Combined Single-Cell and Spatial Transcriptomics Unveil the Complex Organization of the Non-Immune Human Bone Marrow Microenvironment during Aging

Hematopoietic stem cells (HSCs), reside in a specialized bone marrow (BM) microenvironment known as the hematopoietic stem niche. Despite the critical role of the niche in tightly controlling the processes of normal and malignant hematopoiesis, its cellular composition remains only partially understood. Recent studies, including our own, using single-cell RNA sequencing (scRNA-seq) approaches have provided valuable insights into the profiling of the human BM niche. However, a comprehensive effort to describe the cellular heterogeneity and regulatory circuitry of the aged BM microenvironment in elderly humans is still lacking. As individual age, multiple systems and organs experience a progressive loss of anatomical and physiological integrity. Aging of the HSC niche is accompanied by a reduction in the numbers and function of its constituents and a decrease in the levels of HSC-supporting factors. Whether niche aging can contribute to the defects observed in aged hematopoiesis, such as the clonal shift towards myelopoiesis, the decrease in immune surveillance, or age-associated metabolic diseases, remains unresolved. To address this, we utilized scRNA-seq and spatial transcriptomics to provide a detailed characterization of the molecular landscape and stromal interactions in the aged non-immune BM microenvironment. First, we performed scRNA-seq profiling on fluorescence-activated cell sorting-purified endothelial cells (ECs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD31 +/CD9 +) and mesenchymal stromal cells (MSCs, TO-PRO-3 -/CD45 -/CD235 -/Lin -/CD271 +) from human BM samples of young (n=4) and elderly healthy donors (n=5). We analyzed a total of 1514 ECs and 3848 MSCs from older adults, grouped into 7 and 10 subclusters, respectively, defining distinct functional cell states. ECs and MSCs cells from young individuals were annotated using SingleR, utilizing the gene signature of each functional state in the elderly as a reference. Our results revealed significant shifts in the distribution of the functional states of BM niche cells during aging. Regarding ECs, we noticed reduced pathways associated with the cell cycle and RNA transcription pathways, indicating impaired cell cycle activity, coupled with decreased antioxidant defense (Figure 1A). In contrast, there was an increase in subgroups related to the response to foreign molecules, immune system activation, and vascular remodeling, suggesting an inflammatory response and increased vascular remodeling processe