Single‐Cell Transcriptomes of Immune Cells from Multiple Compartments Redefine the Ontology of Myeloid Subtypes Post‐Stroke

Abstract The activation and infiltration of immune cells are hallmarks of ischemic stroke. However, the precise origins and the molecular alterations of these infiltrating cells post‐stroke remain poorly characterized. Here, a murine model of stroke (permanent middle cerebral artery occlusion [p‐MCAO]) is utilized to profile single‐cell transcriptomes of immune cells in the brain and their potential origins, including the calvarial bone marrow (CBM), femur bone marrow (FBM), and peripheral blood mononuclear cells (PBMCs). This analysis reveals transcriptomically distinct populations of cerebral myeloid cells and brain‐resident immune cells after stroke. These include a novel CD14 + neutrophil subpopulation that transcriptomically resembles CBM neutrophils. Moreover, the sequential activation of transcription factor regulatory networks in neutrophils during stroke progression is delineated, many of which are unique to the CD14 + population and underlie their acquisition of chemotaxis and granule release capacities. Two distinct origins of post‐stroke disease‐related immune cell subtypes are also identified: disease inflammatory macrophages, likely deriving from circulating monocytes in the skull, and transcriptionally immature disease‐associated microglia, possibly arising from pre‐existing homeostatic microglia. Together, a comprehensive molecular survey of post‐stroke immune responses is performed, encompassing both local and distant bone marrow sites and peripheral blood.