Defining Periosteal Skeletal Stem Cell Heterogeneity and Age-associated Change

项目摘要/摘要 骨骼干细胞(SSCs)存在于骨髓和骨膜(骨的外层)中， 对于终生再生的骨骼和软骨，使它们成为有希望的退行性疾病的治疗目标 骨病和骨缺损症。这些广泛分布的骨形成干细胞很可能是 异质性，但目前还没有分子标记来明确定义它们在体内的种群。因此， 从基因上定义、表征和操纵SSCs一直是一个巨大的挑战。功能界别 不同SSCs亚群的差异，以及调节不同SSCs亚群的特定因素和分子 在不同组织位置的SSC亚群，基本上是未知的。这些障碍限制了我们的 有能力发现更好的方法来操纵和改善内源性SSC功能，目标是逆转 退行性骨病和陈旧性骨缺损的情况。 年龄是许多骨骼和软骨疾病的重要危险因素，如骨质疏松症和关节炎。 尽管临床上骨和软骨随年龄的变化已经被广泛研究，但潜在的 原因仍然难以捉摸。像其他与年龄相关的功能衰退一样，至少部分骨骼和 老年人的软骨形成归因于SSCs数量和功能的变化。然而，由于 对于上述挑战，SSC亚种群构成的年龄相关变化以及 SSC种群内的细胞和分子变化仍然知之甚少。 这项提议的目标是从分子上定义SSc群体的活体身份，以 表征SCC亚群(SCC异质性)，并检查SSC种群的变化 与老鼠的年龄有关。我们先前的研究表明骨膜SSCs可以由以下基因定义 粘病毒抗性-1(MX1)标志物和α-平滑肌肌动蛋白(α)间充质标志物。 此外，我们还发现Mx1+GFP SMA+骨膜SSCs，而不是α-GFP+骨髓SSCs 对损伤作出反应，为体内损伤修复提供新的成骨细胞。因此，骨膜间充质干细胞对骨膜间充质干细胞的生长至关重要。 体内损伤修复成骨细胞的终生补充。使用这些SSCs作为模型和积极对照， 我们计划通过以下目标来实现这个项目的目标。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 小鼠骨膜组织中的菌落。细胞类型、骨膜细胞的层次结构和分子 骨膜SSCs及其亚群的特性将由单细胞RNA序列确定。纯净的 以Mx1+αSMA+骨膜SSCs作为参照和阳性对照。为了进行比较，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分子差异 SSC的种群。其次，为了确定SSc及其亚群的年龄相关变化，我们将 从老年(2岁)小鼠分离骨膜细胞，进行单细胞RNA-SEQ。转录组的变化 青年和老年骨膜SSCs的特征，包括年龄相关的转录特征 就像神秘的抄写一样。在这项工作完成后，我们将获得对该法规的新的生物学见解 在不同部位发现不同的SSC亚群，为骨病的逆转和治疗提供新的靶点 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.