分离外体富集的细胞外囊泡携带颗粒细胞-巨噬细胞-胚胎干细胞的细胞外刺激因子
1Department of Pharmacology and Toxicology,University of Louisville, 2Experimental Therapeutics Program, Brown Cancer Center,University of Louisville, 3Department of Medicine,University of Louisville, 4Department of Surgery,University of Louisville, 5Immuno-Oncology Program, Brown Cancer Center,University of Louisville, 6Department of Microbiology and Immunology,University of Louisville
Summary
这项研究描述了一种将携带免疫刺激性粒细胞巨噬细胞刺激因子的外细胞富集细胞外囊泡从胚胎干细胞中分离的方法。
Abstract
胚胎干细胞（ESCs）是多能干细胞，能够自我更新和分化到所有类型的胚胎细胞。像许多其他细胞类型一样，ESC 将小膜囊泡（如外显子）释放到细胞外环境中。外显子是细胞间交流的重要中介，在许多（病理）生理过程中发挥着基本作用。粒细胞-巨噬细胞群刺激因子（GM-CSF）作为细胞因子调节免疫反应。GM-CSF在外显子中的存在有可能增强其免疫调节功能。在这里，GM-CSF在穆林ESC细胞系ES-D3中被稳定地过度表达。开发了一个协议，将高质量的外体富集细胞外囊泡（EV）与ES-D3细胞分离出来，从而过度表达GM-CSF。孤立的外体丰富的电动汽车的特点是各种实验方法。重要的是，在富含外显子的电动汽车中发现了大量GM-CSF。总的来说，来自ESC的具有转基因-CSF的外生丰富型电动汽车可以作为无细胞囊泡来发挥其免疫调节作用。
Introduction
ESC来自植入前胚胎1的胚泡阶段。作为多能干细胞，ESC有能力自我更新并分化成任何类型的胚胎细胞。由于其显著的发展潜力和长期增殖能力，ESC对生物医学研究具有极其宝贵的价值。目前的研究工作主要集中在ESC治疗各种重大病理疾病的潜力，包括糖尿病，心脏病和神经退行性疾病2，3，4。
众所周知，哺乳动物细胞，包括ESC，会向细胞外环境释放大小不一的囊泡，这些EV由于在细胞间通信中的作用而具有许多生理和病理功能。在不同的子类型EV中，外显子是小膜囊从不同细胞类型释放到细胞外空间后融合的中间内分泌室，多血管体（MVB），与等离子膜6。据报道，外显体可以调解细胞间的交流，并严重参与许多（病理）生理过程7，8。外显子从它们自己的母细胞中继承了一些生物功能，因为外体含有从细胞溶胶中获取的生物材料，包括蛋白质和核酸。因此，相关的抗原或刺激特定疾病免疫反应的因素被封装在特定类型的细胞9的外源中。这为探索肿瘤衍生外显体作为抗癌疫苗10的临床试验铺平了道路。
GM-CSF是由不同类型的免疫细胞11分泌的细胞因子。新出现的证据表明，GM-CSF激活和调节免疫系统，并在抗原呈现过程中发挥着至关重要的作用。例如，一份临床报告表明，GM-CSF刺激免疫反应肿瘤作为疫苗辅助剂13。在14日的临床试验中，研究了几种基于GM-CSF的癌症免疫治疗策略，以利用GM-CSF强大的免疫刺激活性。其中，由辐照GM-CSF分泌肿瘤细胞组成的癌症疫苗通过诱导细胞和幽默抗肿瘤反应以及随后转移肿瘤15中的坏死，在晚期黑色素瘤患者中显示出一定的希望。
由于来自ESC的外显子具有与原始ESC相似的生物活动，因此可能来自ESC的转基因-CSF携带的外体可以起到无细胞囊泡的作用，以调节免疫反应。本文介绍了从ESC中生产出高质量外体浓缩电动汽车的详细方法，该方法表达了GM-CSF。这些富含外体的电动汽车有可能作为免疫调节囊泡来调节免疫反应。
Protocol
1. 培养ES-D3细胞 要生成无外显子的母牛血清 （FBS），在 4 °C 下以 100，000 x g 的速度将 FBS 装载到超中心和离心机中，持续 16 小时。 离心后，收集血清超高纳特作为无外奥体的FBS，用于培育粘膜ESC细胞系ES-D3和获得富含外体的EV。 在电镀ES-D3细胞之前，在室温下使用明胶（0.1%）涂抹15厘米的组织培养皿30分钟。 按照先前描述的协议16，培养ES-D3细胞没?…
Representative Results
GM-CSF 在穆林 ESC 中表达过度。为了在ES-D3细胞中稳定地过度表达GM-CSF，将MM-CSF cDNA克隆成一个转染载体，以生成表达载体pEF1+-mGM-CSF-IRES-hrGFP（图1A）。通过转染，GM-CSF在ES-D3细胞中表达过度，约20%的瞬态瞬态瞬态ES-D3细胞为GFP阳性。细胞克隆稳定过度表达GM-CSF或空矢量控制被FACS收购。如图1 B</stron…
Discussion
本研究展示了一种高效的方法，可以产生携带免疫刺激蛋白GM-CSF的富含外体的EV，可用于研究富含外体的电动汽车的免疫调节作用。一些研究表明，外显体表现出免疫调节和抗肿瘤功能22。因此，来自表达转基因-CSF的ESC的外显子可能也具有调节免疫反应的生物活动。在本协议中，外源性粘液GM-CSF通过转染在木质ES-D3细胞中稳定过度表达（图1）。重要的是，?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢阿尔卡迪乌斯·斯卢萨尔奇克先生和肯塔基生物医学研究基础设施网络（KBRIN，P20GM103436）获得传输电子显微镜图像。这项工作得到了NIH AA018016-01（J.W.E.）、肯塔基联邦研究挑战信托基金（J.W.E.）、NIH CA106599和CA175003（C.L.）、NIH CA198249（K.Y.）和自由呼吸研究补助金（K.Y.）的资助。
