Abstract
Abstract
The proximate obstacle to realizing the potential benefits of stem cell-based regenerative therapeutics is to safely and efficiently deliver the cells where they are needed/required. Though transplantation of culture-expanded human mesenchymal stem cells (MSC) holds the potential for cure of generalized skeletal diseases such as osteogenesis imperfecta, clinical effectiveness has been constrained to date by the poor osteotropism (migration to bone) of infused MSCs. Tissue-specific migration is initiated by adhesive interactions mediated by molecules on the surface of blood-borne cells that are specialized to resist the shear forces of blood flow, the “homing receptors”. The recruitment of circulating cells to bone occurs within the marrow, at specialized marrow vessels that constitutively express E-selectin, a lectin that recognizes sialofucosylated determinants on its respective ligand(s). To gain insight on the expression and function of homing receptors on human MSC, we performed flow cytometry and Western blot analysis, together with parallel plate flow chamber studies under physiologic shear conditions. Whereas human hematopoietic progenitor cells (HPC) characteristically display the E-selectin ligands CLA and HCELL, which are α(2,3)-sialyl-α(1,3)-fucosylated glycoforms of PSGL-1 and CD44, respectively, biochemical and functional studies showed that human MSC express no E-selectin ligands and are devoid of PSGL-1. We also found that human MSC did not express many effectors of cellular trafficking to marrow present on HPC, such as LFA-1, LPAM-1 (α4β7), nor the chemokine receptor CXCR4, but did express VLA-4 and, notably, display a CD44 glycoform bearing α(2,3)-sialyllactosamine modifications that can serve as an acceptor for exogenous fucosylation. Employing an α(1,3)-fucosyltransferase preparation and enzymatic conditions specifically designed for treating live cells, the native human MSC surface CD44 glycoform was converted to the E-selectin ligand HCELL with commensurate induction of potent E-selectin-dependent shear-resistant rolling interactions on activated endothelium, without effects on MSC viability or multipotency. As observed by real-time intravital microscopy in immunocompromised (NOD/SCID) mouse hosts, intravenously infused HCELL+ human MSCs homed robustly to bone with marrow infiltrates evident within hours of infusion, whereas unmodified MSC and HCELL+ MSC treated with sialidase (which eliminates E-selectin binding) showed negligible rolling on marrow vessels and did not extravasate. Alignment of extravasated HCELL+ human MSC along the endosteal surface was observed, with rare differentiation to osteoblasts as evidenced by immunohistochemical staining for human osteocalcin. These results indicate that despite absence of CXCR4 and many other effectors of cellular trafficking to marrow, osteotropism of systemically administered human MSC was conferred by ex vivo cell surface carbohydrate modification of a single glycoprotein, CD44, rendering the potent E-selectin ligand HCELL. Our findings unveil a readily translatable roadmap for programming MSC trafficking, thus overcoming a critical barrier in use of these cells for regenerative therapeutics of systemic skeletal diseases.