Spatial transcriptomics of murine bone marrow megakaryocytes at single-cell resolution

BACKGROUND: While megakaryocytes are known for making platelets, recent single-cell RNA sequencing data have revealed subpopulations of megakaryocytes with predicted immunoregulatory and bone marrow niche-supporting roles. Although these studies uncovered interesting information regarding the transcriptional variation of megakaryocytes, the generation, localization, and regulation of these subsets have not yet been studied and therefore remain incompletely understood. Considering the complex organization of the bone marrow, we reasoned that the application of spatial transcriptomic approaches could help dissect megakaryocyte heterogeneity within a spatiotemporal context. OBJECTIVES: The aim of this study was to combine spatial context and transcriptomics to assess the heterogeneity of murine bone marrow megakaryocytes in situ at a single-cell level. METHODS: Bone marrow sections were obtained from femurs of C57BL/6J mice. Using the murine whole transcriptome array on the Nanostring GeoMx digital spatial profiling platform, we profiled 44 individual megakaryocytes (CD41(+) by immunofluorescence) in situ throughout the bone marrow, both adjacent and nonadjacent to the endothelium (directly in contact with vascular endothelial-cadherin–positive cells). RESULTS: Principal component analysis revealed no association between transcriptomic profile and adjacency to the vasculature. However, there was a significant effect of proximal vs distal regions of the bone. Two and 3 genes were found overexpressed in the proximal and distal sides, respectively. Of note, proplatelet basic protein and platelet factor 4, 2 genes associated with platelet production, had higher expression in proximal megakaryocytes. CONCLUSION: This study indicates a possible effect of spatial location on megakaryocyte heterogeneity and substantiate further interest in investigating megakaryocyte subpopulations in the context of their spatial orientation.