Bone marrow transplantation, frequently used in the treatment of hematological malignancy and solid tumors, is increasingly being replaced by transplantation of semi-purified hematopoietic stem cells (HSC) derived from bone marrow or mobilized peripheral blood and purified on the basis of the expression of the CD34 cell surface molecule. In our work on mouse and rhesus HSC, we discovered compelling evidence that a CD34- negative precursor to CD34positive stem cells resides in adult bone marrow. We have substantial preliminary data to show that equivalent cells are also present in adult human bone marrow. The overall goal of this proposal is to establish this conclusively and to determine the function of these cells. CD34negative stem cells are identified by low Hoechst dye fluorescence and lack of CD34 expression. Our specific aims are 1) to confirm the hypothesis that the Hoechst-low cells are CD34negative in both bone marrow and mobilized peripheral blood, to establish a definitive phenotype for Hoechst-low cells, and to determine their prevalence in the CD34positive-enriched tissue given to patients; 2) to discover whether human CD34-negative Hoechst-low cells, like rhesus and mouse Hoechst-low cells, can proliferate and differentiate into lymphoid and myeloid cells in vitro and in vivo, and whether they give rise to CD34positive cells; and 3) to transduce Hoechst-low cells so that their fate can be followed in vitro and in vivo in order to confirm that individual clones give rise to multiple hematopoietic cell types, retain their multipotency during culture and marking, and can be genetically modified to express genes of therapeutic relevance. Physical characterization will be accomplished by flow cytometry. Hematopoietic potential will be determined with in vitro and in vivo assays including the long-term-initiating-cell assay (LTCIC) and NOD/SCID mouse xenotransplantation. Hoechst-low cells will be transduced with retroviral vectors and their hematopoietic potential will be tracked in vitro and in vivo. Together, these experiments will enable use to determine whether Hoechst-low cells in humans have functional characteristics of hematopoietic stem cells. We will determine whether CD34negative Hoechst-low cells are multi-potent precursors of CD34positive cells, as they are in Rhesus bone marrow. Conclusive evidence for CD34negative stem cell would transform our view of human hematopoietic stem cell biology, lead to improved bone marrow transplantation methods, and provide an important target for gene therapy.