使用人类脐带血干细胞（CB-SC）治疗后，单核细胞分化成表状独特的巨噬细胞-衍生的外体
Summary
外显体应用是药物开发和再生医学的新兴工具。我们建立了一种高纯度的外体分离方案，将外体与称为CB-SC的新鲜干细胞分离出来，用于机械研究。我们还与人类单核细胞共同培养CB-SC衍生的外体，导致它们分化成表型的分形巨噬细胞。
Abstract
干细胞教育者（SCE）疗法是治疗1型糖尿病和其他自身免疫性疾病的一种新型临床方法。SCE 治疗通过密码机循环分离患者的血液单核细胞（如淋巴细胞和单核细胞），在 SCE 设备中用粘附脐带血源干细胞 （CB-SC） 共同培养患者的血液单核细胞，然后将这些”受过教育的”免疫细胞返回到患者的血液中。外显子是纳米大小的细胞外囊泡，在30\u2012150 nm之间，存在所有生物流体和细胞培养基。为了进一步探索SCE治疗的分子机制，并确定从CB-SC释放的外体的行为，我们调查哪些细胞在用CB-SC治疗期间对这些外体进行噬菌体。通过与人类外周血单核细胞（PBMC）共同培养具有Dio标签的CB-SC衍生外体，我们发现CB-SC衍生的外显体主要由人类CD14阳性单细胞组成，导致单细胞分化为2型巨噬细胞（M2），具有主轴样形和M2相关表面分子标记的表达。在这里，我们提出了一个协议，用于分离和表征CB-SC衍生外体，以及CB-SC衍生外体与人类单核细胞共同培养和M2分化监测的协议。
Introduction
脐带血干细胞（CB-SC）是从人脐带血中鉴定的独特干细胞类型，与其他已知类型的干细胞（如中生血缘干细胞）和造血干细胞（HSC）1等不同。基于其独特的免疫调节特性和紧贴培养皿表面的能力，我们在临床试验2、3中开发出一种被指定为干细胞教育者（SCE）疗法的新技术。在SCE治疗期间，患者的外周血单核细胞（PBMC）通过细胞分离器收集和循环，并在体外与粘附的CB-SC共同培养。这些”受教育”细胞（CB-SC治疗的PBMC）然后返回到患者的循环在闭环系统。临床试验已经证明了SCE疗法治疗自身免疫性疾病的临床安全性和有效性，包括1型糖尿病（T1D）2、4和脱发（AA）5。
外显体是一个纳米粒子系列，直径范围为30\u2012150 nm，存在于所有生物流体和细胞培养介质6中。外显子富含许多生物活性分子，包括脂质、mRNA、蛋白质和微RNA（miRNA），并在细胞到细胞之间的通信中发挥重要作用。最近，外显体已经变得更加有吸引力的研究人员和制药公司，因为他们的治疗潜力在诊所7，8，9。最近，我们的机械研究表明，CB-SC释放的外体有助于SCE治疗10的免疫调节。
在这里，我们描述了通过CB-SC释放的外细胞来探索针对单细胞的SCE治疗机制的协议。首先，使用超离心方法从CB-SC衍生的调节介质中分离出CB-SC释放的外体，并经流式细胞学、西印（WB）和动态光散射（DLS）验证。其次，CB-SC衍生的外体被标记为绿色荧光嗜白染料：迪奥。第三，他们与PBMC共同培养，通过流细胞学检查Dio标记的CB-SC衍生外显子体在PBMC的不同亚细胞的阳性百分比。该协议为研究干细胞免疫调节的外体作用提供了指导。
Protocol
该协议遵循发现和创新中心机构人类研究伦理委员会的指导方针，哈肯萨克子午线健康。从纽约血液中心（纽约，纽约）购买了人类布衣血单位。人体脐带血单位是从健康的献血者那里收集的，从 Cryo-Cell 国际血库（佛罗里达州的奥德斯马尔）购买。纽约血液中心和 Cryo-Cell 都获得了所有血液收集和分配的认证，并获得 IRB 的批准，并签署了捐赠者的同意书。 1. 细胞培养和制备C…
Representative Results
最初，CB-SC的表型和纯度通过流式细胞学与CB-SC相关标记物（如白细胞常见抗原CD45、ES细胞特异性转录因子OCT3/4和SOX2）进行检验。CB-SC 显示高级别的 CD45、OCT3/4、SOX2、CD270 和 galectin 9 表达式，但没有 CD34 的表达式（图 1A）。流细胞学分析证实，包括CD9、CD81和CD63在内的外体特异性标记的表达在CB-SC衍生外体（图1B）上。外体形态和大小分布的特点是TEM和DL…
Discussion
外显体的应用是临床诊断、药物开发、再生医学的新兴领域。在这里，我们提出了一个详细的协议，关于CB-SC衍生的外体的准备和外体的功能研究，人类单细胞的分化。目前的方案表明，功能性CB-SC衍生的外显体通过顺序离心和高纯度的超离心分离，并表现出对单核细胞的免疫调节。
与其他常规协议相比，超滤是一种基于不同于其他细胞外囊泡（EV）的分子量和排除尺寸的分?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢波达尔先生和路德维希先生通过哈肯萨克UMC基金会的慷慨资助。我们感谢赵老师的英文编辑。
Materials
| 1.5 ml Microcentrifuge tube | Fisher Scientific | 05-408-129 | |
| 15 ml conical tube | Falcon | 352196 | |
| 24-well plate | Falcon | 351147 | Non-tissue culture plate |
| 3,3'-Diostadecyloxacarbocyanine perchlorate(Dio) | Millipore sigma | D4292-20MG | Store at 4 °C |
