Name: differentiation of monocytes into phenotypically distinct macrophages after treatment with human cord blood stem cell (cb-sc)-derived exosomes
Uploaded: 2020-11-12T13:00:00
Description: Watch this Scientific Journal Video about Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes at JoVE.com

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。

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...

<ol>
	<li>Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+educator+therapy+and+induction+of+immune+balance.">Stem cell educator therapy and induction of immune balance.</a> Current Diabetes Reports. 12 (5), 517-523 (2012).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+type+1+diabetes+via+islet+beta+cell+regeneration+following+immune+modulation+by+cord+blood-derived+multipotent+stem+cells.">Reversal of type 1 diabetes via islet beta cell regeneration following immune modulation by cord blood-derived multipotent stem cells.</a> BMC Medicine. 10 (1), 3 (2012).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+insulin+resistance+in+type+2+diabetes+via+immune+modulation+of+cord+blood-derived+multipotent+stem+cells+(CB-SCs)+in+stem+cell+educator+therapy:+phase+I/II+clinical+trial.">Targeting insulin resistance in type 2 diabetes via immune modulation of cord blood-derived multipotent stem cells (CB-SCs) in stem cell educator therapy: phase I/II clinical trial.</a> BMC Medicine. 11, 160 (2013).</li><li>Delgado, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+autoimmune+T-cell+memory+by+stem+cell+educator+therapy:+phase+1/2+clinical+trial.">Modulation of autoimmune T-cell memory by stem cell educator therapy: phase 1/2 clinical trial.</a> EBioMedicine. 2 (12), 2024-2036 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hair+regrowth+in+alopecia+areata+patients+following+Stem+Cell+Educator+therapy+BMC.">Hair regrowth in alopecia areata patients following Stem Cell Educator therapy BMC.</a> Medicine. 13 (1), 87 (2015).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annual Review of Cell And Developmental Biology. 30, 255-289 (2014).</li><li>Abak, A., Abhari, A., Rahimzadeh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+cancer:+small+vesicular+transporters+for+cancer+progression+and+metastasis,+biomarkers+in+cancer+therapeutics.">Exosomes in cancer: small vesicular transporters for cancer progression and metastasis, biomarkers in cancer therapeutics.</a> PeerJ. 6, 4763 (2018).</li><li>Adamiak, M., Sahoo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+myocardial+repair:+advances+and+challenges+in+the+development+of+next-generation+therapeutics.">Exosomes in myocardial repair: advances and challenges in the development of next-generation therapeutics.</a> Molecular Therapy. 26 (7), 1635-1643 (2018).</li><li>Akyurekli, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+of+preclinical+studies+on+the+therapeutic+potential+of+mesenchymal+stromal+cell-derived+microvesicles.">A systematic review of preclinical studies on the therapeutic potential of mesenchymal stromal cell-derived microvesicles.</a> Stem Cell Review and Report. 11 (1), 150-160 (2015).</li><li>Hu, W., Song, X., Yu, H., Sun, J., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Released+exosomes+contribute+to+the+immune+modulation+of+cord+blood-derived+stem+cells+(CB-SC).">Released exosomes contribute to the immune modulation of cord blood-derived stem cells (CB-SC).</a> Frontiers in Immunology. (11), 165 (2020).</li><li>Jacobson, G., K&#229;rsn&#228;s, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Important+parameters+in+semi-dry+electrophoretic+transfer.">Important parameters in semi-dry electrophoretic transfer.</a> Electrophoresis. 11 (1), 46-52 (1990).</li><li>Dykstra, M. J., Reuss, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biological Electron Microscopy: Theory, Techniques, and Troubleshooting. , (2011).</li><li>Konoshenko, M. Y., Lekchnov, E. A., Vlassov, A. V., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+extracellular+vesicles:+general+methodologies+and+latest+trends.">Isolation of extracellular vesicles: general methodologies and latest trends.</a> BioMed Research International. 2018, 8545347 (2018).</li><li>Witwer, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+sample+collection,+isolation+and+analysis+methods+in+extracellular+vesicle+research.">Standardization of sample collection, isolation and analysis methods in extracellular vesicle research.</a> Journal of Extracellular Vesicles. 2, (2013).</li><li>Orecchioni, M., Ghosheh, Y., Pramod, A. B., Ley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+polarization:+different+gene+signatures+in+M1(LPS+)+vs.+classically+and+M2(LPS-)+vs.+alternatively+activated+macrophages.">Macrophage polarization: different gene signatures in M1(LPS+) vs. classically and M2(LPS-) vs. alternatively activated macrophages.</a> Frontiers in Immunology. 10, 1084 (2019).</li></ol>

