The hematopoietic stem cell population constitutes a small percentage of the blood-forming tissues. By means of a number of developmental steps coupled with coordinate amplification, however, these few cells maintain homeostasis of the large populations of disparate blood cell types. Using recombinant DNA technology, it is now possible to uniquely mark the genomes of individual stem cells with identifiable sequences carried and inserted into the genome by retroviral vectors. The uniqueness of the marking derives from random retrovirus insertion into the genome. The proposed studies make use of this technique in mice to study several aspects of stem cell proliferation and function. Two mouse strains phenotypically different with respect to stem cell kinetics have been identified. In one strain a significantly larger proportion of stem cells is in cell cycle. This phenotypic difference is predicted to affect the number of simultaneously active stem cell clones and/or the rate of their serial usage. Two experimental mouse models will be used to study this question. The first is to mark stem cell in vitro and transplant them into lethally irradiated hosts to make radiation chimeras. The second is to mark stem cells in mid-gestation fetuses in utero to make somatic transgenic mice. Mice representing each experimental model, and of the two genotypes, will be serially bled, as adults, over their lifetime. Since genetically marked blood cells can be ascribed to individual stem cell clones, the number of clones represented in blood simultaneously, their lineage restrictions, and the temporal succession of clones can be established. To determine if the phenotype is intrinsic to the stem cells, or is extrinsically regulated, allophenic (chimeric) mice compounded from early embryos of the tow strains have been constructed and are available in significant numbers. Marrow cells from allophenic mice which contain a mixture of stem cells of the two different genotypic derivations will be infected with virus in vitro and subsequently will be used to engraft radiation chimeras. The genotypic ratio in the donor marrow is known; the question is whether the same ratio is recapitulated in the radiation chimeras. Stem cells in mid-gestation allophenic fetuses will be genetically marked and the subsequent clonal expression of stem cells of the two genotypes will be determined. By marking the developing hematopoietic systems of fetuses on sequential days during mid- gestation it should be possible construct a fate map. It is predicted that marking at early times in the gestational development of the hematopoietic system will result in less diversity of stem cell clones, since at that time, cells acting as founders for multiple clones may be marked.
