The Principal Investigator seeks to study the properties of hematopoietic stem cells using a newly-described technology in which stem cells are selected based on their quiescence in the presence of cytokines. As such, prolonged exposure of marrow cells to high concentrations of a cell cycle-specific cytotoxic agent, 5-fluorouracil results in the selective death of all cycling progenitors. What is left, approximately 1 per 105 starting cells, has a high incidence of LTCIC, the virtual absence of immediate clonogenic progenitor cells, and a population which is nearly entirely CD34 and c-Kit positive. With these techniques in hand, the Investigator proposes four Specific Aims to study both basic aspects and clinical applications of stem cell biology. In the first Specific Aim, the Investigator plans to optimize the functional isolation of stem cells by testing other combinations of cytokines and comparing results to those previously obtained with IL-3 and KL. In addition, other tissue sources of hematopoietic stem cells including peripheral blood progenitors and umbilical cord blood will be studied. The goal is to identify methods to tailor the output stem cell population. For example, cells more likely to generate lymphoid or myeloid populations might be obtained. In the second Specific Aim, the Investigators plan to determine whether the stem cell isolation strategy may be adapted to deplete malignant cells or HIV-infected cells from normal populations of enriched stem cells. The Investigators plan to spike HIV or malignant cells into a normal starting population, perform selection using their functional isolation techniques, and then assay for residual HIV or malignant cells by RT-PCR techniques. The enriched cells will be assayed for LTCIC to confirm the absence of contaminating tumor or virally infected cells. It is hoped that this technique will allow important clinical applications in AIDS and malignancy. In the third Specific Aim, the Investigators plan to characterize the pattern of expression of specific transcription factors, cytokines, cytokine receptors and viral receptors in their selected cell populations and their progeny. Finally, in the fourth Specific Aim, the Investigators plan to clone novel genes from their selected stem cell populations. The goal is to generate a technique in which stem cell CDNA will be enriched for sequences not present in more differentiated progeny using a subtraction method based on the incorporation of biotinylated nucleotidase into PCR-generated subtractor CDNA from CD34 positive, CD33 positive cells. Once obtained, such clones will be sequenced to determine the class into which they fall (transcription factor, cytokine, cytokine receptor, cell cycle active protein, kinases, phosphotases are all of interest to the investigator) and novel genes will be further characterized. New methods of subtraction will be developed in the laboratory in order to accomplish this aim.
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