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

Megakaryocytes are bone marrow (BM) resident cells that derive from hematopoietic stem cells. A pivotal function of megakaryocytes is the generation of platelets through the release of long protrusions called proplatelets into sinusoidal vessels. single-cell RNA-sequencing on murine BM megakaryocytes has previously revealed transcriptional heterogeneity with segmentation into four distinct categories. These studies postulated functions beyond platelet production with evidence for immunoregulatory and stem cell niche supporting subtypes, as well as a cycling population. The spatial context and transcriptional heterogeneity of megakaryocytes is of great interest as localization of for instance the vasculature is a necessity for platelet production. For single-cell RNA sequencing this spatial orientation is however lost due to the dissociation of tissues. Recent technological advances have enabled the interrogation of gene expression profiles of tissues in situ. This enables the integration of morphological, situational and transcriptional information to classify cells in the context of their microenvironment. In the following we present, for the first time, the application of this technology to BM megakaryocytes at a single cell level. The femur of a healthy 3 and 18 month old mice was collected and parafin sections were prepared. Using the Nanostring GeoMx spatial transcritomics platform individual megakaryocytes (guided by CD41 IF staining) were selected as regions of interest and their transcriptome was determined using the murine whole transcriptome atlas.