Unbiased mapping of skeletal stem cell function at single cell resolution in homeostasis and injury.

项目摘要 组织完整性由专门的成体干细胞维持，这些干细胞补充陈旧和受损的细胞。在老化过程中 在疾病状态下，干细胞功能受损，导致结构进行性退化 和重要器官的功能。这一点在骨骼中尤为明显，骨骼变得越来越脆弱 随着年龄的增长容易骨折。揭示和对抗骨骼退行性变的机制，增强 骨修复，我们必须首先定义骨骼干细胞和祖细胞(SSPC)在体内的行为，并确定 细胞功能的基本调节器。这在技术上是一项具有挑战性的壮举，因为与其他器官相比， 干细胞驻留在特定的物理位置，很容易通过基因和蛋白质的表达来识别， 具有祖细胞活性的细胞在整个骨骼中都可以找到，但没有特定的标志物来识别这些细胞 SITE。因此，我们仍然对SSPC知之甚少，包括是否存在多潜能干细胞。 如果骨骼是由一群血统受限的祖细胞维持的，则为活体。此外，目前还不清楚是否 来自不同地点的祖先在功能上是相同的。最近，我们发现了几具截然不同的骨骼 具有干细胞特征的种群对伤害表现出明显的动态反应。我们假设 这些与骨骼不同解剖区域的SSPC亚型相对应，并且它们发挥着独特的作用 骨骼再生在动态平衡和损伤修复中的作用。我们将利用尖端技术来 研究这一假设。在这项提议的第一部分，我们将并行地在体内进行克隆功能和 转录分析以不偏不倚的方式识别具有祖细胞活性的细胞，询问 SSPC在特定骨骼区域的分化潜能和区分基因表达特征 与精确的细胞行为相关。这些实验将第一次确定表型 各个SSPC在其本地环境中和跨不同的本地利基环境中的功能，提供了一个整体 SSPC概况，以确定干细胞活性和标志物的新调控因子 以实现对治疗有用的细胞群的分离。在这份提案的第二部分，我们将绘制图表 个体祖细胞对不同类型骨修复的动态反应和贡献 受伤。这些数据将揭示驱动骨愈合不同阶段的关键细胞群及其 对损伤的转录反应，包括在祖细胞中丰富的信号通路信号。同舟共济 这项研究将剖析骨骼干细胞状态的复杂网络，以促进靶向 改善骨骼健康和骨折愈合的策略。

Project Summary Tissue integrity is maintained by specialized adult stem cells that replenish old and damaged cells. During aging and in disease states, stem cell function is compromised leading to progressive degeneration of the structure and function of vital organs. This is particularly apparent in the skeleton, which becomes increasingly fragile and prone to fractures as we age. To uncover and counteract the mechanisms of skeletal degeneration and enhance bone repair, we must first define skeletal stem and progenitor cell (SSPC) behavior in vivo and determine the essential regulators of cellular function. This is a technically challenging feat as, in contrast to other organs where stem cells reside within a defined physical location and are readily identified by gene and protein expression, cells with progenitor activity can be found throughout bone and there are no specific markers to identify these in situ. As a result, we still know remarkably little about SSPCs, including whether a multipotent stem cell exists in vivo or if bone is maintained by a pool of lineage-restricted progenitors. Additionally, it is unclear whether progenitors from different locations are functionally equivalent. Recently, we identified several distinct skeletal populations with stem cell characteristics that demonstrate distinct dynamic responses to injury. We hypothesize that these correspond to SSPC subtypes from different anatomical regions of bone and that they play unique roles in skeletal regeneration in homeostasis and injury repair. We will leverage cutting-edge technologies to investigate this hypothesis. In the first part of this proposal, we will perform parallel in vivo clonal functional and transcriptional analysis to identify cells with progenitor activity in an unbiased manner, interrogate the differentiation potential of SSPCs in defined bone regions and distinguish gene expression signatures associated with precise cellular behaviors. These experiments will, for the first time, establish the phenotype and function of individual SSPCs in their native environment and across distinct local niches, providing a holistic overview of the SSPC landscape to enable the identification of novel regulators of stem cell activity and markers to enable the isolation of therapeutically useful cell populations. In the second part of this proposal, we will chart the dynamic response and contribution of individual progenitors to the repair of various types of bone injury. These data will reveal the key cell populations driving discrete stages of bone healing and their transcriptional response to injury, including the signaling pathway signatures enriched in progenitors. Together this study will dissect the complex web of skeletal stem cell states to facilitate the development of targeted strategies to improve bone health and fracture healing.