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?...

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。

<table>
	<tbody>
		<tr>
			<td>1.5 ml Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Falcon</td>
			<td>352196</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Falcon</td>
			<td>351147</td>
			<td>Non-tissue culture 
			plate</td>
		</tr>
		<tr>
			<td>3,3&#39;-Diostadecyloxacarbocyanine perchlorate(Dio)</td>
			<td>Millipore sigma</td>
			<td>D4292-20MG</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>300 Mesh Grids</td>
			<td>Ted Pella</td>
			<td>1GC300</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Falcon</td>
			<td>353046</td>
			<td>Tissue culture plate</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Falcon</td>
			<td>353072</td>
			<td>Tissue culture plate</td>
		</tr>
		<tr>
			<td>ACK Lysis Buffer</td>
			<td>Lonza</td>
			<td>10-548E</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Amicon-15 10kDa Centrifuge Fliter Unit</td>
			<td>Millipore sigma</td>
			<td>UFC901024</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Human Alix</td>
			<td>Biolegend</td>
			<td>634501</td>
			<td>store at 4 &#176;C, RRID: AB_2268110</td>
		</tr>
		<tr>
			<td>Anti-Human Calnexin</td>
			<td>Biolegend</td>
			<td>699401</td>
			<td>store at 4 &#176;C, RRID: AB_2728519</td>
		</tr>
		<tr>
			<td>Anti-Human CD11c antibody, Pe-Cy7</td>
			<td>BD Bioscience</td>
			<td>561356</td>
			<td>store at 4 &#176;C, RRID: AB_10611859</td>
		</tr>
		<tr>
			<td>Anti-Human CD14 antibody, Karma Orange</td>
			<td>Beckman Coulter</td>
			<td>B36294</td>
			<td>store at 4 &#176;C, RRID: AB_2728099</td>
		</tr>
		<tr>
			<td>Anti-Human CD163 antibody, PE</td>
			<td>BD Bioscience</td>
			<td>556018</td>
			<td>store at 4 &#176;C, RRID: AB_396296</td>
		</tr>
		<tr>
			<td>Anti-Human CD19 antibody, PC5</td>
			<td>Beckman Coulter</td>
			<td>IM2643U</td>
			<td>store at 4 &#176;C, RRID: AB_131160</td>
		</tr>
		<tr>
			<td>Anti-Human CD206 antibody, FITC</td>
			<td>BD Bioscience</td>
			<td>551135</td>
			<td>store at 4 &#176;C, RRID: AB_394065</td>
		</tr>
		<tr>
			<td>Anti-Human CD209 antibody, Brilliant Violet 421</td>
			<td>BD Bioscience</td>
			<td>564127</td>
			<td>store at 4 &#176;C, RRID: AB_2738610</td>
		</tr>
		<tr>
			<td>Anti-Human CD270 antibody, PE</td>
			<td>Biolegend</td>
			<td>318806</td>
			<td>store at 4 &#176;C, RRID: AB_2203703</td>
		</tr>
		<tr>
			<td>Anti-Human CD3 antibody, Pacfic Blue</td>
			<td>Biolegend</td>
			<td>300431</td>
			<td>store at 4 &#176;C, RRID: AB_1595437</td>
		</tr>
		<tr>
			<td>Anti-Human CD34 antibody, APC</td>
			<td>Beckman Coulter</td>
			<td>IM2427U</td>
			<td>store at 4 &#176;C, RRID: N/A</td>
		</tr>
		<tr>
			<td>Anti-Human CD4 antibody, APC</td>
			<td>BD Bioscience</td>
			<td>555349</td>
			<td>store at 4 &#176;C, RRID: AB_398593</td>
		</tr>
		<tr>
			<td>Anti-Human CD45 antibody, Pe-Cy7</td>
			<td>Beckman Coulter</td>
			<td>IM3548U</td>
			<td>store at 4 &#176;C, RRID: AB_1575969</td>
		</tr>
		<tr>
			<td>Anti-Human CD56 antibody, PE</td>
			<td>Beckman Coulter</td>
			<td>IM2073U</td>
			<td>store at 4 &#176;C, RRID: AB_131195</td>
		</tr>
		<tr>
			<td>Anti-Human CD63 antibody,PE</td>
			<td>BD Bioscience</td>
			<td>561925</td>
			<td>store at 4 &#176;C, RRID: AB_10896821</td>
		</tr>
		<tr>
			<td>Anti-Human CD8 antibody, APC-Alexa Fluor 750</td>
			<td>Beckman Coulter</td>
			<td>A94686</td>
			<td>store at 4 &#176;C, RRID: N/A</td>
		</tr>
		<tr>
			<td>Anti-Human CD80 antibody, APC</td>
			<td>Beckman Coulter</td>
			<td>B30642</td>
			<td>store at 4 &#176;C, RRID: N/A</td>
		</tr>
		<tr>
			<td>Anti-Human CD81 antibody, FITC</td>
			<td>BD Bioscience</td>
			<td>561956</td>
			<td>store at 4 &#176;C, RRID: AB_394049</td>
		</tr>
		<tr>
			<td>Anti-Human CD86 antibody, APC-Alexa Fluor 750</td>
			<td>Beckman Coulter</td>
			<td>B30646</td>
			<td>store at 4 &#176;C, RRID: N/A</td>
		</tr>