| 300 Mesh Grids | Ted Pella | 1GC300 | |
| 50 mL conical tube | Falcon | 352070 | |
| 6-well plate | Falcon | 353046 | Tissue culture plate |
| 96-well plate | Falcon | 353072 | Tissue culture plate |
| ACK Lysis Buffer | Lonza | 10-548E | 100 ml |
| Amicon-15 10kDa Centrifuge Fliter Unit | Millipore sigma | UFC901024 | |
| Anti-Human Alix | Biolegend | 634501 | store at 4 °C, RRID: AB_2268110 |
| Anti-Human Calnexin | Biolegend | 699401 | store at 4 °C, RRID: AB_2728519 |
| Anti-Human CD11c antibody, Pe-Cy7 | BD Bioscience | 561356 | store at 4 °C, RRID: AB_10611859 |
| Anti-Human CD14 antibody, Karma Orange | Beckman Coulter | B36294 | store at 4 °C, RRID: AB_2728099 |
| Anti-Human CD163 antibody, PE | BD Bioscience | 556018 | store at 4 °C, RRID: AB_396296 |
| Anti-Human CD19 antibody, PC5 | Beckman Coulter | IM2643U | store at 4 °C, RRID: AB_131160 |
| Anti-Human CD206 antibody, FITC | BD Bioscience | 551135 | store at 4 °C, RRID: AB_394065 |
| Anti-Human CD209 antibody, Brilliant Violet 421 | BD Bioscience | 564127 | store at 4 °C, RRID: AB_2738610 |
| Anti-Human CD270 antibody, PE | Biolegend | 318806 | store at 4 °C, RRID: AB_2203703 |
| Anti-Human CD3 antibody, Pacfic Blue | Biolegend | 300431 | store at 4 °C, RRID: AB_1595437 |
| Anti-Human CD34 antibody, APC | Beckman Coulter | IM2427U | store at 4 °C, RRID: N/A |
| Anti-Human CD4 antibody, APC | BD Bioscience | 555349 | store at 4 °C, RRID: AB_398593 |
| Anti-Human CD45 antibody, Pe-Cy7 | Beckman Coulter | IM3548U | store at 4 °C, RRID: AB_1575969 |
| Anti-Human CD56 antibody, PE | Beckman Coulter | IM2073U | store at 4 °C, RRID: AB_131195 |
| Anti-Human CD63 antibody,PE | BD Bioscience | 561925 | store at 4 °C, RRID: AB_10896821 |
| Anti-Human CD8 antibody, APC-Alexa Fluor 750 | Beckman Coulter | A94686 | store at 4 °C, RRID: N/A |
| Anti-Human CD80 antibody, APC | Beckman Coulter | B30642 | store at 4 °C, RRID: N/A |
| Anti-Human CD81 antibody, FITC | BD Bioscience | 561956 | store at 4 °C, RRID: AB_394049 |
| Anti-Human CD86 antibody, APC-Alexa Fluor 750 | Beckman Coulter | B30646 | store at 4 °C, RRID: N/A |
| Anti-Human CD9 antibody, FITC | ThermoScientic | MA5-16860 | store at 4 °C, RRID: AB_2538339 |
| Anti-Human Galectin 9 antibody, Brilliant Violet 421 | Biolegend | 348919 | store at 4 °C, RRID: AB_2716134 |
