Absence of E-Selectin at the Vascular Niche Delays Hematopoietic Stem Cell Turn-Over.

Hematopoietic stem cells (HSC) reside in specialized niches in the bone marrow (BM), that regulate their survival, proliferation and differentiation. Two types of HSC niches have been reported:

1. endosteal niches in close contact with osteoblasts, and

2. endothelial niches near vascular sinuses.

Whether these niches have distinct functions in controlling HSC fate remains unknown. One difference between these two niches is the constitutive expression of E-selectin and P-selectin by BM endothelial cells. E- and P-selectin are two cell adhesion molecules that modulate hematopoietic progenitor cell (HPC) survival, proliferation and differentiation in vitro. We now show that deletion of E-selectin, but not P-selectin, delays HSC turn-over in the BM in vivo. Mice lacking either E-selectin (E^{−/ −}), P-selectin (P^{−/ −}) or both (PE^{−/ −}) were given bromo-deoxyuridine (BrdU) in their drinking water for up to 14 days. Lineage-negative c-KIT⁺ Sca-1⁺ CD34⁻ (LKS34) cells were sorted from the BM and stained for BrdU incorporation into genomic DNA. Although it took only 3.6 days for 50% of LKS34 cells from wild-type (WT) and P^{−/ −} mice to incorporate BrdU, 9 days were required for 50% BrdU incorporation in LKS34 cells from E^{−/ −} and PE^{−/ −} double KO mice. Thus, HSC cycling time is 2.5 times slower in the absence of E-selectin. To confirm these findings, LKS cells were stained with rhodamine123, a vital dye that is retained by metabolically active cells but effluxed from quiescent HSC. A higher proportion of LKS cells from E^{−/ −} mice were rhodamine dull (34±2%) than WT LKS (23±1%; p=0.037) confirming that a greater proportion of HSC from E^{−/ −} mice are quiescent. To further support these findings, we determined the effect of E-selectin deletion on HSC recovery following cytotoxic stress with a single dose of 5-fluorouracil (5FU 150mg/kg). As KIT is strongly down-regulated in the BM of 5FU-treated mice, we examined frequency and BrdU incorporation in Lin⁻ Sca1⁺ CD41⁻ CD48⁻ CD150⁺ long-term reconstituting HSC. We found HSC recovery to be enhanced in E^{−/ −} mice with a 5-fold increase in HSC numbers per femur compared to WT mice at day 7 post-5FU. Despite the more rapid recovery of E^{−/ −} HSC, BrdU incorporation remained significantly lower in E^{−/ −} HSC on days 3 and 7 post-5FU suggesting the decreased HSC turn-over in the absence of E-selectin protects them from the cytotoxic effect of 5FU. To determine whether this effect was mediated by the two described E-selectin receptors PSGL-1 and/or CD44, BrdU incorporation experiments were repeated with mice lacking both the PSGL-1 and CD44 genes. LKS cell turnover in these mice was identical to that of WT suggesting that the effect is mediated by a distinct unknown receptor(s) on HSC. The fact that a novel E-selectin receptor on HSC/HPC is involved was confirmed both using flow cytometry with selectin-IgM chimeras as well as cell adhesion assays using plastic-adsorbed selectin-IgG chimeras. In both assays, 90-95% of LKS cells from CD44^{−/ −} PSGL-1^{−/ −} double KO mice bound E-selectin whereas adhesion to P-selectin was completely lost. Taken together our findings suggest that

1. E-selectin, whose constitutive expression is restricted to BM endothelial cells, plays an important role in the regulation HSC turnover in vivo,

2. endothelial niches, where E-selectin is expressed, support more rapid HSC turn-over within the BM, and

3. this effect is mediated by unknown E-selectin receptors distinct from PSGL-1 or CD44.