Successful In Vitro Expansion and Differentiation of Cord Blood Derived CD34+Cells into Early Endothelial Progenitor Cells Reveals Highly Differential Gene Expression
Successful In Vitro Expansion and Differentiation of Cord Blood Derived CD34+Cells into Early Endothelial Progenitor Cells Reveals Highly Differential Gene Expression
复制标题
DOI：
10.1371/journal.pone.0023210
复制
发表时间：
2011-08-12
期刊：
影响因子：
3.7
通讯作者：
Peter, Karlheinz
中科院分区：
文献类型：
--
作者：
Ahrens, Ingo;Domeij, Helena;Peter, Karlheinz
内皮祖细胞(EPC)可以从外周血、骨髓或脐带血中纯化，通常由有限数量的细胞表面标志物和一些功能测试确定。循环内皮祖细胞的低细胞数往往限制了其详细的体外鉴定。因此，应用体外培养和扩增的方法，至少可以区分两种不同类型的内皮祖细胞，早期和晚期的内皮祖细胞。在此，我们描述了一种体外培养技术，目的是从人脐血中产生大量表型、功能和基因定义的早期内皮祖细胞。用流式细胞仪、免疫荧光显微镜、集落形成单位(CFU)实验和内皮细胞形成实验对内皮祖细胞进行鉴定。EPC数量平均增加了48倍。内皮祖细胞表达VEGFR-2、CD144、CD18和CD61，乙酰化低密度脂蛋白摄取和Ulex凝集素结合阳性。只有在与成熟的内皮细胞共培养时，这些细胞才能刺激内皮管的形成，并形成CFU。微阵列分析揭示了高度上调的基因，包括LL-37(CAMP)、PDK4和α-2-巨球蛋白。此外，已知的与心脏保护(GDF15)或促血管生成(Galectin-3)特性相关的基因在纤维连接蛋白72小时的分化期后也显著上调。我们提出了一种新的方法，允许产生大量的表型、功能和遗传特征的早期内皮祖细胞。此外，我们还发现了几个与EPC分化相关的新基因，其中最上调的基因是LL-37(CAMP)。
Endothelial progenitor cells (EPCs) can be purified from peripheral blood, bone marrow or cord blood and are typically defined by a limited number of cell surface markers and a few functional tests. A detailed in vitro characterization is often restricted by the low cell numbers of circulating EPCs. Therefore in vitro culturing and expansion methods are applied, which allow at least distinguishing two different types of EPCs, early and late EPCs. Herein, we describe an in vitro culture technique with the aim to generate high numbers of phenotypically, functionally and genetically defined early EPCs from human cord blood. Characterization of EPCs was done by flow cytometry, immunofluorescence microscopy, colony forming unit (CFU) assay and endothelial tube formation assay. There was an average 48-fold increase in EPC numbers. EPCs expressed VEGFR-2, CD144, CD18, and CD61, and were positive for acetylated LDL uptake and ulex lectin binding. The cells stimulated endothelial tube formation only in co-cultures with mature endothelial cells and formed CFUs. Microarray analysis revealed highly up-regulated genes, including LL-37 (CAMP), PDK4, and alpha-2-macroglobulin. In addition, genes known to be associated with cardioprotective (GDF15) or pro-angiogenic (galectin-3) properties were also significantly up-regulated after a 72 h differentiation period on fibronectin. We present a novel method that allows to generate high numbers of phenotypically, functionally and genetically characterized early EPCs. Furthermore, we identified several genes newly linked to EPC differentiation, among them LL-37 (CAMP) was the most up-regulated gene.