OC 09.4 - Spatial transcriptomics reveals megakaryocyte heterogeneity in mouse bone marrow

Background: Megakaryocytes (MKs) are bone marrow resident cells with the pivotal role of platelet production. Recent endeavors using single-cell RNA sequencing on murine MKs revealed that functions of megakaryocytes go beyond platelet production with evidence for immunoregulatory and stem cell niche supporting subpopulations. However, the relationship between the spatial organization of MKs and transcriptional heterogeneity is unclear and thus remains to be elucidated whether localization of MKs near sinusoids or at the proximal versus the distal side of bones might influence cell function.

Aims: The aim of this study was to systematically map the molecular, cellular, and spatial composition of MKs in the murine femur by combining transcriptomics with in situ spatial orientation.

Methods: Serial sections were obtained from a 3-month-old C57BL/6J mouse femur. Using the Nanostring GeoMx digital spatial profiling platform and the mouse whole transcriptome array we profiled 44 individual MKs divided over different regions throughout the bone marrow.

Results: Principle component analysis revealed clustering based on the proximal versus distal axes of the bone (P=0.000175), while an association with adjacency to the vasculature was not observed (P=0.1667). Pearson correlation on the distance to the proximal side of the bone showed 5 genes of interest (Fold Chance of >2 and P-value of < 0.05) with two and three genes overexpressed in the proximal and distal side, respectively (Figure 1). Of note, these genes included pro-platelet basic protein (Pbpp) and platelet factor 4 (Pf4) with a higher expression in proximal MKs.

Conclusion(s): Transcriptional heterogeneity was observed in compartments previously thought to be homogeneous, with evidence for Pbpp and Pf4 being expressed more prominently in the proximal side of the femur. Moreover, we present a novel methodology to measure the whole transcriptome of individual MKs in situ and reveal that spatial organization should be considered while assing the MK transcriptome.