Mechanisms and applications of selectin-mediated hematopoietic stem cell adhesion under flow

Hematopoietic stem and progenitor cells (HSPC) give rise to all mature blood cells and have a range of therapeutic uses. However, current understanding of the adhesion molecules and mechanisms regulating HSPC homing to the bone marrow is limited. We studied the interaction of human HSPC with E-, P-, and L-selectin substrates in a flow chamber. We find that primitive CD34 + and CD34+CD38− bone marrow cells roll to a greater extent and more slowly than more differentiated CD34 − and CD34+CD38+ cells, respectively, especially on P- and L-selectin. Our results provide direct evidence of selectin ligands on HSPC under flow and suggest a mechanism by which HSPC subpopulations may differentially home to the bone marrow. These findings also suggest that HSPC can be enriched from bone marrow by exploiting differences in selectin-mediated rolling adhesion. To understand the fundamentals by which different cell populations roll on selectins, and to develop methods for enriching HSPC by rolling adhesion, we extend the use of a previously developed cell-free system to study the interactions between L-selectin and sialyl Lewisx (sLe x) coated microspheres under flow. We find that the rolling flux of sLex microspheres on L-selectin goes through a maximum with respect to shear stress at 0.7 dyne/cm2 and that the magnitude of this shear threshold effect, in which the maintenance and promotion of rolling interactions on selectins requires shear stress above a threshold value, diminishes with increasing L-selectin site density. Our ability to recreate the cellular shear threshold effect in a cell-free system suggests the origin of the effect is in the physical chemistry of L-selectin interaction with its ligand, and not cellular features such as deformability or signaling. We also show that populations of sLex microspheres designed to roll with different velocities on L-selectin can be separated by differences in rolling adhesion and that recovery and purity can be estimated from rolling velocity data. Finally, we demonstrate an enrichment of HSPC from bone marrow by differential rolling on L-selectin that does not adversely affect cell viability. We believe that our novel enrichment method which emulates physiology has several advantages over commercially available HSPC separation technologies