Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol.
Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol.
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DOI：
10.1371/journal.pone.0184895
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Sacchetti A
中科院分区：
文献类型：
--
作者：
Huizer K;Mustafa DAM;Spelt JC;Kros JM;Sacchetti A
循环内皮祖细胞（EPCs）的特性是血管生成相关研究的基础。不幸的是，目前的文献缺乏一致性的EPC子集的定义，由于分离策略的变化和不一致的使用谱系标记。在这里，我们解决的关键点，在确定造血祖细胞（HPC），循环内皮细胞（CEC），培养产生的生长内皮细胞（OECs）从健康成人（AB）和脐带血样本（UCB）。使用基于Ficoll的梯度富集外周血单核细胞（PBMC），然后使用优化的染色和门控策略富集靶细胞。对分选的EPC群体进行RT-PCR，以追踪超出基于细胞表面的免疫表型分析限度的标记物的表达。使用CD 34、CD 133和c-kit染色，结合FSC和SSC，我们成功地准确和可重复地鉴定了4个HPC亚组，并发现AB与UCB在各自人群中存在显著差异。对HPC上内皮标志物的共表达分析揭示了以各种亚群为特征的复杂模式。通过使用CD 34、KDR、CD 45和其他内皮标记物鉴定CEC，并根据其凋亡状态和c-kit表达进行细分。UCB-CEC与AB-CEC的比较显示CD 34和KDR水平存在显著差异。OEC从PBMC-级分生长。我们发现活的c-kit+ CEC是CEC的候选循环前体。血管生成因子和受体的RT-PCR显示，所有EPC亚群均表达血管生成相关分子。总之，免疫分型和门控策略的改进导致在单个程序中准确识别和比较更好定义的细胞群。
The characterization of circulating endothelial progenitor cells (EPCs) is fundamental to any study related to angiogenesis. Unfortunately, current literature lacks consistency in the definition of EPC subsets due to variations in isolation strategies and inconsistencies in the use of lineage markers. Here we address critical points in the identification of hematopoietic progenitor cells (HPCs), circulating endothelial cells (CECs), and culture-generated outgrowth endothelial cells (OECs) from blood samples of healthy adults (AB) and umbilical cord (UCB). Peripheral blood mononuclear cells (PBMCs) were enriched using a Ficoll-based gradient followed by an optimized staining and gating strategy to enrich for the target cells. Sorted EPC populations were subjected to RT-PCR for tracing the expression of markers beyond the limits of cell surface-based immunophenotyping. Using CD34, CD133 and c-kit staining, combined with FSC and SSC, we succeeded in the accurate and reproducible identification of four HPC subgroups and found significant differences in the respective populations in AB vs. UCB. Co-expression analysis of endothelial markers on HPCs revealed a complex pattern characterized by various subpopulations. CECs were identified by using CD34, KDR, CD45, and additional endothelial markers, and were subdivided according to their apoptotic state and expression of c-kit. Comparison of UCB-CECs vs. AB-CECs revealed significant differences in CD34 and KDR levels. OECs were grown from PBMC-fractions We found that viable c-kit+ CECs are a candidate circulating precursor for CECs. RT-PCR to angiogenic factors and receptors revealed that all EPC subsets expressed angiogenesis-related molecules. Taken together, the improvements in immunophenotyping and gating strategies resulted in accurate identification and comparison of better defined cell populations in a single procedure.
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影响因子：
2.2
作者：
Hristov, Mihail;Schmitz, Susanne;Weber, Christian
通讯作者：
Weber, Christian
影响因子：
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作者：
Ackermann, Bradley L.;Berna, Michael J.;Murphy, Anthony T.
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作者：
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Partanen, Jukka
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作者：
Kraan, J.;Strijbos, M. H.;Gratama, J. W.
通讯作者：
Gratama, J. W.
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作者：
Alvarez P;Carrillo E;Vélez C;Hita-Contreras F;Martínez-Amat A;Rodríguez-Serrano F;Boulaiz H;Ortiz R;Melguizo C;Prados J;Aránega A
通讯作者：
Aránega A