Single-cell transcriptomic analysis reveals age-associated alterations in bone marrow niche-related cells

Hematopoietic stem cells (HSCs) depend on the bone marrow (BM) niche for self-renewal and differentiation. Within this niche, CXCL12-abundant reticular (CAR) cells are key regulators of hematopoiesis and bone homeostasis. Single-cell RNA sequencing of adult and old mice revealed that old Adipo-CAR cells form a distinct cluster with reduced expression of ossification- and stem cell maintenance–related genes. RNA velocity and pseudotime analyses indicated impaired osteogenic differentiation, which was confirmed by in vitro assays showing diminished osteogenesis and increased adipogenesis. Structural and immunohistochemical analyses further demonstrated sinusoidal and endothelial abnormalities with age. These results suggest that age-related changes in Adipo-CAR cells and the sinusoidal niche underlie defective osteogenesis and hematopoietic decline. Overall design: Flushed long bone from C57BL6 mice was digested with collagenase IV, dispase, and DNase in HBSS for 30 min at 37 °C. After lysing red blood cells with NH4Cl, cells were filtered through a 70 µm filter.