Reversing Skeletal Aging by Restoring Functional Skeletal Stem Cell Diversity

年龄的增长与全球骨骼疾病发病率的上升密切相关。骨质疏松症是超过5400万美国人的主要公共健康威胁，因为它与高骨折率有关。骨质疏松相关的髋部骨折总是与显著的发病率和惊人的58%的死亡率相关，在受伤的第一年内。这一问题由于缺乏有效的预防和无重大副作用的与年龄有关的骨病的医学治疗而变得更加复杂。最近的研究表明，骨内的成体干细胞群可能作为维持和恢复骨骼健康的再生来源。然而，由于无法分离出真正的干细胞群，干细胞再生策略的突破一直受到阻碍。我们的团队已经帮助描述了高度纯化的骨骼干细胞（SSC）谱系，这些谱系对于维持正常的骨骼稳态和损伤后的再生至关重要。我的最新研究结果表明，衰老改变了干细胞的谱系决定，引发了生态位动力学的改变，从而导致了再生能力的下降，并提供了骨骼衰老是由SSC功能障碍引起的理论依据。本研究的初步结果为肢体长骨中存在多种SSC亚型（SSC多样性）提供了证据。单细胞水平的转录组学分析表明，ssc经历了衰老诱导的分子和组成变化，与功能异质性相一致。我已经确定了Wnt1诱导信号通路蛋白2 (Wisp2)，在衰老的SSC谱系中特异性上调。Wisp2在体外应用于SSC或体内幼龄小鼠骨折时显著损害骨形成。Aim1已经确定了年龄相关的SSC多样性变化的作用，包括SSC亚型的相对比例及其对骨骼完整性的功能异质性，现在将完成时空单细胞分析，以确定SSC的解剖变化及其生态位细胞相互作用。在ai2中，我将通过Wisp2研究干细胞骨骼衰老的机制，我假设Wisp2可能调节SSC的多样性和异质性。拟议的实验还将解决基于干细胞的表观遗传漂移的年龄依赖性作用，以及与干细胞衰老新概念（如不良克隆骨骼生成）的潜在联系。重要的是，我将通过高度敏感的邻近依赖标记来确定ssc中Wisp2受体的未探索身份，以确定参与ssc介导的骨骼衰老的可靶向途径。我的新宿主机构为我的独立研究阶段提供的指导和研究环境是前沿的，与本提案的目的高度相关，允许实施最新的转录组学，表观遗传学和蛋白质组学方法，包括单细胞RNA和atac测序，RNAScope， NanoString单细胞成像和TurboID，来研究提出的目标。这些研究将为从SSC多样性的角度理解骨骼疾病建立一个新的范式，并将促进新的预防、诊断和治疗方法的发展，以解决骨骼疾病。

Advancing age is tightly linked to the increasing global incidence of skeletal diseases. Osteoporosis 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. 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 triggering altered niche dynamics thereby contributing to a decline of regenerative capacity and providing a rationale that skeletal aging is caused by SSC dysfunction. The initial findings of this proposal provide 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. Aim1 has determined the role of age-related changes in SSC diversity including the relative proportion of SSC subtypes and their functional heterogeneity to skeletal integrity and will now be completed with spatiotemporal single cell analysis to define anatomical changes of SSCs and their niche cell interactions. 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 the age-dependent role of stem cell based epigenetic drift and a 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 my new host institution for my independent research phase is cutting-edge and highly relevant to the purpose of this proposal allowing implementation of the latest transcriptomic, epigenetic and proteomic methods, including single-cell RNA- and ATAC-sequencing, RNAScope, NanoString single cell imaging 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.