Materials
| Alkaline phosphate, Calf Intestinal | New England Biolabs | M0290S | Dephosphorylating DNA plasmid |
| anti-Annexin V mAb | Santa Cruz Biotechnology | clone H-3, sc-74438 | Western blot, RRID:AB_1118989 |
| anti-CD81 mAb | Santa Cruz Biotechnology | clone B-11, sc-166029 | Western blot, RRID:AB_2275892 |
| anti-cytochrome c mAb | Santa Cruz Biotechnology | clone A-8, sc-13156 | Western blot, RRID:AB_627385 |
| anti-Flotillin-1 mAb | Santa Cruz Biotechnology | clone C-2; sc-74566 | Western blot, RRID:AB_2106563 |
| anti-GAPDH pAb | Rockland | 600-401-A33S | Western blot, RRID:AB_11182910 |
| anti-mouse IgG, goat, peroxidase-conjugated | Thermo Fisher | 31430 | Western blot, RRID:AB_228307 |
| anti-Oxphos COX IV-subunit IV mAb | Thermo Fisher | clone 20E8C12 A21348 | Western blot, RRID:AB_221509 |
| anti-protein disulfide isomerase (PDI) pAb | Enzo | ADI-SPA-890 | Western blot, RRID:AB_10616242 |
| anti-rabbit IgG, goat, peroxidase-conjugated | Thermo Fisher | 31460 | Western blot, RRID:AB_228341 |
| BCA (bicinchoninic acid) assay | Thermo Fisher | 23223 | Determining protein concentrations |
| Bis-Tris PAGE Gel, ExpressPlus, 4-20% | Genscript | M42015 | Western blot |
| Carbenicillin, Disodium Salt | Thermo Fisher | 10177012 | Selecting E. coli colonies |
| Centrifuge, Avanti J-26 XPI | Beckman Coulter | Low speed centrifugation | |
| Centrifuge rotor, JA-10 | Beckman Coulter | 09U1597 | Low speed centrifugation |
| Centrifuge bottle, Nalgene PPCO | Thermo Fisher | 3120-0500PK | Low speed centrifugation |
| Cu grids with carbon support film | Electron Microscopy Sciences | FF200-Cu | Acquiring electron microscopy images |
| EcoRI | New England Biolabs | R0101 | Digesting DNA plasmid |
| Enhanced chemiluminescence detection system | Thermo Fisher | 32106 | Western blot |
| FACScalibur flow cytometer | Becton Dickinson | Examining GFP levels of ES-D3 cells | |
| Fetal bovine serum | ATCC | SCRR-30-2020 | Medium for ES-D3 cells |
| Fisherbrand Sterile Cell Strainers; Mesh Size: 40μm | Thermo Fisher | 22-363-547 | Filtering ES-D3 cells for FACS sorting |
| Gelatin (0.1%) | Thermo Fisher | ES006B | Culturing ES-D3 cells |
