Multiple pathways of CD34⁺ cell differentiation during embryogenesis

CD34 has long been defined as a canonical marker for endothelial progenitors as well as hematopoietic stem cells, implicating its role in vascular development and hematopoiesis. However, the precise developmental hierarchy and lineage potential of CD34⁺ cells remain controversial. In this study, we integrated inducible genetic lineage tracing techniques, proteomics and single-cell RNA-seq (scRNA-seq) analyses to elucidate the dynamic developmental trajectory of CD34⁺ cells during various embryonic periods in both humans and mice. Remarkably, our analyses indicated that the progeny of CD34⁺ cells marked distinct, spatiotemporally restricted progenitor waves with divergent fates, at which point cells adopted endothelial, hematopoietic and fibroblastic fates, respectively. During gastrulation (E6.5-E8.5), an initial wave of CD34⁺ progenitors predominantly orchestrates vasculogenesis via a Kdr-dependent mechanism. Subsequently, from E9.5 to E14.5, cell cycle activation serves as a molecular switch, facilitating the endothelial-to-hematopoietic transition (EHT) of CD34⁺ progenitors. Unexpectedly, we identify a wave of CD34⁺ progenitors in late embryogenesis that gives rise to fibroblasts, distinct from earlier endothelial or hematopoietic lineages. Furthermore, because umbilical cord blood is a valuable source of different circulating stem/progenitor cells, we distinguish circulating endothelial progenitors from fibroblast progenitors in human cord blood by unique molecular signatures, with GFPT2 specifically marking the fibroblast progenitors. Collectively, our study provides a high-resolution spatiotemporal atlas of CD34⁺ cells during embryogenesis, redefining the temporal shifts of CD34⁺ cells in cell states and offering a precise framework for manipulating CD34⁺ cells in regenerative medicine.