Transcriptome Dynamics of Hematopoietic Stem Cell Formation Revealed Using a Combinatorial Runx1 and Ly6a Reporter...

Area of Science:

• Developmental Biology
• Hematopoiesis
• Stem Cell Biology

Background:

• Studying hematopoietic stem cell (HSC) development is challenging due to the rarity of precursor cells and overlapping marker expression.
• Hemogenic endothelial cells (HECs) give rise to HSCs, but their precise identification and maturation stages remain unclear.

Purpose of the Study:

• To develop a novel reporter mouse model for visualizing and studying the emergence and maturation of pre-HSCs from HECs.
• To identify the earliest sites of HSC activity and characterize the molecular changes during HSC maturation.

Main Methods:

• Generation of a Tg(Runx1-mKO2; Ly6a-GFP) dual reporter mouse.
• Flow cytometry and single-cell RNA sequencing (scRNA-seq) for cell identification and transcriptional profiling.
• Analysis of HSC activity in different developmental niches, including the placenta and fetal liver.

Main Results:

• The dual reporter system successfully marked intra-arterial HECs, hematopoietic cluster cells (HCCs), and HSCs.
• Hematopoietic stem cell and lymphoid potential were primarily identified in double-positive (DP) reporter cells.
• Significant HSC activity was first observed in placental DP cells, indicating its importance for early HSC maturation.
• scRNA-seq revealed that maturing pre-HSCs into fetal liver HSCs exhibit interferon exposure, multi-lineage differentiation gene expression, and a prolonged cell cycle.

Conclusions:

• The Tg(Runx1-mKO2; Ly6a-GFP) mouse is a valuable tool for dissecting HSC development.
• The placenta serves as a critical niche for early HSC maturation and expansion.
• Maturation of HSCs involves specific transcriptional and cell cycle changes, preparing them for their roles in adult hematopoiesis.