| Anti-Human OCT3/4 antibody, eFluor660 | ThermoScientic | 50-5841-82 | store at 4 °C, RRID: AB_11218882 |
| Anti-Human SOX2 antibody, Alexa Fluor 488 | ThermoScientic | 53-9811-82 | store at 4 °C, RRID: AB_2574479 |
| BCA Protein Assay Kit | ThermoFisher Scientific | 23227 | |
| Bovine Serum Albumin | Millipore Sigma | A1933 | |
| Buffy coat | New York Blood Center | 40-60 ml/unit | |
| Cell scraper | Falcon | 353085 | |
| Disposable semi-micro cuvette | VWR | 97000590 | |
| Dissociation buffer | Gibco | 131510014 | 100 ml |
| Dual-Chanber cell counting slides | Bio-Rad | 1450015 | |
| Exosome-Human CD63 Detection reagent | ThermoFisher Scientific | 10606D | store at 4 °C |
| Ficoll-Paque PLUS density grandient media | GE Health | 17-1440-03 | 500 ml |
| Fixed-Angle Rotor(25°) | Thermo Scientifc | 75003698 | Maxium 24,700 x g |
| Gallios flow cytometer | Beckman Coulter | 3 lasers 10 Color Max |
|
| Human cord blood | Cryo-Cell International | 40-100 ml/unit | |
| Human Fc Block | BD Bioscience | 564220 | store at 4 °C |
| Immun-Blot PVDF membrane | Bio-Rad | 1620177 | |
| Millex-GP Syringe Filter Unit, 0.22 µm | Millipore Sigma | SLGP033RS | |
| Optima XE-90 Ultracentriguge | Beckman Coulter | ||
| Orbital Shaker MP4 | BioExpress | S-3500-1 | |
| PBS | ThermoFisher Scientific | 10010049 | 500 ml |
| Propidium Iodide | BD Bioscience | 56-66211E | store at 4 °C |
| Nikon Eclipse Ti2 microscope | Nikon instruments Inc | Eclipse Ti2 | NIS-Elements software version 5.11.02 |
| Hochest 33342 | Thermo Scientifc | 62249 | 5 ml |
| Revert microsopy | Fisher scientific | 12563518 | |
| Rotor 41 Ti | Beckman Coulter | 331362 | Maxium 41,000 rpm |
| Sorvall St 16R Centrifuge | Thermo Scientifc | 75004381 | |
| Swinging Bucket Rotor | Thermo Scientifc | 75003655 | |
| TC-20 cell counter | Bio-Rad | ||
| ThermoScientific Forma 380 Steri Cycle CO2 Incubator | ThermoFisher Scientific | ||
| Transmission electron microscopy | JEOL | JEM-2100 PLUS | |
| Ultracentrifuge tube | Beckman Coulter | 331372 | |
| X'VIVO 15 Serum-free medium | Lonza | BEBP04-744Q | 1000 ml Culture medium, store at 4 °C |
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Cite This Article
Hu, W., Song, X., Yu, H., Sun, J., Zhao, Y. Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes. J. Vis. Exp. (165), e61562, doi:10.3791/61562 (2020).