We demonstrated in sheep that the transplantation of hematopoietic stem cells (HSC) from a preimmune fetal donor into an immunologically immature fetal recipient results in stable hematopoietic chimerism without rejection, or GVHD. Under NIH #HL39875-02 we then developed an in utero model of fetal HSC transplantation in the rhesus monkey and demonstrated sustained hematopoietic chimerism. This is a unique model in which the study of fetal immunotolerance and hematopoietic chimerism is directly applicable to man. Our studies have defined the basic techniques of fetal HSC transplantation and have optimized donor and recipient gestational age, donor cell number, and route of administration to achieve chimerism. The stable engraftment of multiple cell lineages has been confirmed, documenting the survival and renewal of donor pluripotential HSC. To characterize the immune function of our chimeric animals we have demonstrated normal in vitro and in vivo response to specific antigens. We have analyzed the cell surface phenotypes, in vitro response to antigen, cytokine production and cell mediated cytotoxicdy of the chimeric lymphocyte population. We have documented donor specific tolerance by one way MLR. Finally, we have documented a normal response of the donor cell population, to the regulatory signals of the recipient, in response to an anemic stress. Compared to conventional methods of postnatal bone marrow transplantation, the level of donor cell engraftment in these otherwise normal "unprepared" recipients is relatively low (less than l2%), perhaps reflecting competition with native stem cells for recipient marrow sites. Although, perhaps adequate for treatment of some hematopoietic deficiencies, i.e. SCIDS, treatment of many other hematopoietic diseases (e.g. hemoglobinopathies) will probably require higher levels of engraftment. In addition, our analysis of the chimeric state and the mechanism of fetal transplantation tolerance is incomplete. We now propose further studies to: 1) improve engraftment by manipulating donor HSC, including stem cell enrichment, and cytokine stimulation; 2) further characterize the immunologic state of our established chimeric animals and define the mechanism of fetal transplantation tolerance in the primate. 3) Perform parallel in vitro manipulations of human fetal stem cells to optimize methods of culture, enrichment, and expansion, and 4) Define the origin and transplantability of bone marrow stromal cells and CNS macrophages utilizing a new method for detection of engraftment. These studies will further our understanding of immuno-hematopoietic ontogeny and the mechanisms of fetal transplantation tolerance, and thus allow effective (and cost effective) treatment of a variety of severely disabling diseases before birth.