Distinct Stress Responses of Bone Marrow Stromal Cell Subgroups Defined by Unbiased Single-Cell Mass-Cytometry Analysis

The bone marrow niche is a heterogeneous tissue comprised of multiple cell types that collectively regulate hematopoiesis. It is thought to be a critical stress sensor, integrating information at the level of the organism down to signals at the level of the single cell. In so doing, the niche orchestrates hematopoietic stem and progenitor cell (HSPC) responses to organismal stress. However, most studies of the niche have depended on genetic marker or deletion studies that inherently limit analysis to the selected indicator genes or cells. While this has greatly enhanced our understanding of bone marrow function, it does not permit systems level evaluation of subgroups of cells and their relative response to a particular challenge.

We therefore sought a less biased strategy to study bone marrow stromal cells and the cytokines they elaborate under homeostatic and stress conditions. We used Mass-Cytometry (CyTOF) to resolve protein levels at single cell resolution in mouse bone marrow. We established a panel of 36 antibodies: 20 surface and intracellular phenotypic markers, 12 cytokines regulating hematopoiesis, 1 marker of proliferation, 1 marker for DNA damage, 1 viability marker and 1 nucleated cell marker. We intentionally selected antibodies that recognize antigens already defined by others as bone marrow stromal markers. Freshly isolated non-hematopoietic cells from long bones and pelvis were analyzed and clustered into subgroups based on their protein expression signature.

We applied k-means clustering using common markers to group bone marrow stromal cells into phenotypical subtypes. At steady state, analysis of over 20.000 mouse bone marrow stromal single-cells negative for the hematopoietic markers CD45 and Ter119 revealed 4 large clusters: an endothelial population expressing CD31, Sca1 and CD105, a mesenchymal stromal cell population expressing Sca1, CD140a, Nestin and LeptinR, a bone marrow stromal progenitor population expressing CD105, CD271 and Runx2 and a mature bone cell population expressing Osteocalcin and CD140a. Within these clusters, sub-populations were evident by adding CD106, CD90, CD73, Embigin, CD29, CD200, c-Kit and CD51. In total, 28 distinct populations of bone marrow stromal cells were identified based on their phenotypic signature. Only one cluster of cells was negative for all the markers we selected. Therefore, the complex heterogeneity of the bone marrow niche cells can be resolved to 28 subpopulations by single-cell protein analysis. Assessing the response of these groups to systemic challenges of medical relevance, we evaluated cells prior to whole body lethal irradiation (9.5Gy), one hour and one day later (the time of transplantation) and 3 days after irradiation (2d post transplantation) with and without transplanted cells. Notably, LeptinR+CD106+Sca1+ cells putatively essential for hematopoiesis and stem cell support were highly sensitive to and largely killed by irradiation. In contrast, endothelial cells and osteoblastic cells were resistant to irradiation. In particular, osteoblastic cells expressing osteocalcin (GFP+), embigin, NGFR and CD73 increased their expression of multiple hematopoietic cytokines including SDF-1, kit ligand, IL-6, G-CSF and TGF-b one day after irradiation. These data indicate that LeptinR+CD106+Sca1 stromal cells are unlikely to participate in HSPC engraftment post-irradiation while a subset of osteoblastic cells are. Unbiased single cell analysis can resolve subsets of bone marrow cells that respond differently to organismal stress. This method enables comprehensively quantifying subpopulation changes with specific challenges to begin defining the systems biology of the bone marrow niche.