Bulk RNA sequencing of LEPR+CD45-CD31-TER-119- skeletal stem cells from middle-aged Ncstnfl/fl (control) and LeprCre; Ncstnfl/fl (cKO) mouse bone

Skeletal stem and progenitor cells (SSPCs) perform bone maintenance and repair. With age, they produce fewer osteoblasts and more adipocytes leading to a loss of skeletal integrity. The molecular mechanisms that underlie this detrimental transformation are largely unknown. Single-cell RNA sequencing revealed that Notch signaling becomes elevated in SSPCs during aging. To examine the role of increased Notch activity, we deleted Nicastrin, an essential Notch pathway component, in SSPCs in vivo. Middle-aged conditional knockout mice displayed elevated SSPC osteo-lineage gene expression, increased trabecular bone mass, reduced bone marrow adiposity, and enhanced bone repair. Thus, Notch regulates SSPC cell fate decisions, and moderating Notch signaling ameliorates the skeletal aging phenotype, increasing bone mass even beyond that of young mice. Finally, we identified the transcription factor Ebf3 as a downstream mediator of Notch signaling in SSPCs that is dysregulated with aging, highlighting it as a promising therapeutic target to rejuvenate the aged skeleton.骨骼干细胞与祖细胞（Skeletal stem and progenitor cells, SSPCs）负责骨骼的维持与修复。随着机体衰老，这类细胞生成的成骨细胞减少、脂肪细胞增多，最终导致骨骼完整性丧失。介导这一有害转变的分子机制在很大程度上仍未明确。单细胞RNA测序结果显示，衰老过程中SSPCs内的Notch信号通路活性上调。为探究Notch活性上调的作用，我们在体内SSPCs中敲除了Notch通路的必需组分尼卡斯汀（Nicastrin）。中年条件性敲除小鼠表现出SSPCs成骨谱系基因表达上调、骨小梁骨量增加、骨髓脂肪含量降低以及骨修复能力增强。由此可见，Notch信号通路调控SSPCs的细胞命运决定，而下调Notch信号通路可改善骨骼衰老表型，甚至可使骨量高于年轻小鼠。最后，我们鉴定出转录因子早期B细胞因子3（Ebf3）是SSPCs中Notch信号通路的下游介导因子，其表达随衰老发生失调，这使其成为复壮衰老骨骼的极具潜力的治疗靶点。