Optimizing Osteotropism of Human Mesenchymal Stem Cells

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): The proximate obstacle to realizing the enormous potential benefits of adult stem cell-based regenerative therapeutics is to safely and efficiently deliver the cells where they are needed/required. Though direct injection of adult stem cells into site(s) of disease/injury is feasible for certain organs with defined anatomic boundaries (e.g., heart), direct insertion is impractical for systemic conditions such as generalized bone diseases (e.g., Osteogenesis Imperfecta). Tissue-specific migration of blood-borne cells is initiated by adhesive interactions at target tissue endothelium mediated by molecules on circulating cells that are specialized to resist the shear forces of blood flow, the "homing receptors". Recruitment of cells from the vascular compartment to bone occurs within marrow, mediated by homing receptors bearing sialofucosylated lactosaminoglycans that engage E-selectin, a Cadependent lectin that is constitutively expressed on marrow sinusoidal vessels. In the prior funding period, we analyzed the expression of homing receptors on subsets of human hematopoietic stem/progenitor cells (HSPCs) and on human bone marrow-derived mesenchymal stem cells (BMSCs). We found that whereas human HSPCs express multiple homing receptors, including several glycoproteins that function as E-selectin ligands, BMSCs express no E-selectin ligands and only two characterized homing receptors, VLA-4 and CD44. Importantly, however, we observed that CD44 on BMSCs possesses a(2,3)-sialyllactosamine carbohydrate modifications that can serve as an acceptor for exogenous fucosylation. Using a novel alpha-(1,3) fucosyltransferase enzyme and attendant buffer conditions specifically formulated by us for treating BMSCs without affecting cell viability or multipotency, we converted the native CD44 glycoform into HCELL, a highly potent E-selectin ligand. In flow-based assays, HCELL* BMSCs displayed profound adhesive interactions on E-selectin, and, as observed by real-time intravital microscopy in immunocompromised mouse hosts, intravenously infused HCELL* BMSCs homed robustly to bone, with infiltrates evident within hours of infusion. Thus, stereoselective glycan engineering of a single membrane glycoprotein can direct navigation of BMSCs to a predetermined anatomic compartment. In this renewal application, we will extend these exciting findings to further optimize adhesive interactions and transmigration of human BMSCs on endothelium expressing E-selectin. We will also examine the biological effects of improved trafficking of BMSCs to marrow, and will analyze the discrete glycan structures created by enforced fucosylation. We anticipate that results of these studies will unveil a readily translatable roadmap for programming osteotropism of human BMSCs, thereby facilitating clinical use of these cells for treatment of generalized skeletal diseases and for enhancing engraftment following hematopoietic stem cell transplantation.