Reversing Skeletal Aging by Restoring Functional Skeletal Stem Cell Diversity

项目总结 高龄和肥胖与全球骨骼疾病发病率的增加密切相关。骨质疏松 尤其是对5400多万美国人的公共健康构成重大威胁，因为它与高位骨折相关 费率。与骨质疏松症相关的髋部骨折总是与显著的发病率相关，令人惊讶的是，58% 老年人受伤第一年内的死亡率。这个问题因缺乏效率而变得更加复杂 无重大副作用的老年性骨病的预防性和内科治疗。最近的研究表明 骨中发现的成体干细胞群体可能作为再生来源的潜在靶点 保持和恢复骨骼健康。然而，基于干细胞的再生策略的突破已经 由于无法分离真正的干细胞群体而受到阻碍，这可以作为 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 用于维持正常的骨骼稳态和损伤后的再生。我的最新研究结果表明，衰老 改变干细胞的谱系决定，从而导致再生能力的下降，并提供 骨骼老化是由SSC功能障碍引起的。我的初步数据进一步提供了证据 四肢长骨中存在多种SSc亚型(SSc多样性)。转录分析，网址： 单细胞水平显示SSCs经历了衰老诱导的分子和成分变化 具有功能异构性。我已经鉴定出Wnt1诱导信号通路蛋白2(Wisp2)，即 特别是在年老的SSC谱系中上调。Wisp2显著损害骨形成，当应用于 SSC体外实验或幼鼠体内骨折实验。因此，这项建议的目的是确定与年龄相关的作用 SSC多样性的变化，包括SSC亚型的相对比例及其功能异质性 使用多种方法对骨骼完整性进行时空单细胞分析。在AIM2中，我将研究 我推测，干细胞通过Wisp2调节SSC的骨骼老化机制 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，如不利的克隆性骨骼形成。重要的是，我将确定 SSCs中Wisp2受体的高灵敏邻近标记识别靶向通路 参与了SSC介导的骨骼老化。专家提供的指导和研究环境 来自斯坦福大学和美国国立卫生研究院的导师是尖端的，与这项提案的目的高度相关，允许 实施最新的转录和蛋白质组学方法，包括SmartSeq2单细胞RNA- 测序、RNAScope和TurboID，以询问拟议的目标。这些研究将建立一个新的 从SSc多样性的角度理解骨骼疾病的范例，并应有助于 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.