Defining periosteal skeletal stem cells and novel migration mechanisms in bone regeneration and repair in vivo

项目总结/摘要 在与年龄相关的骨疾病中，显著的骨丢失和骨愈合缺陷会导致严重的疼痛和高血压。 发病率和死亡率，并且是美国的主要健康问题。 需要骨骼干细胞（SSC），骨膜中的骨骼干/祖细胞亚群（P-SSC） 在骨修复中起着重要作用。然而，由于SSC的广泛组织分布， 缺乏特异性标记物来区分体内罕见的P-SSC，内源性P-SSC如何对骨产生反应， 损伤以及它们如何在骨愈合中起作用是未知的。因此，我们建议的目标是界定 P-SSC的体内特性和功能以及调节P-SSC以控制骨的机制 在生理条件下的再生和修复。我们还将探索内源性P-的诱导 SSC迁移和增殖，目的是改善年龄相关性骨损伤的恢复。使用 结合活体成像技术，我们最近 确定SSC标记物、粘液病毒抗性-1（Mx 1）和α平滑肌肌动蛋白的组合 （αSMAGFP）可以选择性地标记仅存在于骨膜形成层中的内源性P-SSC， 表达Prx 1GFP的细胞层。此外，连续体内成像显示Mx 1 αSMAGFP P-SSC， 而不是BM-SSCs，对损伤迅速做出反应，并不断提供新的成骨细胞用于损伤修复， vivo.这些P-SSC显示出趋化因子（C-C基序）配体5的受体CCR 3/CCR 5的较高表达 （CCL 5），比BM-SSC和其他骨细胞。此外，我们首次发现， CCL 5的施用刺激这些P-SSC向体内损伤部位的实时迁移。人类 原代骨膜细胞也表达CCR 5并进行CCL 5介导的迁移。因此，我们假设， P-SSCs及其迁移，在CCL 5信号传导后，对于骨修复是必需的，并且外源性 提供CCL 5可改善年龄相关性骨缺损的愈合。我们的新型活体成像技术 各种动物模型使我们能够追踪骨膜和骨骼中的内源性干细胞 骨髓以及它们在活体动物中单细胞水平的分化。因此，我们计划继续执行 具体目标。在目标1中，我们将确定是否需要炎性刺激物如CCL 5 通过在免疫细胞中产生CCL 5的条件性消融，用于P-SSC迁移和骨愈合。我们将 还检查CCL 5是否特异于P-SSC迁移。在目标2中，我们将确定P-SSC及其 通过进行P-SSC的局部消融和通过产生条件性的 P-SSC中CCR 5的缺失。在目标3中，我们将确定本地提供外源性CCL 5 在修复过程的早期诱导内源性P-SSC的迁移和活化，导致加速的 并改善老年小鼠的骨愈合。这项工作完成后，我们将获得新的生物学见解 并将为逆转骨疾病和骨缺损定义新的治疗靶点。

PROJECT SUMMARY/ABSTRACT Significant bone loss and defective bone healing in age-related bone diseases cause severe pain and high morbidity and mortality, and are a major health issue in the U.S. Lifelong regeneration of bone and cartilage requires skeletal stem cells (SSCs), and a skeletal stem/progenitor cell subset in periosteum (P-SSCs) has been known to play an important role for bone repair. However, due to the wide tissue distribution of SSCs and the lack of specific markers to distinguish rare P-SSCs in vivo, how endogenous P-SSCs respond to bone injury and how they function in bone healing are unknown. Therefore, the goal of our proposal is to define the in vivo identity and function of P-SSCs and the mechanisms that regulate P-SSCs in order to control bone regeneration and repair under physiologic circumstances. We will also explore the induction of endogenous P- SSC migration and proliferation with the goal of improving recovery from age-related bone injuries. Using newly generated reporter mouse models in combination with intravital imaging technology, we recently determined that a combination of SSC markers, myxovirus resistance-1 (Mx1) and alpha smooth muscle actin (αSMAGFP) can selectively label endogenous P-SSCs that are exclusively present in the periosteal cambial layer with Prx1GFP expression. In addition, sequential in vivo imaging revealed that Mx1 αSMAGFP P-SSCs, rather than BM-SSCs, rapidly respond to the injury and continually supply new osteoblasts for injury repair in vivo. These P-SSCs show higher expression of CCR3/CCR5, the receptors for chemokine (C-C motif) ligand 5 (CCL5), than BM-SSCs and other bone cells. Moreover, we found for the first time that periosteal administration of CCL5 stimulates the real-time migration of these P-SSCs toward injury sites in vivo. Human primary periosteal cells also express CCR5 and conduct CCL5-mediated migration. We thus hypothesize that P-SSCs and their migration, upon CCL5 signaling, are necessary for bone repair, and that exogenous CCL5 provision improves the healing of age-related bone defects. Our novel intravital imaging technology and the various animal models enable us to track endogenous stem cells present in the periosteum and bone marrow as well as their differentiation at the single-cell level in living animals. We thus plan to pursue the following specific aims. In aim 1, we will determine whether inflammatory stimuli such as CCL5 are necessary for P-SSC migration and bone healing by generating a conditional ablation of CCL5 in immune cells. We will also examine if CCL5 is specific for P-SSC migration. In aim 2, we will determine whether P-SSCs and their migration are required for injury repair by performing local ablation of P-SSCs and by generating a conditional deletion of the CCR5 in P-SSCs. In aim 3, we will determine whether the local provision of exogenous CCL5 induces the migration and activation of endogenous P-SSCs early in the repair process, leading to accelerated and improved bone healing in aged mice. Upon completion of this work, we will achieve new biological insights into periosteal SSCs and will define new therapeutic targets for reversing bone diseases and defects.