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(CA)的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也是唯一的方法，因此被认为是模仿体内正常的结构性造血。我们应用该培养体系作为白血病发生的模型，包括白血病干细胞在各种治疗后的维持和持续，并以MS5为支持基质，HEL和TF-1为模型白血病细胞株进行了优化。虽然这两种白血病细胞系起源于同一类型的红白血病，但它们彼此之间有很大的不同，即基本上每个HEL细胞似乎都保持着CA形成的能力，而只有10%的TF-1群体有CA形成，这表明后一种细胞系在分化过程中存在等级关系。与原始悬浮培养的…相比，形成CA的细胞中某些分化抗原如糖蛋白A和CD42b的表达减少相反，在CA形成细胞中，CD44的表达比最初的悬浮培养更高，CD44是白血病干细胞归巢到骨髓巢所必需的。此外，在CA形成后，细胞自我更新的频率增加，这从重新填充实验的结果中可以明显看出，与流式细胞仪显示的静息细胞比例的增加相反。这些结果表明，CA形成细胞可能保留了白血病干细胞的特性。与同源细胞的悬浮培养相比，这些CA形成细胞对各种化疗药物的敏感性也大大降低。特别是，柔红霉素是一种常用的抗白血病药物，其荧光特性可用于显示其在细胞内的定位，它显示出明显的积聚到与CA形成细胞的溶酶体相对应的隔室，这与在相同细胞的药物敏感培养中观察到的亚细胞分布完全不同。综上所述，白血病细胞通过形成CA而获得耐药性，而这种耐药性背后的机制之一可能涉及药物在细胞内的运输和/或代谢的改变，而不仅仅是物理上阻止药物渗透到白血病干细胞所在的造血细胞的缝隙中。目前描述的利用小鼠MS5基质细胞形成异种CA的系统被认为是有用的，特别是在即将到来的个性化药物时代作为理想药物的评估系统。较少

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