Reassessing endothelial-to-mesenchymal transition in postnatal mouse bone marrow using single-cell RNA sequencing

Conflicting reports exist on whether endothelial cells (ECs) serve as a source of bone marrow stromal cells (BMSCs). In studies concerning the endothelial-to-mesenchymal transition (EndoMT), ECs expressing mesenchymal markers, as well as BMSCs expressing endothelial markers, are typically considered intermediates of EndoMT. To understand the lineage relationship between ECs and BMSCs in postnatal mouse bone marrow, we performed single-cell RNA sequencing (scRNA-seq) on ECs and BMSCs obtained from 5-week-old wild-type C57BL/6J mice, and analyzed the potential intermediates of EndoMT. Subsequently, BMSC and EC lineage tracing models, flow cytometry, and immunostaining techniques were used to validate the findings from scRNA-seq analysis. We profiled bone marrow cells from mouse tibiae and femurs using scRNA-seq. Overall, bone marrow cells and bone fragments from tibiae and femurs of 5-week-old wild-type C57BL/6J mice were collected and digested with a mixture of collagenase, dispase and DNase. Subsequently, samples were subjected to lineage positive cell depletion by magnetic- and fluorescence-activated cell sorting (MACS/FACS). Cells positive for B220, Ter119, Gr-1, CD11b, CD3e, CD45, CD71, CD3, CD19 were depleted. Finally, we analyzed ECs expressing mesenchymal markers Prrx1, Lepr, Pdgfra, and Col1a1, as well as BMSCs expressing endothelial markers Pecam1, Cdh5, Tek, and Emcn in the scRNA-seq analysis.