In this revised application, Dr. Sharkis proposes to study stem cell biology using a technique developed in his laboratory to produce highly enriched populations of candidate stem cells. A research plan of three specific aim is proposed. In the first specific aims, the investigators will extend their studies of purified populations of candidate stem cells by assessing their previous best population, FR25/AA4.1_/lin_/ALDH+ for expression of murine CD34 and binding to wheat germ agglutinin. As only ten of the previously sorted cells are required for reliable reengraftment and stem cell self-renewal, the investigators propose that if these populations of cells are homogeneous with respect to CD34 and WGA, they have achieved a homogeneous pure stem cell population. The Investigators will prove that their cells are indeed stem cells by limiting dilution transplantation into sex mismatched recipient animals, and then following the sorted cells into secondary recipient animals. Only by showing long-term reconstitution and serial transplant ability will the Investigators conclude that their sorted cells are true stem cells. Once idealized, the methodology to produce pure stem cells will be used in subsequent specific aims. Along with purification of stem cells, the investigators will attempt to follow these properties, long-term repopulation and self-renewal, in a long-term marrow culture setting. The ultimate hope is to develop an in vitro system which reliably mirrors in vivo biology (as determined by long-term repopulation and secondary transplant ability) so that in the long-term, the shorter, less expensive in vitro long- term marrow culture system will serve as a suitable surrogate for transplantation studies. In the second specific aim, the Investigators plan to co-culture their purified stem cells with different fractions of thymocytes. Clinical observation has provided information that T cells may play an important role in re-engraftment following marrow transplantation. As such, the basis for this clinical observation will be determined by co-culturing sorted stem cells with different fractions of T cells and determining their effects on long-term repopulation and stem cell self-renewal. In a related series of experiments, neonatal thymectomy will be performed, as preliminary data has shown that engraftment potential is lost if stem cells from neonatal thymectomized mice are transplanted into a similar recipient host. Such stem cells will be incubated with T cell fractions to determine the precise cell type responsible for the defect. Parallel studies will be performed in long-term marrow culture, again to attempt to develop a surrogate assay. An integral hypothesis of the investigators is that because of their quiescent nature, stem cells are resistant to apoptosis. The Investigators will test this directly using a new methodology, combining individual cell assays for cell division, and apoptosis. Purified hematopoietic stem cells will be subjected to manipulations designed to trigger apoptosis in other cell types (4HC, RT, use of stem cells from 5FU-treated mice) and the effects on induction of stem cell apoptosis compared to normal cells will be determined. Finally, in the third specific aim, the use of ES cells for hematopoietic reconstitution will be explored. Preliminary data suggests that day 4 ES cells have long-term repopulating capacity. However, the degree of chimerism in the initial experiments was low. Therefore, initial experiments will be directed to improving sorting efficiency. The precise timing of collection and the method of growth of ES cells (different cytokines will be attempted and growth on a stromal cell line derived from osteopetrotic mice) will be tested. Cells from different culture condition will then be transplanted into mice and tested for long-term repopulating ability and self-renewal by serial transplantation. Once established, the system will be used to test whether genes can be manipulated in such repopulating cells. The model gene will be Rb1. Different vector systems for introducing different amounts of foreign DNA into the ES cells will be determined and again an in vitro system will be developed in parallel to determine whether surrogate assays for transplant ability can be established.
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