Critical Reagents that Support Fetal Hematopoiesis

The overall goal is to develop serum-free medium for the proliferation and differentiation of fetal liver, spleen and bone marrow. The culture system will be comprised of serum-free medium and various cytokines. Serum-free medium will allow the direct effect the cytokines have on the fetal hematopoietic cells to be free of the extraneous components found in fetal bovine serum. Such an in vitro system will be valuable in assessing the changes in fetal blood populations during ontogeny. Fetal sheep will be used in these studies because of the well defined sequence of events in maturation of the blood system. In sheep, the liver is hematopoietic from day 20 to gestation, spleen from day 50 and bone marrow at about 70 days of gestation. Cells isolated from these tissues at various times will be placed in liquid culture containing serum-free medium and various cytokines. From these, cultures cells will be removed, both immediately and intermittingly, placed in soft-agar assays containing various cytokines and evaluated for colony formation (CFU-Mix, CFU-GM, and BFU-E). Such an approach will allow the characterization of the fetal hematopoietic populations during ontogeny.