The analysis of bone marrow failure states is complicated by limited models of hematopoietic stem cell physiology. The in vitro culture of committed progenitor cells (CFU-GM, CFU-E) may or may not reflect early events in hematopoiesis. Murine bone marrow failure states (W/Wv, CBA-N, etc) and bone marrow transplantation studies can provide some insight into the physiology of human hematopoiesis, especially if parallel models with human stem cells can be developed. Recently, primitive hematopoietic stem cells (CFU-Blast) have been cultured from both murine and human samples; these cells may represent a cell close to the totipotential stem cell in their capacity for proliferation and differentiation. Human and murine (from animals pre-treated with 5-fluorouracil [5-FU]) hematopoietic cells will be harvested and cultured in semi-solid media. Ten to sixteen days later the colonies of primitive, agranular, medium-sized, refractile, undifferentiated """"""""blast"""""""" cells will be plucked from the methylcellulose, and their functional capacity will be determined. Using defective-retrovirus-mediated gene transfer as a marker of clonality, the self-renewal capacity and proliferative potential of this early stem cell will be ascertained. For in vivo studies the harvested primitive cells will be evaluated for their ability to reconstitute lethally-irradiated syngeneic or allogeneic mice, and defective mutant mice. The stage of differentiation and commitment of the CFU-Blast will be compared to that of the CFU-Spleen. Blasts will be harvested from in vitro cultures and injected into lethally irradiated mice and the number and lineage commitment of any detectable spleen colonies will be determined. Similarly, spleen colonies derived from 5-FU treated donor bone marrow cells will be harvested and plated in semi-solid media to determine whether CFU-Blast-derived colonies can be obtained. Although similar human in vivo studies cannot be pursued, parallel in vitro studies of function will be performed. Short-term cultures in semi-solid media under lymphoid and myeloid conditions will asses the totipotential properties of this cell. Human and murine Blast colony cells will also be used to initiate and seed existing long-term continuous marrow cultures, and their recovery from these cultures will be determined. The goals of these studies are to determine the true proliferative and self-renewal capacity of the CFU-Blast, to explore its role in hematopoiesis, and to develop parallel models for future studies of normal and disordered hematopoiesis.
