The goal of this proposal is to delineate the culture conditions using pharmaceutical grade serum-free medium that will support the ex vivo expansion of cryopreserved umbilical cord blood for use in adult transplantation. The studies presented here will evaluate clinically relevant culture conditions necessary to retain the short-term and long-term hematopoietic reconstituting cells using the recently developed sheep in utero transplantation model for human hematopoiesis. Feasibility data presented in this proposal shows that l) cryopreserved cord blood can be ex vivo expanded in a pharmaceutical grade serum-free medium specifically designed for the ex vivo expansion of CD34+ cells from cord blood, and 2) maintain their long-term engrafting ability in the sheep model for human hematopoiesis. In the Phase I effort, we propose to extend these studies to evaluate the role various cytokine cocktails, concentration effects, and culture periods have on ex vivo expansion of cryopreserved cord blood cells. In particular, we propose to analyze the cultured cells in in vitro hematopoietic assays for I) cell cycle status, 2) viable cell number, 3) immunophenotype, 4) colony formation, 5) long- term initiating cells, and 6) long-term engrafting ability using the sheep model for human hematopoiesis. The Phase II will focus on other cytokine combinations, concentrations and culture times in the in vitro hematopoietic assays and in vivo for long-term engraftment and quantitation of the expansion potential of the engrafting cells in the sheep model for human hematopoiesis. Using the above approach we will be able to address, in a clinically relevant manner, a number of important issues concerning the use of ex vivo expansion of cryopreserved umbilical cord blood for adult transplantation. PROPOSED COMMERCIAL APPLICATION: The ex vivo expansion of cryopreserved cord blood with pharmaceutical grade serum-free reagents and conditions will be essential in its use in adult transplantation.