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. Overall design: Hindlimbs were isolated with subsequent bone and bone marrow isolation with fluorescence-activated cell sorting to isolate LEPR+CD45-CD31-TER-119– skeletal stem cells from middle-aged control and cKO mice. N=4 mice per group.

骨骼干细胞和祖细胞（Skeletal stem and progenitor cells，SSPCs）负责骨骼稳态维持与损伤修复。随着机体衰老，此类细胞生成的成骨细胞减少、脂肪细胞增多，进而导致骨骼完整性丧失。介导这一有害转变的分子机制目前尚未完全明确。单细胞RNA测序结果显示，衰老过程中SSPCs内的Notch信号通路（Notch signaling）活性上调。为探究Notch信号活性上调的作用，我们在体内SSPCs中敲除了尼克斯特林（Nicastrin）——一种Notch信号通路的必需组分。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、骨髓脂肪堆积减少以及骨修复能力增强的表型。由此可见，Notch信号调控SSPCs的细胞命运决定，而下调Notch信号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Ebf3（Ebf3）是SSPCs中Notch信号通路的下游介导分子，其表达随衰老发生失调，这使其成为恢复衰老骨骼活力的极具前景的治疗靶点。整体实验设计：分离后肢，随后提取骨骼与骨髓，通过荧光激活细胞分选（Fluorescence-activated cell sorting, FACS）从中年对照小鼠与条件性敲除（cKO）小鼠中分离LEPR阳性、CD45阴性、CD31阴性、TER-119阴性的骨骼干细胞。每组设置4只小鼠。