SRA

Hematopoietic stem cells (HSCs) in adult are specified early from the endothelium-derived precursors (e.g., hemogenic endothelium, and pre-HSCs) in mouse mid-gestation embryos, the detailed process, however, is still largely unknown due to their rareness, transience, and current inability to prospectively isolate them efficiently . Here we developed a potent set of surface markers that could capture the earliest emerging HSCs, the CD45- pre-HSCs with high accuracy and purity, as rigorously and functionally verified by single-cell-initiated serial transplantation assays. Then we applied single-cell RNA-Seq technique to analyze five populations related to HSC formation: the CD45- (type 1) and CD45+ (type 2) pre-HSCs as well as endothelial cells in the E11 AGM region; and later mature HSCs in the E12 and E14 fetal livers. Compared to other cell populations, both type 1 and type 2 pre-HSCs have their unique signatures of transcription machinery, transcription factor network, signaling pathway, cell cycle status, metabolism state, and lncRNA expression patterns. Our work paves the way for dissection of the complex molecular mechanisms regulating the step-wise formation of HSCs from endothelial cells, thus informing future efforts on engineering HSCs for clinical application. Overall design: RNA-Seq of 35 10-cell pooled samples from 5 FACS sorted cell types, i.e., endothelial cells (ECs), T1 and T2 pre-HSCs from E11 AGM region, as well as mature HSCs from E12 and E14 fetal liver