Early hematopoietic differentiation is regulated by complex interactions between hematopoietic cells, stromal cells and cytokines. The studies outlined in this proposal are designed to reduce the complexity of the system to render it amenable to critical analysis of the regulation of differentiation. We will isolate murine precursors for hematopoietic and stromal cells from bone marrow by fluorescence activated cell sorting using lineage specific monoclonal antibodies. Previously, we have isolated a bone marrow population highly enriched for pluripotent hematopoietic stem cells and multipotent progenitors, as well as a population enriched for several restricted myeloid and B cell precursors. The developmental regulation of these populations by defined cytokines and stromal cells was analyzed and revealed novel differentiation stages in the myeloid and B cell lineages. These findings lead us to propose three questions: Can markers be found that will allow further separation of hematopoietic precursors and progenitors? Can we identify the precursors for stromal cells that support hematopoietic cells? What are the requirements for growth and differentiation for these hematopoietic and stromal cell precursors? We will approach these questions by separating bone marrow according to the staining pattern obtained with a panel of antibodies. Several of these antibodies have been previously described, others have been recently derived by us. B cell precursors in the isolated bone marrow populations will be identified by their response to the stromal elements in Whitlock- Witte cultures. Myeloid precursors will be assessed by their ability to form colonies upon stimulation with lL-3, GM-CSF, M-CSF, G-CSF, and lL-5. All precursor populations will also be tested for their differentiation capacity in vivo in lethally irradiated mice. We will then analyze the interaction of isolated hematopoietic precursors with stromal cultures obtained from isolated stromal cell precursors. A limiting dilution system that we have developed allows the proliferation and differentiation of stromal cells precursors in vitro. Thus, the proposed studies will define more precisely early myeloid and B cell differentiation stages and will establish the origin of stromal cells. We also expect that these studies will lead to a better understanding of the interactions of these cells and their regulation by cytokines.
