CORE--CONTROL OF STEM CELL DIFFERENTIATION IN THE THYMUS

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Description (taken directly from the application): Understanding the mechanism of hematopoietic stem cell function requires in vitro analysis of lineage commitment. For the T lineage, this is only available by ex vivo thymic organ culture. Our goal is to provide our documented expertise in this technology to Program Investigators. In addition, we plan to optimize and extend this technology to make it more useful for future studies of T lineage commitment. I. We will perform thymic organ culture assays to evaluate the T lymphoid potentials of hematopoietic stem cells. Our laboratory has optimized standard thymic organ culturing techniques, and is currently developing new approaches to increase the experimental utility of thymic organ culture. For example, we have developed a technique for retroviral marking that will allow the clonal analysis of progenitors. We will perform fetal thymic organ reconstitutions, and in addition, limiting dilution assays and thymic reaggregation studies. II. We will provide reagents and expertise for the evaluation of T lymphoid development and function in vivo model systems. T lineage development is characterized by changes in expression of a wide variety of morphological markers. We will use antibody staining and multicolor FACS analysis to assess the differentiative status of T cells and their progenitors. In addition, T cell function will be evaluated by in vivo and in vitro stimulation followed by tests of cytokine production, T cell proliferation, and cytotoxicity. III. Current and future projects aim to evaluate the function of specific genes in hematopoietic lineage commitment. We will provide vectors, cell lines, cell lines, and technical support for retrovirus-mediated gene transfer in hematopoietic stem cells and lymphoid progenitors, and stroma, for expression of exogenous genes in thymus.