		<tr>
			<td>Anti-Human CD9 antibody, FITC</td>
			<td>ThermoScientic</td>
			<td>MA5-16860</td>
			<td>store at 4 &#176;C, RRID: AB_2538339</td>
		</tr>
		<tr>
			<td>Anti-Human Galectin 9 antibody, Brilliant Violet 421</td>
			<td>Biolegend</td>
			<td>348919</td>
			<td>store at 4 &#176;C, RRID: AB_2716134</td>
		</tr>
		<tr>
			<td>Anti-Human OCT3/4 antibody, eFluor660</td>
			<td>ThermoScientic</td>
			<td>50-5841-82</td>
			<td>store at 4 &#176;C, RRID: AB_11218882</td>
		</tr>
		<tr>
			<td>Anti-Human SOX2 antibody, Alexa Fluor 488</td>
			<td>ThermoScientic</td>
			<td>53-9811-82</td>
			<td>store at 4 &#176;C, RRID: AB_2574479</td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Millipore Sigma</td>
			<td>A1933</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffy coat</td>
			<td>New York Blood Center</td>
			<td></td>
			<td>40-60 ml/unit</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Falcon</td>
			<td>353085</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable semi-micro cuvette</td>
			<td>VWR</td>
			<td>97000590</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissociation buffer</td>
			<td>Gibco</td>
			<td>131510014</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Dual-Chanber cell counting slides</td>
			<td>Bio-Rad</td>
			<td>1450015</td>
			<td></td>
		</tr>
		<tr>
			<td>Exosome-Human CD63 Detection reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>10606D</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS density grandient media</td>
			<td>GE Health</td>
			<td>17-1440-03</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Fixed-Angle Rotor(25&#176;)</td>
			<td>Thermo Scientifc</td>
			<td>75003698</td>
			<td>Maxium 24,700 x g</td>
		</tr>
		<tr>
			<td>Gallios flow cytometer</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>3 lasers 
			10 Color Max</td>
		</tr>
		<tr>
			<td>Human cord blood</td>
			<td>Cryo-Cell International</td>
			<td></td>
			<td>40-100 ml/unit</td>
		</tr>
		<tr>
			<td>Human Fc Block</td>
			<td>BD Bioscience</td>
			<td>564220</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Immun-Blot PVDF membrane</td>
			<td>Bio-Rad</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GP Syringe Filter Unit, 0.22 &#181;m</td>
			<td>Millipore Sigma</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 Ultracentriguge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital Shaker MP4</td>
			<td>BioExpress</td>
			<td>S-3500-1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>ThermoFisher Scientific</td>
			<td>10010049</td>
			<td>500 ml</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>BD Bioscience</td>
			<td>56-66211E</td>
			<td>store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti2 microscope</td>
			<td>Nikon instruments Inc</td>
			<td>Eclipse Ti2</td>
			<td>NIS-Elements software version 5.11.02</td>
		</tr>
		<tr>
			<td>Hochest 33342</td>
			<td>Thermo Scientifc</td>
			<td>62249</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>Revert microsopy</td>
			<td>Fisher scientific</td>
			<td>12563518</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotor 41 Ti</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td>Maxium 41,000 rpm</td>
		</tr>
		<tr>
			<td>Sorvall St 16R Centrifuge</td>
			<td>Thermo Scientifc</td>
			<td>75004381</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging Bucket Rotor</td>
			<td>Thermo Scientifc</td>
			<td>75003655</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-20 cell counter</td>
			<td>Bio-Rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoScientific Forma 380 Steri Cycle CO2 Incubator</td>
			<td>ThermoFisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>JEOL</td>
			<td>JEM-2100 PLUS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>X&#39;VIVO 15 Serum-free medium</td>
			<td>Lonza</td>
			<td>BEBP04-744Q</td>
			<td>1000 ml Culture medium, store at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

