Reversing Skeletal Aging by Restoring Functional Skeletal Stem Cell Diversity

项目摘要 年龄增长和肥胖与全球骨骼疾病发病率的增加密切相关。骨质疏松 特别是对超过5400万美国人构成重大的公共健康威胁，因为它与高骨折相关 rates.骨质疏松相关的髋部骨折总是与显著的发病率相关， 老年人在受伤后第一年的死亡率。这一问题因缺乏有效的 预防和药物治疗与年龄有关的骨疾病，无主要副作用。最近的研究 揭示了骨骼中的成体干细胞群，这些细胞群可能作为再生来源， 保持和恢复骨骼健康。然而，基于干细胞的再生策略的突破 由于无法分离出真正的干细胞群， 有针对性的方法。我们的团队帮助描绘了高度纯化的骨骼干细胞（SSC）谱系， 用于维持正常的骨稳态和损伤后的再生。我最新的研究结果表明 改变干细胞的谱系决定，从而导致再生能力下降， 骨骼衰老是由SSC功能障碍引起的。我的初步数据进一步证明了 四肢长骨中存在多种SSC亚型（SSC多样性）。转录组学分析. 单细胞水平显示，精原干细胞经历衰老诱导的分子和组成变化， 功能异质性。Wnt 1诱导信号通路蛋白2（Wisp 2）， 在衰老的SSC谱系中特异性上调。当应用于 体外SSC或体内幼龄小鼠骨折。因此，本建议的目的1是确定年龄相关的作用 SSC多样性的变化，包括SSC亚型的相对比例及其功能异质性， 骨骼完整性使用各种方法进行时空单细胞分析。在目标2中，我将检查 通过Wisp 2的干细胞为基础的骨骼衰老机制，我假设，可能会调节SSC 多样性和异质性。拟议的实验还将解决其与新概念的潜在联系 干细胞老化的原因，如不良克隆性骨骼生成。重要的是，我将确定未经探索的身份， 通过高灵敏度的邻近依赖性标记来鉴定SSCs中的Wisp 2受体以鉴定靶向通路 参与SSC介导的骨骼衰老。专家提供的指导和研究环境 来自斯坦福大学和国家卫生研究院的导师是尖端的，与本提案的目的高度相关， 实施最新的转录组学和蛋白质组学方法，包括SmartSeq 2单细胞RNA- 测序，RNAScope和TurboID，以询问拟议的目标。这些研究将建立新的 从SSC多样性的角度理解骨骼疾病的范式，并应促进 开发新的预防、诊断和治疗方法来解决骨骼疾病。

PROJECT SUMMARY Advancing age and obesity are tightly linked to the increasing global incidence of skeletal diseases. Osteoporosis in particular poses a major public health threat for over 54 million Americans as it is interrelated with high fracture rates. Osteoporosis-related hip fractures are invariably associated with significant morbidity and strikingly, a 58% mortality rate in the elderly within the first year of injury. This problem is compounded by a lack of efficient preventive and medical therapies for age-related bone disease free of major side effects. Recent studies have revealed adult stem cell populations within bone that could be potentially targeted as a regenerative source to maintain and restore skeletal health. However, breakthroughs in stem cell based-regenerative strategies have been hampered by the inability to isolate bona fide stem cell populations which could serve as starting points for targeted approaches. Our group has helped delineate highly purified skeletal stem cell (SSC) lineages crucial for maintaining normal bone homeostasis and regeneration following injury. My latest results suggest that aging shifts lineage determination of stem cells thereby contributing to a decline of regenerative capacity and providing a rationale that skeletal aging is caused by SSC dysfunction. My preliminary data further provides evidence for the existence of multiple SSC subtypes (SSC diversity) present in limb long bones. Transcriptomic analysis at the single-cell level shows that SSCs undergo aging-induced molecular and compositional changes coinciding with functional heterogeneity. I have identified Wnt1 Inducible Signaling Pathway Protein 2 (Wisp2) that is specifically upregulated in the aged SSC lineage. Wisp2 significantly impairs bone formation when applied to SSC in vitro or fractures of young mice in vivo. Thus, Aim1 of this proposal is to determine the role of age-related changes in SSC diversity including the relative proportion of SSC subtypes and their functional heterogeneity to skeletal integrity using a variety of methods for spatio-temporal single-cell analysis. In Aim2 I will examine the mechanism of stem cell-based skeletal aging through Wisp2, which I hypothesize, may regulate SSC diversity and heterogeneity. Proposed experiments will also address its potential connection to new concepts of stem cell aging such as adverse clonal skeletogenesis. Importantly, I will determine the unexplored identity of the Wisp2 receptor in SSCs by highly sensitive proximity-dependent labeling to identify targetable pathways involved in SSC-mediated skeletal aging. The guidance and research environment provided by the expert mentors from Stanford and the NIH is cutting-edge and highly relevant to the purpose of this proposal allowing implementation of the latest transcriptomic and proteomic methods, including SmartSeq2 single-cell RNA- sequencing, RNAScope, and TurboID, to interrogate the proposed aims. These studies will establish a new paradigm for understanding skeletal disease from the perspective of SSC diversity and should facilitate the development of new preventive, diagnostic, and therapeutic approaches to tackle skeletal disorders.