Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells

Abstract

According to the multistep model of cell migration, chemokine receptor engagement (step 2) triggers conversion of rolling interactions (step 1) into firm adhesion (step 3), yielding transendothelial migration. We recently reported that glycosyltransferaseprogrammed stereosubstitution (GPS) of CD44 on human mesenchymal stem cells (hMSCs) creates the E-selectin ligand HCELL (hematopoietic cell E-selectin/L-selectin ligand) and, despite absence of CXCR4, systemically administered HCELL + hMSCs display robust osteotropism visualized by intravital microscopy. Here we performed studies to define the molecular effectors of this process. We observed that engagement of hMSC HCELL with E-selectin triggers VLA-4 adhesiveness, resulting in shear-resistant adhesion to ligand VCAM-1. This VLA-4 activation is mediated via a Rac1/Rap1 GTPase signaling pathway, resulting in transendothelial migration on stimulated human umbilical vein endothelial cells without chemokine input. These findings indicate that hMSCs coordinately integrate CD44 ligation and integrin activation, circumventing chemokinemediated signaling, yielding a step 2-bypass pathway of the canonical multistep paradigm of cell migration.T he successful application of adoptive cellular therapies, including stem cell-based regenerative medicine, critically depends on delivering relevant cells to target sites (1). Four distinct steps have been described in the process of cell migration: tethering and rolling mediated principally by the selectin group of adhesion molecules (step 1), chemokine receptor engagement and resultant G-protein-coupled "inside-out" activation of integrins (step 2), firm adhesion by integrins (step 3), culminating in transendothelial migration (TEM) (step 4) (2, 3). The conventional multistep paradigm holds that step 2 is critical for TEM, with engagement of discrete chemokine receptors on the cell surface triggering subsequent integrin-dependent steps. For recruitment of circulating cells to bone marrow, the CXCL12/CXCR4 chemokine axis plays a central role (4). Notably, specialized sinusoidal vessels constitutively express CXCL12 and E-selectin at sites where cells extravasate into the marrow parenchyma (5). Hematopoietic stem cells express multiple E-selectin ligands and abundant CXCR4 (1), and are thus equipped with relevant effectors of step 1 and step 2 events mediating osteotropism.Mesenchymal stem cells (MSCs) can differentiate into a variety of tissues (6, 7) and display potent immunomodulatory effects (8, 9). Although MSCs have shown therapeutic potential in skeletal diseases (10), myocardial injury, and immunologic disorders (11, 12), a critical limitation to the therapeutic use of MSCs is their modest tissue colonization upon systemic administration (13-15). Importantly, human MSCs (hMSCs) lack expression of various adhesion receptors that mediate step 1 interactions, particularly E-selectin ligands (16). Moreover, in contrast to hematopoietic cells, hMSCs display a rather limited repertoire of chemokine receptors, with variable eviden...

Glycoengineering of HCELL, the Human Bone Marrow Homing Receptor: Sweetly Programming Cell Migration

“…Studies of human MSCs undertaken in both static and hydrodynamic flow conditions showed that engagement of CD44 with hyaluronic acid, or of (glycoengineered) HCELL with E-selectin, in each case triggers a Rac1/Rap1-GTPase signaling pathway, resulting in upregulation of VLA-4 firm adhesion to its ligands VCAM-1 and fibronectin. Consistent with these findings, human MSCs underwent transendothelial migration in the absence of exogenous chemokine input on cytokine-stimulated human endothelial monolayers expressing both E-selectin and VCAM-1 71 . Interestingly, we observed that engagement of either CD44 or HCELL induces a bimolecular complex between CD44/HCELL and VLA-4, but that VLA-4 activation is dependent on G-protein-coupled signaling, not on the adhesion molecule co-association 71 .…”

Primary Mesenchymal Stem and Progenitor Cells from Bone Marrow Lack Expression of CD44 Protein

“…On the contrary, primary MSCs showed more efficient homing to BM compared with the cultured MSCs (40). Loss of BM homing capacity of the cultured MSCs might be due to culture-induced changes in expression of adhesion receptors, including CD44 and CXCR4 on the cells, which might lead to unwanted entrapment in other organs (6,38). CD44 exists in multiple isoforms on different types of cells, and CD44 expression as well as its binding capacity toward its ligands are regulated by different cytokine stimulation and other environmental changes (41).…”

“…6). These included cell adhesion receptors, such as integrins (ITGA3, ITGAE, ITGB5, and ITGA6) and CD109, ADAM12, CD151, CD59, and CD248, some of which have been reported to be expressed on cultured MSCs (30,38). Although MSC-associated markers, such as CD73 and CD146, are up-regulated, NGFR/CD271, VCAM1, CD36, and EPOR are down-regulated in the cultured MSCs compared with the fresh ex vivo analyzed CD44…”

Intravascular Stem Cell Transplantation for Stroke

“…Recently, glycosyltransferase-programmed stereosubstitution of CD44 to an E-selectin ligand (HCELL) was used to enhance binding to E-selectin. This modification ultimately promoted CD49d binding to VCAM-1 and enhanced transendothelial migration of human MSCs into bone [87]. Our laboratory has previously shown that injection of CD49d + mouse NPCs selected through FACS results in higher cell engraftment in the cortex, hippocampus, and subventricular zone.…”