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 megakar-yocytes, the generation, localization, and regulation of these subsets have not yet been studied and therefore remain incompletely understood. Considering the complex or-ganization of the bone marrow, we reasoned that the application of spatial tran-scriptomic 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 sub-populations in the context of their spatial orientation.