Bone marrow transplantation is an important clinical procedure for the treatment of malignancies and is an essential component of gene therapy. Yet the details of engraftment, including the dynamics of stem cell seeding, proliferation and differentiation, ate not well understood. A novel transplantation paradigm has been established in which retrovirally marked marrow from allophenic mice compounded from C57BL/6 and DBA/2 embryos serves as graft. Coexistent stem cell pools are thus put in a competitive setting where the genotypes of the repopulating clones are distinguished by isozymes, and individual clones are distinguished by random genomic integration of provirus. A genetically determined difference(s) between the two strains defines two distinct phases of engraftment, with each genotype dominating one of the phases.
The specific aims of the proposal are built on this precise method of determining number, size and genotype of repopulating clones. First, serial transplantation of allophenic marrow is proposed to distinguish between two hypotheses: one proposes that the early domination of repopulation by DBA/2, and its ultimate senescence, is linked to a higher proliferative irate; a second proposes that engraftment is due to genotype-limited differences in the abundance of stem cell subsets, each important in one of the two phases of engraftment. Second, the question of whether or not the genetic difference extends to fetal engraftment of hematopoietic anlage will be asked. Allophenic marrow and fetal liver will be engrafted into stem cell deficient (W/W-v) fetuses. Third, recombinant inbred (RI) mouse strains derived from C57BL/6 and DBA/2 progenitor strains will be used as donors in an innovative donor/recipient strain combination. The use of RI strains win allow us to determine the number of genes involved, and through segregation of the trait in relationship to others already mapped, to determine approximate genomic location. Fourth, engraftment entails cooperation between donor stem cells and host stroma. Allophenic mice will be constructed by combining normal and mutant (Sl/Sld) embryos. Questions to be asked include: How much normal microenvironment is required for normal hematopoiesis? Is hematopoiesis anatomically diffuse or is it restricted to foci which contain wild-type stromal cells, as defined by the CFU-F assay? Answers to these questions may provide insight into the role of the stem cell growth factor elaborated by normal alleles at the Steel locus.
