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Detection, characterization, and genetic modification of acute myeloid leukemia (AML) stem cells

University of British Columbia

The first goal of the current project was to establish reliable assays for the routine detection and quantitation of leukemic stem cells. The development of such assays would, firstly, provide definitive proof for the existence of a primitive cell analagous to a normal pluripotent hematopoietic stem cell within the AML population, and secondly, provide means by which to characterize and manipulate these cells in order to gain an understanding of the mechanisms of leukemogenesis. It was demonstrated that a leukemic longterm culture-initiating cell (LTC-IC) could be routinely detected under appropriate conditions, and also that most AML samples contain a mixture of normal and abnormal LTC-IC. The assay was shown to be quantitative, and calculation of the frequencies of both leukemic and normal LTC-IC in AML peripheral blood samples showed that the frequency of both is considerably higher than the frequency of LTC-IC in normal peripheral blood, suggesting that normal LTC-IC are mobilized as a part of the leukemic process. In addition, differences in the responses of normal and leukemic cells to various cytokines known to be active on normal LTC-IC were revealed. A second and more reliable assay for stem cells is the animal repopulation assay. The ability of a small subset of cells within AML samples to repopulate immunodeficient NOD/SCID mice was demonstrated, again confirming the existence of a rare stem cell in AML that is responsible for the maintenance of the leukemic clone. This cell was quantitated and its high proliferative capacity and self-renewal ability were demonstrated. Like the LTC system, this assay system provides a model in which to assess the manipulation of human AML cells, both for evaluation of therapeutic strategies and for dissecting the mechanisms of leukemogenesis. The second major goal of this work was to perform retroviral-mediated gene transfer into AML stem cells. It was found that colony-forming cells and LTC-IC were easily transduced under a variety of conditions, but that the NOD/SCID repopulation ability was impaired during the in vitro period required for transduction. When a variety of culture conditions, including the presence or absence of several cytokine combinations, were tested, all cases this ability was lost within 24 hours. Strategies for the selection of transduced cells were also investigated. Transfer of the murine heat stable antigen (HSA) gene, a cell surface molecule, enabled the enrichment of transduced cells using an immunomagnetic separation technique. It was also demonstrated that retroviral vectors pseudotyped with a gibbon ape leukemia virus were more efficient than those pseudotyped with the amphotropic envelope from the Moloney murine leukemia virus at transducing AML cells.

Thesis/Dissertation

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2009-06-29

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http://hdl.handle.net/2429/9825

Genetics

University of British Columbia

UBCV

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