Tracing the Origin of the HSC Hierarchy Reveals an SCF-Dependent, IL-3-Independent CD43− Embryonic Precursor

•Pro-HSCs are early embryonic precursors of definitive HSCs in E9.5 dorsal aorta•In contrast to CFU-C, pro-HSCs lack CD43 marker and have low cKIT expression•SCF, but not IL-3, promotes progression of pro-HSCs into definitive HSCs•HSCs develop hierarchically in a four-step process

SummaryDefinitive hematopoietic stem cells (HSCs) develop in the aorta gonad mesonephros (AGM) region in a stepwise manner. Type I pre-HSCs express CD41 but lack CD45 expression, which is subsequently upregulated in type II pre-HSCs prior to their maturation into definitive HSCs. Here, using ex vivo modeling of HSC development, we identify precursors of definitive HSCs in the trunk of the embryonic day 9.5 (E9.5) mouse embryo. These precursors, termed here pro-HSCs, are less mature than type I and II pre-HSCs. Although pro-HSCs are CD41+, they lack the CD43 marker, which is gradually upregulated in the developing HSC lineage. We show that stem cell factor (SCF), but not interleukin-3 (IL-3), is a major effector of HSC maturation during E9–E10. This study extends further the previously established hierarchical organization of the developing HSC lineage and presents it as a differentially regulated four-step process and identifies additional targets that could facilitate the generation of transplantable HSCs from pluripotent cells for clinical needs.