Abstract: The overall goal of this project is to expand human cord blood hematopoietic stem and progenitor cells (HSPC), to commercially available quantities that can be used in the clinic. This would allow the hematopoietic stem cells to be commercially available for use in either the treatment of cancers, autoimmune diseases, and in cases of Acute Radiation Syndrome (ARS). Furthermore, it is well known that umbilical cord blood hematopoietic stem cells are few in number, and cannot be used for an adult transplant or even in allogenic transplants because less than 30% of the patients can find an MHC compatible donor. Quality Biological, Inc (QBI) has been studying this phenomena by developing the necessary ex-vivo technologies that will contribute to the overall cell culture and analysis of these HSPC populations. We have extended these studies to determine the effect of specific cytokine cocktails (Flt-3, Stem Cell Factor, Thrombopotin, and IL-6; FST6) have on human CD34+ cells in 2D static cultures. Using this approach we noted an increase in the overall presence of HSPCs with time in culture as determined by CD34+ cell staining and flow cytometry. To further characterize these cells we took advantage of the xenograft serial transplantation models using either the fetal sheep or the NOD/SCID mice models for human hematopoiesis. The primary recipients of the human CD 34+cell had both short-term (progenitor cells) and long-term engrafting cells (stem cells) present, whereas, the secondary recipients only had the long-term engrafting cells (stem cells). These in vitro and in vivo assays allowed us to modify the serum-free culture conditions and systematically analyze the effect a specific modification had on the stem cell/long-term engrafting population and/or the progenitor cell/short-term engrafting population. CD34+ cells cultured in serum-free medium QBSF-60 with cytokines typically survive for only 7 days while maintaining their long-term engrafting capacity. In comparison, in the presence of the cytokine combination, FST6, we have maintained the viability of the CD34+ cells and their long-term engrafting capacity for up to 14-21 days, after which the cultures consisted of only progenitor cells. We propose to evaluate in the Phase 1 the above cytokine combinations alone and in the presence of other cytokines as bFGF and TGF β in a novel scalable, 3-Dimensional culture system that mimics the fluidic dynamics of the hematopoietic system. In the Phase II study we propose to evaluate these culture conditions with the optimal cytokine combination alone and in the presence of co-cultures comprised of CD34+ cells and cell types associated with the hematopoiesis using a limiting dilution and serial transplantation studies to determine whether the long-term engrafting cells have expanded or only the more committed progenitors. These cell types will include human endothelial cells, mesenchymal stem cells, and fibroblasts that have all been shown to pr...