Dynamic analyses of interactions between leukemic stem cells and hemopoietic stromal cells visualized by molecular imaging technique, and their possible pharmaceutical applications.

鹅卵石区域（CA）形成通常被认为是在体外长期维持正常多能造血干细胞的一种且唯一的方式，因此被认为是模拟体内正常组成型造血。我们已经应用这种培养系统作为白血病发展的模型，涉及白血病干细胞在各种治疗后的维持和持久性，并通过使用MS 5作为支持性基质，HEL和TF-1作为模型白血病细胞系对其进行优化。虽然这两种白血病细胞系来源于相同的红白血病分类，但它们彼此差异很大，即，基本上每个HEL细胞似乎都保持CA形成的能力，而TF-1群体中只有10%显示CA形成，这表明在TF-1细胞系中存在沿着分化的等级。与原始悬浮培养相比，CA形成细胞中的一些分化抗原如血型糖蛋白A和CD 42 b的表达减少。 关于我们 相反，对于这两种细胞系，最近报道白血病干细胞归巢到骨髓小生境所需的CD 44的表达在CA形成细胞中相对于原始悬浮培养物升高。此外，CA形成后，自我更新的频率增加，这是明显的，从再填充试验的结果，与静息细胞比例的升高，通过流式细胞术证明。这些结果表明CA形成细胞可能保留白血病干细胞的性质。与同源细胞的悬浮培养物相比，这些CA形成细胞的特征还在于对各种化疗剂的敏感性大大降低。特别是，柔红霉素，一种经常处方的抗白血病剂，其荧光特性可用于可视化其细胞内定位，显示出明显的积聚到对应于CA形成细胞的溶酶体的隔室中，这与在相同细胞的药物敏感性培养物中观察到的亚细胞分布完全不同。总之，白血病细胞通过形成CA获得耐药性，并且这种获得背后的机制之一可能涉及药物的细胞内转运和/或代谢的改变，而不仅仅是物理阻止药物渗透到白血病干细胞所在的造血生态位中。目前描述的使用鼠MS 5基质细胞的异源CA形成系统被认为特别适用于在即将到来的个性化医学时代中作为所需药物的评价系统。少

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 … More 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