Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient-derived hematopoietic stem cells
Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient-derived hematopoietic stem cells
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DOI：
10.1039/b902083g
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发表时间：
2009-01-01
期刊：
影响因子：
6.1
通讯作者：
Cooper, Jonathan M.
中科院分区：
文献类型：
--
作者：
Faley, Shannon L.;Copland, Mhairi;Cooper, Jonathan M.
干细胞具有自我更新和分化的能力，因此有望成为治疗其他无法治愈的退行性疾病的一种手段。然而，干细胞损伤也可以在疾病中发挥作用，形成实体瘤和白血病，如慢性粒细胞白血病(CML)，一种造血干细胞(HSC)疾病。尽管最近医学上取得了进展，但慢性粒细胞白血病仍然无法通过药物治疗治愈。因此，要了解单个干细胞白血病的化疗耐药机制，可能需要在单细胞水平上进行分析。考虑到分离HSCs困难、昂贵，而且由于患者的细胞产量低，这项任务在目前的技术下并不是微不足道的。此外，造血细胞在很大程度上是非贴壁的，因此很难用传统的细胞培养技术随着时间的推移进行研究。因此，需要新的微流控平台，其允许并行地对数百个未附着的单个细胞进行功能询问。我们展示了在微流控平台中使用最少的试剂和少量的原代细胞进行分析的能力，通常是在宏观范围内进行的。我们研究了正常和慢性粒细胞白血病干细胞对酪氨酸激酶抑制剂达沙替尼的反应，达沙替尼是一种被批准用于治疗慢性粒细胞白血病的药物。动态芯片三色细胞活性分析显示，正常细胞和慢性粒细胞白血病干/祖细胞对达沙替尼的反应甚至在暴露的早期阶段也有差异，在此期间，正常细胞的细胞死亡率显著高于对照组和慢性粒细胞白血病细胞。然而，进一步的研究表明，达沙替尼确实显著减少了慢性粒细胞白血病干/祖细胞的原位迁移。
Stem cells hold great promise as a means of treating otherwise incurable, degenerative diseases due to their ability both to self-renew and differentiate. However, stem cell damage can also play a role in the disease with the formation of solid tumors and leukaemias such as chronic myeloid leukaemia (CML), a hematopoietic stem cell (HSC) disorder. Despite recent medical advances, CML remains incurable by drug therapy. Understanding the mechanisms which govern chemoresistance of individual stem cell leukaemias may therefore require analysis at the single cell level. This task is not trivial using current technologies given that isolating HSCs is difficult, expensive, and inefficient due to low cell yield from patients. In addition, hematopoietic cells are largely non-adherent and thus difficult to study over time using conventional cell culture techniques. Hence, there is a need for new microfluidic platforms that allow the functional interrogation of hundreds of non-adherent single cells in parallel. We demonstrate the ability to perform assays, normally performed on the macroscopic scale, within the microfluidic platform using minimal reagents and low numbers of primary cells. We investigated normal and CML stem cell responses to the tyrosine kinase inhibitor, dasatinib, a drug approved for the treatment of CML. Dynamic, on-chip three-color cell viability assays revealed that differences in the responses of normal and CML stem/progenitor cells to dasatinib were observed even in the early phases of exposure, during which time normal cells exhibit a significantly elevated cell death rate, as compared to both controls and CML cells. Further studies show that dasatinib does, however, markedly reduce CML stem/progenitor cell migration in situ.