The goal of this project is to investigate the early stages of hematopoiesis in a mouse model system, utilizing methodology for obtaining highly enriched populations of hematopoietic stem cells (HSC) from mouse bone marrow tissue. These cells are then transplanted into irradiated recipient animals in a congenic strain combination which allows identification and quantitation of graft-derived cells over the lifetime of the transplant recipients. Using this approach, one can ask a variety of questions regarding the nature of HSC in the mouse, including the identification of surface antigens expressed by these cells, the functional heterogeneity of the stem cell compartment at a single cell level, and the susceptibility of HSC populations to infection by pathogenic mouse viruses and by retroviral constructs which amy be exploited as vectors to introduce new genetic material into the stem cell compartment. Further, in vitro culture systems and growth factors can be tested for the ability to propagate and perhaps influence self-renewal of the enriched HSC populations. In order to realize these goals, it is critical to develop confidence in the methodology utilized to enrich the HSC population. In particular, the enrichment method should select for all primitive HSC present in the bone marrow, and not simply a subpopulation which may or may not include the most primitive members of the HSC compartment. This possibility has been considered in some detail, starting from the hypothesis that mouse HSC can be identified by immunofluorescent staining as those cells which express the Ly6A/E and Thy-1 antigens in the absence of antigens characteristic of differentiated lineages of blood cells (Lineage negative). Surprisingly, while mouse HSC seem largely to fit the phenotypic description of Ly6A/E positive and Lineage negative, in mouse strains expressing the Thy-1.2 allele a significant level of HSC activity was demonstrated within both Thy-1 positive and Thy-1 negative populations. In contrast, the Thy-1.1 allele was expressed by the majority of HSC. Therefore, some variability exists in the cell surface antigenic phenotype of mouse HSC.
