The proposed experiments are aimed at elucidating the in vivo properties of hematopoietic stem cells with respect to developmental potential and temporal organization. The use of retroviral-mediated gene transfer provides a means of making in a unique and benign fashion each stem cell in a bone marrow explant. Genetically transduced bone marrow is used to engraft lethally irradiated recipient mice. Stem cells in a bone marrow inoculum are defined by a quantitative Southern blot analysis of myeloid and lymphoid cell populations isolated from engrafted mice. The existence and proliferative capacity of each stem cell is therefore retroactivity defined by the presence and magnitude of a given integrant in a mature cell population. The developmental potential of such cells is defined by the distribution of an integrant in cell populations of different lineages. Analysis of numerous mice should permit the derivation of a developmental fate-map which may, for the first time in a mammalian system, describe decisions and regulatory phenomena governing a complex program of cellular differentiation. Further studies begin to address the relative importance of intrinsic properties and host environment in the in vivo behavior of stem cell clones. The representation of individual members of a stem cell population as mature cell progeny over time will be assessed by periodic DNA analysis of the blood. This will result in a direct test of clonal succession models which predict that only a subset of the entire stem cell pool is actively engaged in hematopoiesis at any point in time. The experiments will further define kinetic parameters of hematopoiesis in response to artificial stress, thus providing an estimate of the flexibility inherent in this system. Further experiments will attempt to develop systems to allow engraftment with physically isolated stem cell clones. Focusing on in vivo as well as in vitro isolation of such clones, it should be possible to precisely evaluate the properties of the CFU-S as well as to begin interrelating the pluripotent stem cell, the CFU-S and thee clonogenic progenitor cell. The results obtained will provide a framework for future experimentation aimed at a mechanistic understanding of hematopoietic differentiation. The similarity of the murine and human blood systems implies that the proposed experiments will lead to a better understanding of hematopoiesis in man, and thus allow for the design of more effective therapeutic strategies in the treatment of genetic as well as acquired syndromes.
