ACTIVATION OF PURIFIED HEMOPOIETIC STEM CELLS

描述：在修订后的提案中，调查人员提出了三个要求

DESCRIPTION: In this revised proposal, the Investigators request three additional years of funding to continue their work into characterizing hematopoietic stem cells. In previous studies, the Investigators generated monoclonal antibodies against two novel cell surface proteins which are displayed by hematopoietic stem cells. These two markers, CD34 and Thy-1, along with other cell surface markers were used to sort human bone marrow, fetal liver and cord blood into pools of stem cell candidates. The Investigators have also described an apparent shortening of telomeric DNA in adult stem cell populations or fetal stem cells which have been pass aged in vitro. On the basis of these previous findings, the Investigators plan to further characterize hematopoietic stem cells with a long-term goals of obtaining pure populations and developing the culture techniques that will sustain proliferation and self-renewal of highly purified stem cells. They propose three Specific Aims. First, using immunomagnetic techniques and multiparameter flow cytometry, the Investigators plan to generate new monoclonal antibodies and to screen a large panel of antibodies available through the 5th Workshop on Leukocyte Surface Antigens. Hopefully, new reagents will be obtained which may be additionally useful to sort candidate stem cell populations into more highly purified cells. The second Specific Aim is to define culture conditions for the survival activation, proliferation and self-renewal of candidate stem cells from hematopoietic tissues of various stages of ontogeny. Both stimulatory and inhibitory cytokines will be tested for their effects on these parameters, the Investigators plan to follow both in vitro (long-term culture initiating cell) and in vivo (SCID-hu) assays. Finally, in the third Specific Aim, the Investigators plan to further delineate the relationship between proliferative potential of hematopoietic cells and the length of their telomeric DNA. Using a telomere specific probe, the Investigators will use Southern blotting to calculate the mean length of telomeric segments. This will be correlated with proliferative capacity of cells taken from various hematopoietic sources.Hopefully, with the availability of an enzyme called telomerase, the investigators plan to manipulate telomere length in culture, and thereby test the hypothesis that fixed genetic changes dictate stem cell potential.