Methodological Development of a Clonogenic Assay to Determine Endothelial Progenitor Cell Potential
Methodological Development of a Clonogenic Assay to Determine Endothelial Progenitor Cell Potential
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DOI：
10.1161/circresaha.110.231837
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发表时间：
2011-06-24
影响因子：
20.1
通讯作者：
Asahara, Takayuki
中科院分区：
文献类型：
--
作者：
Masuda, Haruchika;Alev, Cantas;Asahara, Takayuki
由于缺乏能够评估 EPC 分化级联的检测系统，对循环内皮祖细胞 (EPC) 动力学的精确和概念性了解受到阻碍。开发了 EPC 集落形成的测定系统来描绘循环 EPC 分化。使用半固体培养基和来自脐带血的单个或大量CD133(+)细胞进行的EPC集落形成测定显示，由具有内皮谱系潜力和特性的小或大细胞组成的两种类型的贴壁细胞集落的形成，分别称为小EPC集落形成单位和大EPC集落形成单位。每个 EPC 集落形成单位细胞的体外和体内测定揭示了从小 EPC 集落到大 EPC 集落的分化层次，分别表明具有高度增殖活性的原始 EPC 阶段和具有血管生成特性的最终 EPC 阶段。与传统 EPC 培养测定系统的实验比较揭示了 EPC 集落形成单位细胞分化为非集落形成早期 EPC。通过将该测定系统与造血祖细胞测定相结合，实现了单个 CD133(+) 细胞向内皮和造血谱系的命运分析，并证明了从单个造血干细胞发展为集落形成 EPC 和造血祖细胞。 EPC 集落形成测定可以根据分级 EPC 集落形成的评估，确定单个或大量细胞的循环 EPC 动力学。该测定进一步能够正确探索 EPC 起源和造血干细胞之间可能的联系，代表了研究 EPC 生物学中涉及的分子信号传导途径的一种新颖而强大的工具。 （Circ Res. 2011；109:20-37。）
The precise and conceptual insight of circulating endothelial progenitor cell (EPC) kinetics is hampered by the absence of an assay system capable of evaluating the EPC differentiation cascade. An assay system for EPC colony formation was developed to delineate circulating EPC differentiation. EPC colony-forming assay using semisolid medium and single or bulk CD133(+) cells from umbilical cord blood exhibited the formation of two types of attaching cell colonies made of small or large cells featuring endothelial lineage potential and properties, termed small EPC colony-forming units and large EPC colony-forming units, respectively. In vitro and in vivo assays of each EPC colony-forming unit cell revealed a differentiation hierarchy from small EPC to large EPC colonies, indicating a primitive EPC stage with highly proliferative activity and a definitive EPC stage with vasculogenic properties, respectively. Experimental comparison with a conventional EPC culture assay system disclosed EPC colony-forming unit cells differentiate into noncolony-forming early EPC. The fate analysis of single CD133(+) cells into the endothelial and hematopoietic lineage was achieved by combining this assay system with a hematopoietic progenitor assay and demonstrated the development of colony-forming EPC and hematopoietic progenitor cells from a single hematopoietic stem cell. EPC colony-forming assay permits the determination of circulating EPC kinetics from single or bulk cells, based on the evaluation of hierarchical EPC colony formation. This assay further enables a proper exploration of possible links between the origin of EPC and hematopoietic stem cells, representing a novel and powerful tool to investigate the molecular signaling pathways involved in EPC biology. (Circ Res. 2011;109:20-37.)