Engraftment of hematopoietic stem or progenitor cells during bone marrow transplantation is dependent on the preferential homing of progenitors to the bone marrow microenvironment. Within the hematopoietic micro-environment, whether it is embryonic yolk sac, fetal liver, or adult bone marrow, microvascular endothelium not only provides cellular contact and secrete cytokines that allows for the preservation of the steady state hematopoiesis, but also acts as a gatekeeper controlling the trafficking and homing of the HSC. Most studies in the role of human bone marrow endothelial cells (BMEC) in the regulation of hematopoiesis had been stymied by the lack of techniques to isolate homogenous population of BMEC monolayers. We have developed a reproducible technique for isolation and cultivation of BMEC monolayers. We have shown that direct cellular contact between BMEC and CD34+ progenitors is critical for the long-term proliferation, commitment and terminal-differentiation of the hematopoietic stem cells. BMEC monolayers support the adhesion and transmigration of a subset of HSC cells with high proliferative potential. This binding which is dependent on divalent cations, can be partially blocked by antibodies to B1 and B2 integrins. The goal of this project is to define BMEC membrane bound factor(s) that regulate trafficking, expansion and in this proposal we intend to explore the role of membrane bound factors, including CD34/L- selectin, B1, B2 integrins, and membrane bound KL/c-kit receptor that may regulate trafficking as well as proliferation of progenitor cells. We have recently shown that BMEC also express membrane bound delta like protein (DLL1). DLL1 and its receptor human Notch 1/TAN-, which regulate developmental cell fate processes, may also play a major role in adhesion/proliferation of HSC. In this regard, we plan to study the role of these ligand pairs including DLL1/Notch1, by using blocking monoclonal antibodies to either ligand pair in blinding, and transmigration studies as well as in long-term BMEC-CD34+ progenitor coculture studies. In a novel approach, genetically engineered adenoviral vectors to overexpress BMEC membrane bound cytokines and adhesion molecules will be used to examine their function in binding, and long-term coculture studies. We have utilized expression cloning using a BMEC cDNA library to screen for human DLL1 homologue, and as yet unrecognized adhesion/homing receptor that regulates adhesion but also may regulate proliferation of HSC. Disruption of BMEC metabolism within the bone marrow microenvironment may contribute to stem cell dysfunction and progression to aplastic anemias, myelodysplasia, and graft failure during bone marrow transplantation. These studies may culminate in the identification of known or as yet unrecognized adhesion molecule that may regulate selective homing of HSC to the bone marrow.
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