differentiation of monocytes into phenotypically distinct macrophages after treatment with human cord blood stem cell (cb-sc)-derived exosomes

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。

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...

Watch this Scientific Journal Video about Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes at JoVE.com

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell CB-SC-Derived Exosomes

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。

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

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates ...

干细胞教育者疗法用于治疗 I 型糖尿病和其他自身免疫性疾病。该方法被开发出来，通过CB-SC释放的外体来探索干细胞教育者治疗免疫调节的分子机制。该协议提供了指导，如何从人类脐带血干细胞培养物中纯化外体，并确定它们对单核细胞的免疫调制。演示这个程序的将是来自我实验室的博士生魏虎。首先，将CB-SC衍生的调节介质像文本手稿中描述的那样三次离心，每次离心后将上流水线收集到一个新的50毫升锥形管中。第三次离心后，使用0.22微升过滤器过滤获得的上清液，将15毫升介质转移到每个10千元离心过滤器单元。在4000xg的离心机30分钟，以分离浓缩的外显性介质。将浓缩的外显体转移到超离心管。然后在10万xg下对外体进行颗粒，在4摄氏度下80分钟。然后丢弃上一杯，在10毫升PBS中重新暂停颗粒的外体。执行最后的超离心收集外显颗粒，并在 200 微升 PBS 中重新排泄。将 3000 万个人类 PBMC 添加到 15 毫升管中，以 300 x g 的离心机在 4 摄氏度下 10 分钟。将细胞重新在300微升的冷PBS中，并加入60微升CD14微珠。混合好，在冰上孵化细胞15分钟。再加入6毫升的冷PBS，在4摄氏度下以300 x g的离心机10分钟。然后将颗粒细胞重新在500微升的冷运行缓冲液中重新填充。将分离柱放在磁铁分离器中，用两毫升冷运行缓冲液洗涤三次。将重新暂停的单元格转移到分离列中，让它们通过。同样，每次洗涤用两毫升的运行缓冲液清洗柱三次。然后从磁铁分离器上提起柱，将其放在冰上 15 毫升离心管上。用两毫升冷运行缓冲液将CD14正细胞分离，并在4摄氏度下以300 x g离心10分钟，使细胞产生颗粒。将颗粒重新放入两毫升冷化学定义的血清介质中，将其 50 微升转移到 1.5 毫升管中。用10微升的Krome橙结合抗人类CD14单克隆抗体染色获得的细胞20分钟。加入一毫升PBS，以300 x g离心10分钟，对细胞进行颗粒化。将细胞颗粒重新放入200微升PBS中，并转移到5毫升管中。通过流式细胞测定CD14+单细胞的纯度。在经过六井板治疗的组织培养物中，获得100万颗纯化单核细胞，在37摄氏度下孵育2小时，在5%的二氧化碳下。孵育后丢弃上经剂，轻轻加入两毫升37度预热化学定义的无血清培养基，然后将80微克的CB-SC衍生外显体加入到六井板的单细胞培养物中，总体积为2毫升。在37摄氏度下孵育5%二氧化碳，三至四天。然后使用倒置显微镜以 200 倍的放大倍率对细胞形态进行成像。