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

• Published in: European journal of immunology Vol. 47; no. 9; pp. 1477 - 1487
• Main Authors: Tornack, Julia, Kawano, Yohei, Garbi, Natalio, Hämmerling, Günter J., Melchers, Fritz, Tsuneto, Motokazu
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 01.09.2017
• Subjects: Animals; Ataxin; Autophagy - genetics; Bone marrow; Bone Marrow Cells - physiology; c-Kit protein; CD150 antigen; CD34 antigen; Cell Differentiation; Cell Proliferation; Cell Self Renewal; Cells, Cultured; Cytokines; Enrichment; Flow cytometry; Flt3 ligand; FLT3L protein; Genes; Genes, Reporter - genetics; Green Fluorescent Proteins - genetics; Guanine Nucleotide Exchange Factors - genetics; Guanine Nucleotide Exchange Factors - metabolism; Hematopoietic Stem Cells - physiology; Homeodomain Proteins - genetics; Homeodomain Proteins - metabolism; Hypoxia; Hypoxia - genetics; Kinases; Ligands; Long‐term repopulation; Membrane Proteins - genetics; Membrane Proteins - metabolism; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phagocytosis; Protein C; Regulators; Ribonucleic acid; RNA; Rodents; Signaling Lymphocytic Activation Molecule Family Member 1 - metabolism; Stem cell transplantation; Stem cells; Subpopulations; Bone marrow; Cytokines; Flt3 ligand; Long-term repopulation; Stem cells
• ISSN: 0014-2980; 1521-4141
• DOI: 10.1002/eji.201646730

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.