Efficient expansion of hematopoietic stem cells would reduce the complexity of donation, the volume necessary for storage and permit sequential use of single harvests. The genetic alteration of stem cells offers the potential for rendering cells resistant to HIV-1 infection or cytotoxic chemotherapy, permitting correction of genetic defects or directing a targeted immune response. However, current approaches for stem cell expansion have had limited success and the ability to transduce new genes into stem cells is limited in direct proportion to the ability to expand the stem cell population because of the relative dependence of current transduction methods on active cell cycling. Therefore, this project is intended to build upon prior developments from the mentor's laboratory in the isolation, molecular characterization and manipulation of stem cells to focus on: 1) mechanisms by which cell cycle regulation of stem cells is achieved, 2) determine if specific regulatory molecules can be manipulated to alter cell cycle kinetics of stem cells and 3) evaluate whether such manipulation necessarily affects the differentiation state of stem cells. Particularly, we will focus on the roles of cyclin-dependent-kinase inhibitors (CKIs), p2l or p27 in stem cell regulation by evaluating hematopoietic stem cell function using mice genetically engineered to be deficient in p2l or p27. We plan to conduct in vitro assays (long term culture, cell cycle analysis) and in vivo experiments (5-FU exposure and sequential bone marrow transplantation) to assess the impact of p2l or p27 on stem cell regulation. In addition, we will document the functional relationship between CKIs and the known inhibitors of hematopoietic progenitor cells, TGF-beta1 and MIP-1alpha. Furthermore, we will try to achieve human stem cell expansion by altering the negative regulators (CKIs) using an antisense strategy without involving prodifferentiative growth factors. Finally, we will evaluate the intrinsic influence of CKIs on stem cell differentiation by assessing the expression levels of lineage specific transcription factors and cytokine receptors in conjunction with in vitro assays. The central hypothesis to be tested is that stem cell quiescence is actively enforced by mechanisms which can be molecularly dissociated from signals inducing differentiation and can be manipulated to result in controlled self-renewal of hematopoietic stem cells.
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