Flt3 ligand-eGFP-reporter expression characterizes functionally distinct subpopulations of CD150 long-term repopulating murine hematopoietic stem cells.

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 cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporter cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporter cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporter HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporter cells develop to Flt3L-eGFP-reporter cells in vitro, although Flt3L-eGFP-reporter cells remain Flt3L-eGFP-reporter . CD150 Flt3L-eGFP-reporter cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporter 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.