Identification of Myeloid Proliferation Niches in Bone Marrow

Abstract

Unlike most other tissues found in land vertebrates, the bone marrow (BM) has unique characteristics: It is a soft, gelatinous tissue in which cells are freely moving and distributed in a seemingly random manner. In terms of cell components, the BM houses a wide variety of cell types, especially those of the hematopoietic system. These include the hematopoietic stem cells (HSCs), the hematopoietic progenitor stem cells (HPSCs) and all types of downstream myeloid and lymphoid cells. Cells from the myeloid lineage represents the most frequent cell types in the BM, and include billions of red blood cells and platelets, as well as cells from the innate immune system. The BM is located inside the cavities of some of the strongest tissues in the body, the skeletal bones. These are composed of connective tissue rich in mesenchymal cells (osteoblast-lineage cells, chondrogenic cells, marrow stromal cells, stromal fibroblasts, endothelial cells and perivascular cells) and complex extracellular matrix (ECM) components. Hence, the biophysical peculiarities of the bone-BM ecosystem have made studying the in situ hematopoietic anatomy and its physiological spatio-temporal cell dynamics quite challenging. Our group has developed strategies for analyzing entire bone units and their components. These strategies have been optimized and used to preserve the BM of postnatal mice. This enables the present study to focus on the in situ exploration of the spatial niches of the BM. The aims of this study were therefore to characterize the spatial distribution of proliferative hematopoietic-myelopoietic progenitors with respect to their niche; and analyze the inferred communication networks between bone and BM cells using single-cell transcriptomics in silico. First, a proliferation-labelling assay was performed on unperturbed mice. Second, the femurs were collected, processed, and stained using immunofluorescence (IF) targeting markers to distinguish between niche-specific cells and the hematopoietic cells of interest. Third, femur sections were imaged to collect 3D spatial Bone-BM data. Fourth, image cytometry was used to compute the relevant spatial variables of the selected markers and random distributions. Finally, integration and cell communication networks were inferred using publicly available BM single-cell transcriptomic datasets. My results reveal a novel mapping of proliferative myeloid-Gr1+ cells, distributed among privileged BM regions characterized by trabecular bone (TB). These cells preferentially locate at cell-contact distance to the BM-vasculature and at 10-cells distance to the osteogenic niche in a non-random fashion. I used a comprehensive integrated scRNA-seq dataset to identify and infer cell communication networks between niche and GMPs-neutrophils, with relevant reported functions identified through the secreted signaling communication context. This work ultimately advanced the efforts to reach a comprehensive understanding of the unperturbed proliferative myeloid niches in the post-natal BM of mouse femurs.