We propose to continue studies of the cell physiology of hemopoietic stem cells. Despite the strong evidence from in vivo studies, it has not been possible to detect and quantitate lymphohemopoietic progenitors in culture. In our laboratory we have developed a two-step methylcellulose culture assay for adult murine lymphohemopoietic progenitors that are capable of expressing both myeloid and B-lymphoid lineages in culture and have identified several two-factor combinations that are capable of supporting the early stages of lymphohemopoiesis. We now propose to develop an assay for human lymphohemopoietic progenitors capable of yielding progenies in myeloid and B-cell lineages. We will begin by testing combinations of factors that successfully supported maintenance of B-cell potentials in the murine primary myeloid colonies as well as media conditioned by various types of cells and tissues. We also propose to develop clonal culture assay methods for murine and human lymphohemopoietic progenitors that possess both B- and T-cell capabilities. First, we will develop a two-step methylcellulose culture assay for murine lymphohemopoietic progenitors capable of yielding T-cells. Initially, we will again focus on the combinations of cytokines that successfully supported B-cell potentials of the murine lymphohemopoietic progenitors in primary cultures to be followed by secondary culture in media containing combinations of cytokines which have been reported to be involved with T-cell ontogeny. If we are unable to develop a culture assay based on combinations of recombinant cytokines, we will test the use of fetal thymic organ culture and co-culture with thymic stromal cell lines and stromal cells obtained directly from culture of thymus. We will also test media conditioned by these cells and tissues. The third specific aim is to determine the mechanisms of commitment of lymphohemopoietic progenitors to B-cell and T-cell lineages. Although it has been proposed that stem cells lose lymphoid potential prior to commitment to specific myeloid lineages, these is little experimental evidence for such a hierarchy. By using micromanipulation techniques, we will establish genealogy of development of the progenitors for T- and B-cells and monopotent myeloid progenitors.
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