人骨髓间充质干细胞（干细胞）的免疫调节性能评估
Summary
人类骨髓间充质干细胞（MSC）的免疫调节属性显示越来越重要的临床应用。用MSC和外周血白细胞的共同培养系统的预染色的荧光染料羧基琥珀酰亚胺酯（CFSE），我们描述对效应白细胞增殖和特定亚群的体外评估的MSC免疫调节的。
Abstract
The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.
Introduction
人间充质干细胞（MSCs）是体祖细胞可以分化成骨，软骨和脂肪组织1-4的近轴中胚层谱系，以及几extramesodermal谱系5。首先从成人骨髓中分离，这些多谱系祖细胞现已发现在多种组织和6-8，出乎意料的是，显示出具有出现高度适合于临床应用9-12强免疫调节性能。参与免疫调节作用的详细机制正在积极研究对特定病种有效的应用。其中一个最简单的方式来评估免疫调节是通过评估的效应白细胞增殖13的抑制。大多数效应白细胞，如T淋巴细胞和单核细胞增生不歇刺激或激活时。免疫调节功能，可以评估时抑制增殖证明。
传统上，效应白细胞增殖进行了评价通过检测的 [3 H]胸苷掺入DNA中。然而，这种方法具有缺点显著由于辐射和后期使用处置的关注，以及所需要的复杂的设备。虽然有非放射性测定法来评估细胞增殖，所述羧基琥珀酰亚胺酯（CFSE）测定具有其它优点，如允许识别特定细胞群，这是在涉及多种细胞类型的共培养实验中特别有用的。 CFSE是荧光染料的细胞可以通过流式细胞术分析来评估。作为细胞分裂时，这种细胞标记的强度成比例地降低;这不仅使得能够确定的总的细胞增殖，但也允许对细胞分裂的高达8分割的数目评估前的荧光变得难以DET等针对背景信号。此外，该荧光CFSE的稳定性允许在体内跟踪，使得细胞可以可视化长达很多个月14标记的细胞。
该测定也可以改变，以评估特定类型的效应子白细胞或特定人群的免疫调节功能的MSC诱导的免疫调节白细胞，如白介素-10（IL-10）产生的CD14 +单核细胞 15通过执行的磁珠表面标记选择之前或共培养适当的利息后的细胞群。我们的协议描述评估对效应白细胞MSCs的免疫调节作用（ 图 1中所示的流程图）的基本测定，并在此基础测定法对异源CD4 +效应 T淋巴细胞的MSC诱导的白细胞免疫调节评价的变型（图中所示的流程图在图4中）。
Protocol
Representative Results
Discussion
Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application.
One of the most critical technical aspects for success in these assays is having adequate PBMC…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).
Materials
| Ficoll-Paque PLUS | GE Healthcare | 71-7167-00 AG | Density grandient for isolation of peripheral blood mononuclear cells (PBMCs) |
| Vibrant CFDA-SE Cell Tracer Kit (CFSE) | Life Technologies | V12883 | Cellular label for detection of cell division |
| Phytoagglutinin (PHA) | Sigma | L8902 | Activation of human PBMCs |
| Dynabeads Human T-Activator CD3/28 | Life Technologies | 111.32D | Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc. |
| autoMACS™ Separator | Miltenyi Biotec | autoMACS™ Separator | Magnetic based cell separator |
| autoMACS® Columns | Miltenyi Biotec | 130-021-101 | separation columns |
| CD14 microbeads, human | Miltenyi Biotec | 130-050-201 | For positive selection of CD14+ human monocytes and macrophages from PBMCs |
| CD4 microbeads, human | Miltenyi Biotec | 130-045-101 | For positive selection of CD4+ human T lymphocytes from PBMCs |
| RPMI 1640 Medium | Life Technologies | 11875 | Human PBMC/leukocyte culture medium |
| DMEM, Low glucose, pyruvate | Life Technologies | 11885 | Human mesenchymal stem cell (MSC) culture medium |
| L-glutamine | Life Technologies | 25030-081 | Supplementation for MSC complete medium |
| Penicillin/Streptomycin | Life Technologies | 15070-063 | Supplementation for PBMC/leukocyte and MSC complete medium |
| Fetal bovine serum (FBS) | 1) Hyclone, for MSC culture 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations) | 1) SH30070.03M 2) 10091-148 | Pre-test lots for support of MSC in vitro culture |
| 24-well cell culture plate | Corning | COR3524 | Co-culture plate |
| 50 mL centrifuge tube | Corning | 430291 | Isolation PBMCs from whole blood by Ficoll-Paque PLUS |
| 15 mL centrifuge tube | Corning | 430766 | Collection of the labeled and unlabeled cell fractions |
| Round-bottom tubes | BD Falcon | 352008 | Collection of cells for flow cytometric analysis |
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