Hematopoietic stem cells (HSC) in the bone marrow continuously give rise to all blood cell types throughout the entire life. The activity of HSC in aged individuals shows abnormalities such as reduced lymphoid development, enhanced myeloid development, and predisposition to myeloproliferative diseases. Recent evidence suggests that the HSC comprise two distinct subsets: the lymphoid-biased HSC with limited self-renewal capacity, and the myeloid-biased HSC with superior long-term reconstitution capacity. The latter subset becomes predominant with age, limiting lymphopoiesis and predisposing to myeloproliferation. While the HSC heterogeneity model provides essential framework for understanding HSC dynamics and aging, it is limited by our current inability to prospectively identify the two HSC subsets or to trace their progeny in intact organisms. Our preliminary studies characterized transgenic strains expressing green fluorescent protein or Cre recombinase in a fraction of HSC consistent with myeloid-biased HSC. We propose that these reporter strains can be used for the identification and lineage tracing of the two HSC subsets. In Aim 1, expression analysis and adoptive transfers will be used to verify the marking of functional HSC subsets by the fluorescent and Cre reporters. In Aim 2, these reporters will be used for the quantification and lineage tracing of the HSC after mobilization and immune-mediated stress. In addition, the contribution of HSC subsets to the development of myeloid leukemia will be characterized. These experiments would establish a novel genetic system to analyze HSC heterogeneity, and characterize HSC dynamics in intact animals without adoptive transfer. A successful outcome would provide useful tools and initial data for the study of HSC heterogeneity during leukemogenesis. Understanding the cellular basis of HSC activity should facilitate novel approaches to the rejuvenation of age- or stress-impaired hematopoiesis and to the treatment of hematopoietic malignancies. PUBLIC HEALTH RELEVANCE: Hematopoietic stem cells (HSCs) in the bone marrow continuously give rise to all blood cell types throughout the entire life. We will characterize novl genetic systems to identify and trace the functional subsets of HSCs in naive animals and during leukemia development. Understanding the cellular basis of HSCs activity should facilitate novel approaches to the rejuvenation of age- or stress- impaired blood development and to the treatment of blood cancers.