Defining Periosteal Skeletal Stem Cell Heterogeneity and Age-associated Change

项目总结/摘要 骨骼干细胞（SSC）存在于骨髓和骨膜（骨的外层）中， 骨和软骨的终身再生，使它们成为退行性疾病的有希望的治疗靶点。 骨疾病和骨缺损。这些广泛分布的成骨干细胞很可能是 这些细胞是异质性的，但是没有分子标记物特异性地定义它们在体内的群体。因此，我们认为， 从基因上定义、表征和操纵SSC一直是一个巨大的挑战。功能 不同SSC亚群的差异，以及调节不同SSC的特定因子和分子， 不同组织位置的SSC亚群基本上是未知的。这些障碍限制了我们 能够发现更好的方法来操纵和改善内源性SSC功能，以逆转 退行性骨疾病和老年骨缺损的情况。 年龄是许多骨和软骨疾病的重要风险因素，如骨质疏松症和关节炎。 虽然骨和软骨随年龄的临床变化已被广泛研究，但潜在的骨和软骨损伤可能是一个潜在的问题。 原因仍然难以捉摸。像其他与年龄相关的功能衰退一样，至少有一些骨骼缺陷和 老年人的软骨损伤归因于SSC的数量和功能的变化。但由于 对于上述挑战，SSC亚群组成的年龄相关变化，以及 SSC群体内的细胞和分子变化仍然知之甚少。 该提案的目标是从分子上定义SSC群体的体内身份， 表征SCC亚群（SCC异质性），并检查SSC群体的变化 与老鼠的年龄有关。我们以前的研究表明，骨膜SSCs可以通过以下基因来定义： 粘液病毒抗性-1（Mx 1）标志物和α平滑肌肌动蛋白（αSMA）间充质标志物。 此外，我们发现Mx 1 +αSMA+骨膜SSCs，而不是Nestin-GFP+骨髓SSCs，能够快速地诱导成纤维细胞增殖。 对损伤作出反应，并为体内损伤修复提供新的成骨细胞。因此，骨膜SSCs对于 体内损伤修复成骨细胞的终身补充。使用这些SSC作为模型和阳性对照， 我们计划透过以下目标，达到这个计划的目标。首先，使用最新的单细胞RNA 测序（RNA-seq）技术，对干细胞进行全面的分析和鉴定 在小鼠骨膜组织中的种群。细胞类型、骨膜细胞的层次结构和分子生物学 骨膜SSC及其亚群的特征将通过单细胞RNA-seq来定义。纯化 Mx 1 +αSMA+骨膜SSC将用作参考和阳性对照。为了比较，我们将执行一个 对骨髓SSCs进行类似的分析，以进一步确定这两种不同的SSCs之间的分子差异， SSC的人口。其次，为了确定SSC及其亚群的年龄相关变化，我们将 对从老年（2岁）小鼠分离的骨膜细胞进行单细胞RNA-seq。转录组变化 年轻和老年骨膜SSCs之间的特征，包括年龄相关的转录组学特征， 比如神秘的转录这项工作完成后，我们将对生物学的调控实现新的见解 不同部位的不同SSC亚群，为逆转骨疾病提供了新的治疗靶点， 缺陷

PROJECT SUMMARY/ABSTRACT Skeletal stem cells (SSCs) reside in the bone marrow and periosteum (outer layer of bone) and contribute to the lifelong regeneration of bone and cartilage, making them as a promising therapeutic target for degenerative bone diseases and bone defects. These broadly distributed bone forming stem cells are likely to be heterogeneous, and yet there is no molecular marker that specifically defines their population in vivo. Hence, genetically defining, characterizing, and manipulating SSCs has been a tremendous challenge. The functional differences in different subpopulations of SSCs, as well as specific factors and molecules that regulate different SSC subpopulations in different tissue locations, are essentially unknown. These obstacles have limited our ability to discover better ways to manipulate and improve endogenous SSC functions with the goal of reversing conditions of degenerative bone diseases and aged bone defects. Age is a significant risk factor for many disorders of bone and cartilage, such as osteoporosis and arthritis. Although the clinical changes in bone and cartilage with age have been extensively studied, the underlying causes remain elusive. Like other age-associated functional declines, at least some of the defects in bones and cartilage in the elderly have been attributed to changes in the populations and functions of SSCs. However, due to the challenges described above, the age-associated changes in SSC subpopulation composition, as well as cellular and molecular changes within SSC populations remain poorly understood. The goal of this proposal is to molecularly define the in vivo identity of the SSC population, to characterize SCC subpopulations (SCC heterogeneity), and to examine changes in the SSC population associated with age in mice. Our previous studies showed that periosteal SSCs can be genetically defined by the myxovirus resistance-1 (Mx1) marker and the alpha smooth muscle actin (αSMA) mesenchymal marker. Furthermore, we found that Mx1+αSMA+ periosteal SSCs, rather than Nestin-GFP+ bone marrow SSCs, rapidly respond to injury and provide new osteoblasts for injury repair in vivo. Hence, periosteal SSCs are critical for the lifelong replenishment of injury-repairing osteoblasts in vivo. Using these SSCs as a model and positive control, we plan to achieve the goal of this project through the following aims. First, using the latest single-cell RNA sequencing (RNA-seq) technology, to perform a comprehensive profiling and identification of the stem cell population in mouse periosteal tissue. Cell types, the hierarchal structure of periosteal cells, and molecular characteristics of periosteal SSCs and their subpopulations will be defined by single-cell RNA-seq. Purified Mx1+αSMA+ periosteal SSCs will be used as a reference and positive control. For comparison, we will perform a similar analysis for bone marrow SSCs to further define the molecular differences between these two different populations of SSCs. Second, to determine age-associated changes in SSCs and their subpopulations, we will perform single-cell RNA-seq for periosteal cells isolated from aged (2-year old) mice. Changes in transcriptome between young and old periosteal SSCs will be characterized, including age-associated transcriptomics features like cryptic transcription. Upon completion of this work, we will achieve new biological insights into the regulation of different SSC subsets in different locations, providing new therapeutic targets for reversing bone diseases and defects.