Key Points• Human HSPCs expressing CD34 exhibit E-selectin binding activity, whereas those lacking CD34 do not.• CD34 is a unique E-and P-selectin ligand on human HSPCs that binds with kinetics comparable to other known selectin ligands.

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.

In contrast to the short-term(ST)-CD34pos stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSC) found in the CD34neg stem cell pool have trouble migrating and engrafting when introduced intravenously. We set out to fully elucidate the adhesion mechanisms used by ST/LT-HSCs to migrate to the bone marrow in order to understand these deficiencies. Focusing on murine ST-HSCs(Flk2negCD34pos) and LT-HSCs(Flk2negCD34neg), we observed a distinctive expression pattern of bone marrow homing effectors necessary for the first step, namely sialyl Lewis-X(sLex;ligand for E-selectin), and the second step, namely CXCR4 (receptor for SDF-1). sLex expression was higher on Flk2negCD34pos ST-HSCs(>60%) compared to Flk2negCD34neg LT-HSCs(<10%), which correlated to binding to E-selectin. Higher levels of CXCR4 were observed on Flk2negCD34pos ST-HSCs compared to Flk2negCD34neg LT-HSCs. Interestingly, expression of CD26, a peptidase known to deactivate chemokines (i.e.SDF-1), was higher on Flk2negCD34neg LT-HSCs. Given that migration is compromised in Flk2negCD34neg LT-HSCs, we aimed to enhance their ability to migrate using recombinant fucosyltransferase 6 (rhFTVI) and DiprotinA (CD26-inhibitor). We observed that although LT-HSCs expressed low levels of sLex, in vivo engraftment was not compromised. Moreover, although both treaments enhanced migration in vitro, only pre-treatment of LT-HSCs with DiprotinA enhanced engraftment in vivo. Remarkably, fucosylation of Flk2negCD34pos ST-HSCs consistently led to their ability to transplant secondary recipients, the gold standard for testing functionality of LT-HSCs. These data suggest that treatments to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs, differentially influences their abilities to migrate and engraft in vivo and boosts ST-HSCs engraftment in vivo.

Highlights• Human fucosyltransferases expressed and purified from both Pichia pastoris yeast and Bombyx mori silkworm display specific activities > 1000 pmol/min/g that efficiently create sialyl Lewis X epitopes on primary human stem cells capable of binding E-selectin.• Fusing BmApoLpIII to fucosyltransferases allows for the production of highly active human enzymes in large scale from Bombyx mori silkworm.

Exosomes are tiny vesicles released by cells that carry communications to local and distant locations. Emerging research has revealed the role played by integrins found on the surface of exosomes in delivering information once they reach their destination. But until now, little has been known on the initial upstream steps of the migration process. Using biochemical and imaging approaches, we show here that exosomes isolated from both leukemic and healthy hematopoietic stem/progenitor cells can navigate their way from the cell of origin due to the presence of sialyl Lewis X modifications surface glycoproteins. This, in turn, allows binding to E-selectin at distant sites so the exosomes can deliver their messages. We show that when leukemic exosomes were injected into NSG mice, they traveled to the spleen and spine, sites typical of leukemic cell engraftment. This process, however, was inhibited in mice pre-treated with blocking E-selectin antibodies. Significantly, our proteomic analysis found that among the proteins contained within exosomes are signaling proteins, suggesting that exosomes are trying to deliver active cues to recipient cells that potentially alter their physiology. Intriguingly, the work outlined here also suggests that protein cargo can dynamically change upon exosome binding to receptors such as E-selectin, which thereby could alter the impact it has to regulate the physiology of the recipient cells. Furthermore, as an example of how miRNAs contained in exosomes can influence RNA expression in recipient cells, our analysis showed that miRNAs found in KG1a-derived exosomes target tumor suppressing proteins such as PTEN.