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

4 years ago

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

Fritz Melchers, Julia Tornack, Natalio Garbi, Motokazu Tsuneto, Günter J. Hämmerling, Yohei Kawano

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-reporterlow cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporterhigh cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporterlow cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporterhigh HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporterlow cells develop to Flt3L-eGFP-reporterhigh cells in vitro, although Flt3L-eGFP-reporterhigh cells remain Flt3L-eGFP-reporterhigh. CD150+Flt3L-eGFP-reporterlow cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporterhigh 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. Long-term repopulating HSCs in bone marrow of the mouse are in vast majority resting, hypoxic, autophagy-active, Flt3- and Flt3-Ligand-non-expressing LSK cells. The identification of this new marker promises to further improve conditions for enrichment of LT-HSCs and, thus, successful, efficient restoration of all blood cell lineages in transplanted hosts after chemotherapy and irradiation.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/eji.201646730