In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry
In search for cross-reactivity to immunophenotype equine mesenchymal stromal cells by multicolor flow cytometry
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DOI：
10.1002/cyto.a.22026
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发表时间：
2012-04-01
期刊：
影响因子：
3.7
通讯作者：
Meyer, Evelyne
中科院分区：
文献类型：
--
作者：
De Schauwer, Catharina;Piepers, Sofie;Meyer, Evelyne
近年来，利用间充质干细胞(MSC)进行细胞治疗在马兽医中的应用越来越频繁。在大多数情况下，对马MSC的分离和体外分化进行了描述，但很少进行正确的免疫表型鉴定。缺乏针对MSC的单一标记，特别是针对马MSC的单抗的可获得性有限，这些都严重阻碍了这项研究。在这项研究中，使用适当的阳性对照，用流式细胞术对30株商品化的单抗进行筛选，以识别马的表位，以确认它们的特异性。共聚焦显微镜观察到CD45、CD73、CD79a、CD90、CD105、单核细胞标志物MHC-II以及CD29和CD44的两个克隆发生交叉反应。不幸的是，没有一个被评估的CD34克隆识别阳性对照内皮细胞上的马表位。随后，根据细胞表面抗原和胞浆内蛋白的9个标志物组合，采用多色流式细胞术对6匹马的第4代脐带血来源的未分化马的MSC进行了鉴定。此外，还纳入了相应的阳性对照和阴性对照，并用7-氨基放线菌素D排除死亡细胞，对存活的单细胞群进行了分析。分离的第四代马MSC为CD29、CD44、CD90阳性，CD45、CD79a、MHC-II阳性，单核细胞标志物阴性。发现了CD73和CD105的一个变量表达。成功分化为成骨、成软骨和成脂的谱系被用作额外的验证。我们建议，这个筛选出的9个标记物可以用于马MSC的适当免疫表型。(C)2012年国际细胞测量促进会
During recent years, cell-based therapies using mesenchymal stem cells (MSC) are reported in equine veterinary medicine with increasing frequency. In most cases, the isolation and in vitro differentiation of equine MSC are described, but their proper immunophenotypic characterization is rarely performed. The lack of a single marker specific for MSC and the limited availability of monoclonal antibodies (mAbs) for equine MSC in particular, strongly hamper this research. In this study, 30 commercial mAbs were screened with flow cytometry for recognizing equine epitopes using the appropriate positive controls to confirm their specificity. Cross-reactivity was found and confirmed by confocal microscopy for CD45, CD73, CD79a, CD90, CD105, MHC-II, a monocyte marker, and two clones tested for CD29 and CD44. Unfortunately, none of the evaluated CD34 clones recognized the equine epitopes on positive control endothelial cells. Subsequently, umbilical cord blood-derived undifferentiated equine MSC of the fourth passage of six horses were characterized using multicolor flow cytometry based on the selected nine-marker panel of both cell surface antigens and intracytoplasmatic proteins. In addition, appropriate positive and negative controls were included, and the viable single cell population was analyzed by excluding dead cells using 7-aminoactinomycin D. Isolated equine MSC of the fourth passage were found to be CD29, CD44, CD90 positive and CD45, CD79a, MHC-II, and a monocyte marker negative. A variable expression was found for CD73 and CD105. Successful differentiation towards the osteogenic, chondrogenic, and adipogenic lineage was used as additional validation. We suggest that this selected nine-marker panel can be used for the adequate immunophenotyping of equine MSC. (C) 2012 International Society for Advancement of Cytometry