Discovery of embryonically derived bipotent endothelial-macrophage progenitor cells in postnatal aorta

ABSTRACT Background Yolk sac (YS) progenitors are a source of macrophages and endothelial cells in some tissues that are thought to be maintained postnatally by self-renewal in their differentiated states. How this is achieved remains poorly understood. Methods and Results Single-cell digests from mouse aortas selectively formed macrophage colony-forming units (CFU-M) in methylcellulose, which self-renewed in secondary cultures from single cells. CFU-M comprised a homogeneous population of Lin − CD45 +/Lo CD11b − F4/80 − Sca-1 + c-Kit + progenitor cells that co-expressed fractalkine receptor (CX 3 CR1) and colony stimulating factor-1 receptor (CSF1R). These progenitors displayed high proliferative activity from adult aorta even at steady state. Flt3 Cre lineage mapping revealed their independence from Flt3 + bone marrow hematopoietic progenitor cells. They were especially abundant in neonatal aorta, with subsequent age-related decline, suggesting prenatal seeding, which was confirmed by finding their emergence in YS after embryonic day (E) 7.5 and from aorta-gonad-mesonephros by E10.5. Inducible fate-mapping using Csf1r Mer-iCre-Mer and Cx3cr1 YFP-creER mice established that these progenitors originate from an E8.5 CSF1R + and E8.5-9.5 CX 3 CR1 + source, together with macrophages and endothelial cells in the aortic wall. Complementary differentiation studies revealed aortic progenitors to be vasculogenic and bipotent for macrophages and endothelium, contributing to adventitial neovascularization in aortic ring assays and forming perfused blood vessels and macrophages after transfer into ischemic hindlimb. Single-cell RNA sequencing showed their relatively homogeneous myelopoietic and angiogenic gene expression profile without expression of mature myeloid or endothelial genes. Finally, we found that aortic progenitors also express angiotensin converting enzyme (ACE) and angiotensin II receptor, AGTR2, and established regulatory roles for angiotensin II, which augmented their proliferative, self-renewal and differentiation properties in vitro and expansion in aorta in vivo . Conclusion Our discovery of aortic endothelial-macrophage progenitors adds to the recognized fate of YS progenitors in postnatal tissues. These bipotent cells may help explain the local renewal of YS-derived tissue-resident macrophages and endothelial cells after birth.