In order to insure normal lifelong blood function and avoid proliferative disorders such as leukemia, the mechanisms which govern the hematopoietic system must be tightly controlled and flexible. In both mouse and human all mature blood cells are clonally derived from a rare population of stem cells which are totipotent in differentiation ability and self-renewing in their ability to continuously function for long time-periods. The proposed experiments are aimed at directly characterizing and isolating these cells and their immediate clonal progeny, investigating in vitro conditions to facilitate direct manipulation of stem cells and analyzing the functional roles if novel cell surface receptors in mediating stem cell behavior. The main stem cell assay involves transplantation of purified, genetically marked or modified stem cells into irradiated host mice and analysis of in vivo hematopoietic function over time. Stem cell purifications involve a variety of cell surface markers in concert with flow cytometry. Experiments to culture stem cells in vitro will utilize a large panel of stromal cell lines as candidate supporting elements. The combination of cell purification and precise in vivo analysis with in vitro culture should provide a system for direct functional analyses. Parallel studies will investigate the functions of two c-kit related receptor tyrosine kinases isolated from enriched fetal liver stem cells. Several distinct approaches are proposed. First, antibody reagents to these receptors will be utilized as a further possible means to characterize the stem cell population. Second, these receptors will be introduced into stem cells and their effects on in vivo function will be measured in transplantation experiments. Third, one of the receptor genes will be genetically ablated by homologous recombination strategies in Embryonic Stem (ES) cells. These will be used to construct mutant mice and may provide an ideal system for structure-function studies. Taken together the proposal consists of systemic, cellular and molecular approaches to understanding hematopoietic stem cell function. The similarity of the murine and human blood system implies that the experiments will lead to a better understanding of hematopoietic stem cell function in man and thus allow for the design of more effective therapeutic strategies in the treatment of genetic as well as acquired syndromes.
