Technical Abstract: Human peripheral blood contains bone marrow-derived stem cells capable of differentiating into vascular endothelial cells (EC) and smooth muscle cells (SMC), the constituent cells of blood vessels. These progenitors are a potential source of cells for therapeutic tissue regeneration and for tissue engineering. In many instances (e.g., in the elderly and in diabetics who have diminished stem cell numbers or in tissue engineered constructs which need to be pre-fabricated), such cells will have to be provided by unrelated donors, creating the possibility that the recipient's immune system will mount an allogeneic rejection response against progenitor cell-derived vascular cells. In previous funding periods, the applicants have characterized the human allogeneic immune response to EC and SMC derived from differentiated vessels. Specifically, they evaluated the roles of different EC costimulator molecules involved in T cell activation and devised strategies to protect vascular EC from T cell-mediated injury. In this competitive renewal the applicants will shift their attention to stem cell-derived vascular cells (and have retitled the proposal to reflect this change of emphasis). Specifically, the applicants will differentiate circulating progenitor cells into EC and/or SMC in vitro and then compare these cells to vessel derived EC and/or SMC re the molecules they express which are relevant for alloimmunity (aim1);re their capacities to evoke an allogeneic T cell response in vitro (aim 2);and, using novel reconstitution methods in immunodeficient mice, re their capacity to evoke an allogeneic immune response in vivo (aim 3). Finally the applicants will evaluate two molecular strategies developed in the previous funding period to allow human progenitor cell-derived EC and SMC to evade a destructive immune response, namely knockdown of costimulators or expression of Bcl-2. Methods to be used include immunochemistry and immunofluorescence;molecular biology (e.g., transduction, qRT-PCR);cellular immunoassays;morphology and immunohistochemistry. These studies will define the extent and the mechanisms of the alloimmune response vs. progenitor cell-derived blood vessels and evaluate an investigator-initiated strategy to evade this response. Lay Abstract: Circulating stem cells from one individual may be used to repair damaged organs or build replacement organs for another individual. The investigators will study whether or not human stem cells, which form new blood vessels can cause the treated patient's immune system to reject the graft and if so, will test a new method for avoiding this complication.
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