Spatial transcriptomic profiling reveals local and domain-establishing regulatory signaling networks within the bone marrow
The application of single-cell RNA sequencing (scRNAseq) to the bone field has led to significant advancements in our understanding of skeletal stem cell (SSC) heterogeneity, yet disparity remains in the characterization of these cells in the context of their native environment. To resolve these limitations, we combined scRNAseq and spatial transcriptomics in adult femurs to provide endogenous, in vivo context. First, predictive modeling was used to determine the spatial location of SSCs within the bone marrow. These results localized CXCL12-abundant reticular cells to the bone marrow, while PDGFRa+SCA1+ were found to enrich within the outer periosteum. Correlative analyses were next used to define the cellular niche composition, identifying smooth muscle cells, as well as endothelial cell and macrophage subtypes overrepresented within the SSC niche. Using cell-cell communication prediction and spatial transcriptomics, we defined ligand-receptor pairs specifically expressed within the niche and mapped their expression back to niche-resident cells. Finally, these SSC niches were placed in the context of bone microdomains. Signaling gradients derived from the vasculature and bone surfaces were unbiasedly assessed using SpatialTime. This data indicated a striking, spatially-restricted activation of metabolic and major morphogenetic pathways. This project demonstrates the ability of this technique to spatially localize SSC subpopulations, define the cellular components of the stem cell niche, unravel cell-cell communication and place this niche in the context of its bone microdomain to gain a comprehensive understanding of local and global SSC regulatory networks within the bone. Overall design: Femurs from 10 week old C57BL/6 mice