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Defining Periosteal Skeletal Stem Cell Heterogeneity and Age-associated Change

定义骨膜骨骼干细胞异质性和年龄相关的变化

基本信息

批准号：
9807858
负责人：
Weiwei Dang
金额：
$ 24万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-15 至 2021-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9807858
关键词：
2 year old Adult Age Aging American Arthritis Biological Bone Diseases Bone Injury Bone Marrow Bone Regeneration C57BL/6 Mouse Calvaria Cartilage Cell physiology Cells Characteristics Clinical Defect Degenerative Disorder Disease Elderly Expression Profiling Fracture Gene Expression Profiling Genetic Genetic Transcription Goals Heterogeneity Inflammatory Injury Label Location Longevity Mesenchymal Methods Modeling Molecular Mus Myxovirus Natural regeneration Osteoblasts Osteoporosis Periosteal Cell Periosteum Physiological Population Quality of life RNA analysis Regulation Reporter Reporting Resistance Resources Risk Factors Rodent Signal Transduction Site Smooth Muscle Actin Staining Method Stem cells Structure Technology Time Tissues Work aged base bone bone aging bone healing cell age cell type functional decline healing improved in vivo injury and repair insight intravital imaging long bone migration molecular marker mouse model nestin protein new therapeutic target single cell analysis single cell sequencing single-cell RNA sequencing skeletal stem stem cell population therapeutic target transcriptome transcriptomics

项目摘要

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.

项目概要/摘要骨骼干细胞 (SSC) 存在于骨髓和骨膜（骨的外层）中，并有助于骨和软骨的终生再生，使它们成为退行性病变的有前途的治疗靶点骨疾病和骨缺陷。这些广泛分布的骨形成干细胞很可能是异质性，但没有分子标记来具体定义它们在体内的群体。因此，从基因角度定义、表征和操纵 SSC 一直是一个巨大的挑战。功能性的不同SSC亚群的差异，以及调节不同亚群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危险因素，例如骨质疏松症和关节炎。尽管骨和软骨随年龄的临床变化已被广泛研究，但潜在的原因仍然难以捉摸。与其他与年龄相关的功能衰退一样，至少有一些骨骼和功能的缺陷老年人软骨的变化归因于SSC数量和功能的变化。然而，由于为了应对上述挑战，SSC 亚群构成与年龄相关的变化，以及 SSC 群体内的细胞和分子变化仍知之甚少。该提案的目标是从分子水平上定义 SSC 群体的体内身份，以表征 SCC 亚群（SCC 异质性），并检查 SSC 群的变化与小鼠的年龄有关。我们之前的研究表明，骨膜 SSC 可以通过基因定义粘病毒抗性 1 (Mx1) 标记和 α 平滑肌肌动蛋白 (αSMA) 间充质标记。此外，我们发现 Mx1+αSMA+ 骨膜 SSC，而不是 Nestin-GFP+ 骨髓 SSC，迅速对损伤做出反应并为体内损伤修复提供新的成骨细胞。因此，骨膜 SSC 对于体内损伤修复成骨细胞的终生补充。使用这些 SSC 作为模型和阳性对照，我们计划通过以下目标来实现该项目的目标。一、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群体。细胞类型、骨膜细胞的层次结构和分子骨膜 SSC 及其亚群的特征将通过单细胞 RNA 测序来定义。纯化的 Mx1+αSMA+ 骨膜 SSC 将用作参考和阳性对照。为了进行比较，我们将执行对骨髓 SSC 进行类似分析，以进一步明确这两种不同的分子之间的差异 SSC 群体。其次，为了确定 SSC 及其亚群与年龄相关的变化，我们将对从老年（2 岁）小鼠分离的骨膜细胞进行单细胞 RNA 测序。转录组的变化将表征年轻和老年骨膜 SSC 之间的特征，包括与年龄相关的转录组学特征就像神秘的转录一样。完成这项工作后，我们将对调控获得新的生物学见解不同位置的不同SSC亚群，为逆转骨疾病和提供新的治疗靶点缺陷。