Tracing haematopoietic stem cell formation at single-cell resolution.

Area of Science:

• Developmental Biology
• Stem Cell Biology
• Hematopoiesis

Background:

• Haematopoietic stem cells (HSCs) originate from embryonic precursors, including haemogenic endothelial cells and pre-haematopoietic stem cells (pre-HSCs).
• The precise molecular identity of pre-HSCs remains largely undefined, hindering a full understanding of HSC development.
• Understanding pre-HSC biology is critical for advancing regenerative medicine and HSC engineering.

Purpose of the Study:

• To isolate and characterize nascent pre-HSCs using potent surface markers.
• To elucidate the unique molecular signatures and functional properties of pre-HSCs.
• To investigate the role of mechanistic targets of rapamycin (mTOR) signaling in HSC emergence.

Main Methods:

• Isolation of high-purity pre-HSCs validated by single-cell-initiated serial transplantation.
• Single-cell RNA sequencing of endothelial cells, CD45(-) and CD45(+) pre-HSCs, and fetal liver HSCs.
• Transcriptome data-based functional analysis to assess cell-cycle heterogeneity and signaling pathways.

Main Results:

• Pre-HSCs exhibit distinct transcriptional machinery, arterial signature, metabolic state, signaling pathways, and transcription factor networks.
• Mechanistic targets of rapamycin (mTOR) activation is essential for HSC emergence, but not for hematopoietic progenitors.
• Significant heterogeneity in cell-cycle status among pre-HSCs was revealed through transcriptome analysis.

Conclusions:

• Identification of the core molecular signature of pre-HSCs provides a foundation for further research.
• The study clarifies the indispensable role of mTOR in HSC generation from precursors.
• These findings offer insights into the stepwise generation of HSCs in vivo and inform strategies for HSC engineering.