Lgr 6+细胞的性质。Subaims将决定是否（目的1.1）强Lgr 5+细胞是舌后部的味觉干细胞，（目的1.2）Lgr 5+细胞在成熟味蕾细胞的过程中产生Shh+细胞，（目标1.3）单个味蕾是单克隆的（来源于单个祖细胞）或寡克隆（来源于少数祖细胞）和Lgr 5+细胞是多能的（分化成不同的细胞类型）或单能Lgr 6+细胞代表舌前部和后部的成体味觉干/祖细胞。在目的2中，我们将在体外确定Lgr 5+和Lgr 6+细胞的再生能力。单个Lgr 5+肠干细胞能够在体外构建隐窝-绒毛结构。我们将通过FACS分选从舌中分离Lgr 5+和Lgr 6+干细胞，并在超低附着板中的限定培养基中培养它们，以检查它们的生长、扩增和长期传代（目的2.1），以及Lgr 5+和Lgr 6+细胞的相关性。使用基于Matrigel的三维培养系统，我们将确定Lgr 5+和Lgr 6+细胞的再生能力，单个Lgr 5+和Lgr 6+细胞是多能的还是单能的，以及哪些培养组分影响生长、扩增和分化（目标2.2）。这些研究领域是治疗味觉障碍患者（如接受头颈部放疗或化疗的癌症患者）的再生方法的重要第一步，并可能导致开发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.