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

项目摘要/摘要 年龄相关性骨病的显著骨丢失和骨愈合缺陷会导致剧烈疼痛和高 发病率和死亡率，是美国终身骨和软骨再生的主要健康问题 需要骨骼干细胞(SSCs)，骨膜中的骨骼干细胞/祖细胞亚群(P-SSCs) 众所周知，它在骨骼修复中起着重要作用。然而，由于SSCs的广泛组织分布和 缺乏特异性标志物来区分体内罕见的P-SSCs，内源性P-SSCs如何对骨做出反应 损伤及其在骨愈合中的作用尚不清楚。因此，我们建议的目标是定义 P-SSCs在体内的识别和功能及其调控骨的机制 在生理环境下的再生和修复。我们还将探索内源P-的诱导。 SSC迁移和增殖，目的是促进与年龄相关的骨骼损伤的恢复。vbl.使用 新生成的报告鼠模型与活体成像技术相结合，我们最近 确定SSC标志物、粘病毒抗性-1(Mx1)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-SSCs Prx1GFP表达式的层。此外，体内序贯成像显示Mx1αSMAGFP P-SSCs， 而不是BM-SSCs，快速对损伤做出反应，并不断为损伤修复提供新的成骨细胞 活着。这些P-SSC高表达趋化因子(C-C基序)配体5的受体CCR3/CCR5 (CCL5)，高于BM-SSCs和其他骨细胞。此外，我们首次发现骨膜 体内应用CCL5可刺激这些P-SSCs实时向损伤部位迁移。人类 原代骨膜细胞也表达CCR5，并进行CCL5介导的迁移。因此，我们假设 P-SSCs及其在CCL5信号转导下的迁移是骨修复所必需的，而且外源性 CCL5可促进年龄相关性骨缺损的愈合。我们新的活体成像技术 各种动物模型使我们能够追踪存在于骨膜和骨骼中的内源性干细胞 以及在活动物体内的单细胞水平上的分化。因此，我们计划继续推行 遵循特定的目标。在目标1中，我们将确定是否需要CCL5等炎性刺激 通过有条件地消融免疫细胞中的CCL5，促进P-SSC的迁移和骨愈合。我们会 还要检查CCL5是否专用于P-SSC迁移。在目标2中，我们将确定P-SSC及其 损伤修复需要迁移，方法是局部消融P-SSCs和产生条件性 删除P-SSCs中的CCR5。在目标3中，我们将确定本地提供的外源CCL5是否 在修复过程的早期诱导内源性P-SSCs的迁移和激活，导致加速 并改善老年小鼠的骨骼愈合。这项工作完成后，我们将获得新的生物学见解 转化为骨膜干细胞，并将确定逆转骨病和骨缺损的新治疗靶点。

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.