Flt3 ligand-eGFP-reporter expression characterizes functionally distinct subpopulations of CD150⁺ long-term...

The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long-term repopulating HSCs (LT-HSC) are routinely enriched as Lin^- Sca1⁺ c-Kit⁺ CD34^- Flt3^- CD150⁺ CD48^- cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L-eGFP reporter mice, we show that endogenous Flt3L-eGFP-reporter RNA expression correlates with eGFP-protein expression. This Flt3L-eGFP-reporter expression distinguishes two LT-HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L-eGFP-reporter^low cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporter^high cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporter^low cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporter^high HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporter^low cells develop to Flt3L-eGFP-reporter^high cells in vitro, although Flt3L-eGFP-reporter^high cells remain Flt3L-eGFP-reporter^high . CD150⁺ Flt3L-eGFP-reporter^low cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporter^high cells do express EPCR but not CD41. Thus, FACS-enrichment of Flt3/ Flt3L-eGFP-reporter negative, Lin^- CD150⁺ CD48^- EPCR⁺ CD41⁺ HSCs allows a further 5-fold enrichment of functional LT-HSCs.