| Budget Amount *help |
¥49,010,000 (Direct Cost: ¥37,700,000、Indirect Cost: ¥11,310,000)
Fiscal Year 2006: ¥17,680,000 (Direct Cost: ¥13,600,000、Indirect Cost: ¥4,080,000)
Fiscal Year 2005: ¥31,330,000 (Direct Cost: ¥24,100,000、Indirect Cost: ¥7,230,000)
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| Research Abstract |
A cobblestone area (CA) formation is generally regarded as one and only way to maintain normal pluripotent hemopoietic stem cells for long term in vitro, and therefore considered to mimic normal constitutive hematopoiesis in vivo. We have applied this culture system as a model of leukemia development involving maintenance and persistence of leukemic stem cells after various therapies, and optimized it by using MS5 as a supportive stroma, and HEL and TF-1 as model leukemic cell lines. Although the both leukemic cell lines derived from the same classification of erythroleukemia, they differ considerably each other, i.e., essentially every HEL cells seemed to maintain ability of CA formation, whereas only 10% of TF-1 population revealed CA formation, suggesting the presence of hierarchy along the differentiation in the latter cell line. Expression of some differentiation antigens such as Glycophorin-A and CD42b was diminished in the cells forming CA compared with original suspension cultu
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re of both cell lines, conversely, expression of CD44 which has recently been reported to be required for the homing of leukemic stem cells to bone marrow niche, was elevated in CA forming cells relative to the original suspension culture. Furthermore, the frequency of self renewal increased upon CA formation, which was evident from the result of repopulating assay, in contrast to the elevation of resting cell proportion demonstrated by flow cytometry. These results indicate that the CA forming cells may retain the properties of leukemic stem cells. Those CA forming cells were also characterized by greatly reduced sensitivity to various chemotherapeutic agents, in comparison to suspension cultures of the cognate cells. Particularly, Daunorubicin, a frequently prescribed anti-leukemic agent whose fluorescent property can be utilized to visualize its intracellular localization, showed distinct accumulation into the compartment corresponding to lysosome of the CA forming cells, and this was entirely different from the subcellular distribution observed in the drug-sensitive culture of the same cells. In summary, leukemic cells acquire drug resistance through the formation of CA, and one of the mechanisms behind this acquisition may involve alterations in intracellular transport and/or metabolism of the drugs, rather than mere physical prevention of the drug penetration into hemopoietic niche where leukemic stem cells reside. The currently described system of heterologous CA formation using murine MS5 stromal cells was considered to be useful particularly as an evaluating system for desirable drugs in the forthcoming era of personalized medicine. Less
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