Slowly cycling cells and hair follicle stem cells

描述（申请人提供）：我们感兴趣的是阐明组织干细胞自我更新和命运选择的分子机制，在正常组织发育和稳态中。干细胞在未来治疗许多致命疾病方面有着巨大的希望，但它们的基本特性还没有被很好地理解。在缺乏特定生化标记的情况下，很难识别组织干细胞，这阻碍了该领域的进展。越来越多的证据表明，干细胞是不经常分裂的细胞，可以在许多小鼠组织中识别为DMA标签保留细胞（或lrc）。最近，我们开发了一种适用于许多自我再生组织的策略，将绿色荧光蛋白（GFP）融合到组蛋白H2B中，对LRCs进行脉冲标记。我们以小鼠皮肤和毛囊为模型系统，因为lrc与皮肤上皮干细胞之间的关系研究得最深入。在我们的初步工作中，我们追踪并分离了GFP lrc，并证明这些细胞中至少有一部分具有干细胞表型。在本提案中，我们描述了三个具体目标策略，以更详细地研究lrc和干细胞之间的关系。我们将：1)将LRCs划分为不同的亚种群，并研究它们的严酷程度；2)检查它们特殊的细胞周期特性；3)描述它们不同的基因表达模式。为了实现这些目标，我们使用体内细胞跟踪和体外细胞分选方法结合各种检测方法，包括：毛发再生和皮肤损伤；细胞培养和皮肤重建；细胞周期动力学；和微阵列。

DESCRIPTION (provided by applicant): We are interested in elucidating the molecular mechanisms involved in tissue stem cell self-renewal and fate choice, in normal tissue development and homeostasis. Stem cells hold great promises for future therapies of numerous deadly diseases, yet their basic properties are not well understood. Progress in the field has been hampered by the difficulty of identifying tissue stem cells in the absence of specific biochemical markers. Increasing evidence suggested that stem cells are infrequently dividing cells, that could be identified in many mouse tissues as DMA label retaining cells (or LRCs). Recently we developed a strategy applicable to many self-regenerating tissues to pulse-label LRCs with the green fluorescent protein (GFP) fused to histone H2B. We have used the mouse skin and the hair follicle as model systems, since the relation between LRCs and skin epithelial stem cells had been investigated in most depth. In our preliminary work we tracked and isolated GFP LRCs and demonstrated that at least a fraction of these cells had a stem cell phenotype. In this proposal we describe in three Specific Aims strategies to examine in more detail the relation between LRCs and stem cells. We will: 1) classify LRCs into distinct sub- populations and investigate their sternness; 2) examine their special cell cycle properties; 3) characterize their distinct patterns of gene expression. To accomplish these goals we use in vivo cell tracking and in vitro cell sorting methods combined with a variety of assays which include: hair regeneration and skin wounding; cell culture and skin reconstitution; cell cycle kinetics; and microarrays. First, our data will have global relevance for the isolation and characterization of putative stem cells with LRCs properties from many tissues. Although we focus on the skin, our work will lead the way for similar approaches in many systems which lack the specific means to separate stem cells from other cells of their tissue of residence. Second, stem cell proliferation must be tightly controlled in tissues to regulate the balance between normal and abnormal growth. Our studies will unravel some of the mechanisms developed by putative SCs to control their proliferation, an important first step in understanding such perturbation in the diseased tissues. Finally, our work will provide some of the basic knowledge necessary for more efficient manipulation of skin stem cells for clinical applications such as cell transplantation on burn victims.