Single cell transcriptomics analysis of young Ncstnfl/fl (control), middle-aged Ncstnfl/fl (control) and middle-aged LeprCre; Ncstnfl/fl (cKO) bone and bone marrow

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: 5 mice per condition pooled.Hindlimbs were isolated with subsequent bone and bone marrow isolation with fluorescence-activated cell sorting to isolate hematopoietic and endothelial cells (CD45+CD31+TER-119-) and skeletal and stromal lineages (CD45-CD31-TER-119-). Hematopoietic/endothelial and osteolineage/stromal compartments were then mixed at a 1:1 ratio to allow for enrichment of the rarer osteolineage/stromal cells.