Mapping the miRNA landscape of primitive macrophage extracellular vesicles highlights their pro-vasculogenic effects in engineered human cardiac tissue.

Resident cardiac macrophages, derived from primitive yolk sac precursors during embryogenesis, have increasingly been recognized for their distinct phenotype and functions in regulating homeostasis of the human heart. However, the profile of their extracellular vesicles (EVs) in cardiac signaling and regulation remains uncharted. Here, we employ differentiation of human pluripotent stem cell-derived primitive macrophages (Mac), harvesting their secreted EVs and performing in-depth characterization of associated microRNAs (miRNAs). Primitive macrophages secreted nanoscale EVs that expressed canonical EV markers, and miRNA sequencing highlighted a diverse and unique profile of miRNAs when compared to EVs sourced from other principal cardiac cell lineages and published data from monocyte-derived cells. In particular, we noted the abundance and enrichment of vascular-modulatory let-7 miRNAs and miR-126-3p. Functional screening of Mac-EVs in a 3D model of in vitro cardiac vasculogenesis confirmed enhanced early endothelial cell organization and branching. Establishing a reference for the human Mac-EV miRNome enables further hypothesis-driven mechanistic tests of Mac-EV miRNAs in mediating cardiac physiology and disease, opening the door to identification of therapeutic targets and modalities for cardiac repair.