Hemopoietic stem cells can be identified in a variety of tissues. However, the bone marrow is the only site of active blood cell production under normal conditions, indicating that local tissue influences are of importance to hemopoiesis. The nature of these local controls is not understood, but a population of supporting elements known as stromal cells apparently provides a hemopoietic microenvironment for developing blood cells. The specific objectives of this work are to identify stromal cell subpopulations, to determine how stromal cells regulate hemopoiesis, and to examine stromal cell engraftment following bone marrow transplantation. The experimental approach will extensively utilize the Dexter and Whitlock-Witte long-term bone marrow cultures that support myelopoiesis or lymphopoiesis, respectively. The key feature of both these systems is the establishment of an adherent stromal cell layer that duplicates aspects of the in vivo hemopoietic microenvironment. Stromal cell lines and clones have been grown from these cultures and their interactions with hemopoietic cells will be tested by seeding them with stromal cell depleted populations of hemopoietic cells. Potential subpopulations of stromal cells will be identified by determining if the stromal cells in Dexter and Whitlock-Witte constitute restricted microenvironments for myelopoieses and lymphopoiesis respectively, by phenotyping stromal cells, and by testing the hemopoietic support capacity of stromal cell clones. Stromal cell regulation of hemopoiesis will be analyzed by asking whether stromal cells can mediate their effects via diffusable factors or if direct contact with developing blood cells is necessary. A particular focus of this work will examine if stromal cells exhibit a plasticity in response to changes in their external milieu that could contribute to the formation of distinct microenvironments. The final series of experiments will investigate stromal cell engraftment following bone marrow transplantation. A kinetic analysis of stromal cell homing following intravenous injection will be made; whether or not stromal cells that engraft are functional will be tested by analyzing their ability to cure the hereditary macrocytic anemia in microenvironmentally defective S1/S1d mice.