| GM-CSF ELISA kit | Thermo Fisher | 88733422 | Determining GM-CSF concentrations |
| KnockOut Dulbecco’s Modified Eagle’s Medium | Thermo Fisher | 10-829-018 | Medium for ES-D3 cells |
| Leukemia Inhibitory Factor | Thermo Fisher | ESG1106 | Medium for ES-D3 cells |
| L-glutamine | VWR | VWRL0131-0100 | Medium for ES-D3 cells |
| Lipofectamine 2000 transfection reagent | Thermo Fisher | 11668019 | Transfecting ES-D3 cells |
| Microplate reader, PowerWave XS | BioTek | Determining GM-CSF concentrations | |
| MoFlo XDP high-speed cell sorter | Beckman Coulter | Isolating single ES-D3 cell clones | |
| NEB 5-alpha Competent E. coli | New England Biolabs | C2988J | Generating GM-CSF expression plasmid |
| Neomycin | Thermo Fisher | 10-131-035 | Selecting ES-D3 clones |
| Non-essential amino acids | Thermo Fisher | SH3023801 | Medium for ES-D3 cells |
| Non-fat dry milk | Thermo Fisher | NC9022655 | Western blot |
| Opti-MEM I Reduced Serum Medium | Thermo Fisher | 31985062 | Transfecting ES-D3 cells |
| Paraformaldehyde | Electron Microscopy Sciences | 15710 | Acquiring electron microscopy images |
| Penicillin/streptomycin | VWR | sc45000-652 | Medium for ES-D3 cells |
| Plasmid pEF1a-FD3ER-IRES-hrGFP | Addgene | 37270 | Generating GM-CSF expression plasmid |
| PVDF membranes | Millipore EMD | IPVH00010 | Western blot |
| QIAprep Spin Miniprep Kit (250) | QIAGEN | 27106 | Generating GM-CSF expression plasmid |
| QIAquick Gel Extraction Kit (50) | QIAGEN | 28704 | Generating GM-CSF expression plasmid |
| Quick Ligation Kit | New England Biolabs | M2200S | Generating GM-CSF expression plasmid |
| Transmission electron microscope | Hitachi | HT7700 | Acquiring electron microscopy images |
| Trypsin | VWR | 45000-660 | Culturing ES-D3 cells |
| Ultracentrifuge, OptimaTM L-100 XP | Beckman Coulter | High speed centrifugation | |
| Ultracentrifuge rotor, 45Ti | Beckman Coulter | 09U4454 | High speed centrifugation |
| Ultracentrifuge polycarbonate bottle | Beckman Coulter | 355622 | High speed centrifugation |
| UranyLess staining solution | Electron Microscopy Sciences | 22409 | Acquiring electron microscopy images |
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Citazione di questo articolo
Meng, S., Whitt, A. G., Tu, A., Eaton, J. W., Li, C., Yaddanapudi, K. Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells. J. Vis. Exp. (177), e60170, doi:10.3791/60170 (2021).