MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry.
MSC surface markers (CD44, CD73, and CD90) can identify human MSC-derived extracellular vesicles by conventional flow cytometry.
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DOI：
10.1186/s12964-015-0124-8
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发表时间：
2016-01-12
期刊：
影响因子：
--
通讯作者：
del Cañizo C
中科院分区：
文献类型：
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作者：
L Ramos T;Sánchez-Abarca LI;Muntión S;Preciado S;Puig N;López-Ruano G;Hernández-Hernández Á;Redondo A;Ortega R;Rodríguez C;Sánchez-Guijo F;del Cañizo C
人间充质基质细胞（hMSC）是具有再生和免疫调节活性的多能细胞，使其成为细胞治疗的有吸引力的工具。在过去的几年中，已经表明hMSC的有益作用可能是由于旁分泌作用，并且至少部分地由细胞外囊泡（EV）介导。EV已经成为细胞间通讯的重要介质。流式细胞术（FCM）是大多数临床实验室使用的常规技术，可用作hMSC-EV表征的方法。虽然有几份报告已经通过FCM表征了EV，但缺乏针对hMSC衍生EV的特定面板和方案。我们研究的主要目的是使用标准流式细胞仪表征hMSC-EV。使用来自健康供体骨髓的人MSC、间充质细胞系（HS-5和hTERT）和白血病细胞系（K562细胞）获得用于FCM表征的EV。从不同细胞系释放的EV通过超离心分离，并在常规流式细胞仪中使用多参数分析进行表征。还通过透射电子显微镜（TEM）、蛋白质印迹（WB）和纳米颗粒跟踪分析（NTA）进行EV表征。EV膜由特定细胞表面分子（取决于其细胞来源）与特定蛋白质（如四跨膜蛋白（如CD 63））的组合构成。我们用流式细胞术对BM-hMSC释放的EV进行了表征，定义为小于0.9 μm的颗粒，hMSC标志物（CD 90，CD 44和CD 73）阳性，而CD 34和CD 45（造血标志物）阴性。此外，hMSC衍生的EV也对EV的两个特征性标志物CD 63和CD 81呈阳性。为了验证我们的表征策略，还研究了来自间充质细胞系（hTERT/HS-5）的EV，使用白血病细胞系（K562）作为阴性对照。从间充质细胞系释放的EV显示与来自原代BM-hMSC的EV相同的免疫表型特征，而来自K562细胞的EV不显示hMSC标记。我们使用来自用GFP转导的hMSC的EV进一步验证了该组。最后，还通过WB、TEM和NTA表征了源自不同来源（hMSC、hTERT/HS-5和K562）的EV，证明了WB表达外泌体标志物CD 63和CD 81，以及来自MSC来源的那些中的CD 73。EV形态和尺寸/浓度分别通过TEM和NTA确认。我们描述了一种策略，允许通过流式细胞术对hMSC衍生的EV进行鉴定和表征，该方法可在大多数具有标准流式细胞术设施的实验室中常规使用。本文的在线版本（doi：10.1186/s12964-015-0124-8）包含补充材料，可供授权用户使用。
Human mesenchymal stromal cells (hMSC) are multipotent cells with both regenerative and immunomodulatory activities making them an attractive tool for cellular therapy. In the last few years it has been shown that the beneficial effects of hMSC may be due to paracrine effects and, at least in part, mediated by extracellular vesicles (EV). EV have emerged as important mediators of cell-to-cell communication. Flow cytometry (FCM) is a routine technology used in most clinical laboratories and could be used as a methodology for hMSC-EV characterization. Although several reports have characterized EV by FCM, a specific panel and protocol for hMSC-derived EV is lacking. The main objective of our study was the characterization of hMSC-EV using a standard flow cytometer. Human MSC from bone marrow of healthy donors, mesenchymal cell lines (HS-5 and hTERT) and a leukemic cell line (K562 cells) were used to obtain EV for FCM characterization. EV released from the different cell lines were isolated by ultracentrifugation and were characterized, using a multi-parametric analysis, in a conventional flow cytometer. EV characterization by transmission electron microscopy (TEM), western blot (WB) and Nano-particle tracking analysis (NTA) was also performed. EV membranes are constituted by the combination of specific cell surface molecules depending on their cell of origin, together with specific proteins like tetraspanins (e.g. CD63). We have characterized by FCM the EV released from BM-hMSC, that were defined as particles less than 0.9 μm, positive for the hMSC markers (CD90, CD44 and CD73) and negative for CD34 and CD45 (hematopoietic markers). In addition, hMSC-derived EV were also positive for CD63 and CD81, the two characteristic markers of EV. To validate our characterization strategy, EV from mesenchymal cell lines (hTERT/HS-5) were also studied, using the leukemia cell line (K562) as a negative control. EV released from mesenchymal cell lines displayed the same immunophenotypic profile as the EV from primary BM-hMSC, while the EV derived from K562 cells did not show hMSC markers. We further validated the panel using EV from hMSC transduced with GFP. Finally, EV derived from the different sources (hMSC, hTERT/HS-5 and K562) were also characterized by WB, TEM and NTA, demonstrating the expression by WB of the exosomal markers CD63 and CD81, as well as CD73 in those from MSC origin. EV morphology and size/concentration was confirmed by TEM and NTA, respectively. We described a strategy that allows the identification and characterization by flow cytometry of hMSC-derived EV that can be routinely used in most laboratories with a standard flow cytometry facility. The online version of this article (doi:10.1186/s12964-015-0124-8) contains supplementary material, which is available to authorized users.