Haematopoietic stem cells (HSCs) are derived early from embryonic precursors, such as haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs), the molecular identity of which still remains elusive. Here we use potent surface markers to capture the nascent pre-HSCs at high purity, as rigorously validated by single-cell-initiated serial transplantation. Then we apply single-cell RNA sequencing to analyse endothelial cells, CD45(-) and CD45(+) pre-HSCs in the aorta-gonad-mesonephros region, and HSCs in fetal liver. Pre-HSCs show unique features in transcriptional machinery, arterial signature, metabolism state, signalling pathway, and transcription factor network. Functionally, activation of mechanistic targets of rapamycin (mTOR) is shown to be indispensable for the emergence of HSCs but not haematopoietic progenitors. Transcriptome data-based functional analysis reveals remarkable heterogeneity in cell-cycle status of pre-HSCs. Finally, the core molecular signature of pre-HSCs is identified. Collectively, our work paves the way for dissection of complex molecular mechanisms regulating stepwise generation of HSCs in vivo, informing future efforts to engineer HSCs for clinical applications.

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