添加一毫升基于PBS的细胞分离缓冲液，通过上下移液分离细胞，用细胞刮刀收获剩余的细胞。通过以1，690 x g离心5分钟收集细胞，并在200微升PBS中重新产生细胞。添加五微升的FC阻滞剂以阻止非特异性结合，然后加入抗体。在室温下孵育细胞30分钟。将一毫升PBS加入孵化细胞，以300 x g离心10分钟。将颗粒重新在 200 微升 PBS 中，每个样品添加 5 微升碘化丙胺。将细胞转移到新的五毫升流量管中，进行流量细胞学，以评估CD14、CD80、CD86、CD163、CD206和CD209表达的水平。用CB-SC相关标记通过流量细胞学对CB-SC的表型和纯度进行了检查。分析表明，CD45、OCT3/4、SOX2、CD270和galectin9表达水平很高，但未观察到CD34的表达。流细胞学分析还确认了外显标记、CD9、CD 81和CD63在CB-SC衍生外显体上的表达。外体的大小形态以TEM为特征，大小分布由DLS确定。西方印迹进一步证明了外向关联标记 Alix 的表达， 没有 ER 相关标记的表达式， 卡内辛。使用荧光显微镜观察到迪奥标记的CB-SC-Exo与PBMC的直接相互作用。为了更好地定义哪些细胞群体与标有CB-SC-Exo的表盘相互作用，不同的细胞室被封闭，并标有细胞特异性标记，如T细胞的CD3、骨髓树突细胞的CD11C、单细胞的CD14、B细胞的CD19和NK细胞的CD56。单核细胞主要针对CB-SC衍生的外体，因为它们表现出高于其他免疫细胞的中位荧光强度。外显体处理的单核细胞成功地分化成主轴样形态。使用CB-SC衍生的外体治疗后，M2相关标记（如CD163、CD206和CD209）的水平有上升的调节。常规M2巨噬细胞与CB-SC外型诱导M2巨噬细胞之间的表型比较无显著差异。该协议的关键步骤是将CB-SC衍生的外细胞添加到六井板的培养单细胞中，并孵育三到四天，这需要细胞分化到具有主轴状形态的其他2型巨噬细胞。接下来，我们将探讨CB-SC衍生的外体内部的分子成分，这些分子成分有助于将单细胞分化为2型巨噬细胞。

Research

JoVE Journal

Immunology and Infection

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes

Expanding Cytotoxic T Lymphocytes from Umbilical Cord Blood that Target Cytomegalovirus, Epstein-Barr Virus, and Adenovirus

扩大目标，脐血细胞毒性T淋巴细胞，巨细胞病毒，EB病毒，腺病毒

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where ...

科研

免疫与感染

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

临床应用<em&gt;睡美人</em和人工抗原呈递细胞基因修饰T细胞从外设和脐带血中

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer  a biologic product with the potential for ...

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

从脐带血分离的前体B细胞亚群

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation of Human Monocytes by Double Gradient Centrifugation and Their Differentiation to Macrophages in Teflon-coated Cell Culture Bags

人类单核双梯度离心法和其分化为巨噬细胞的特富龙涂层的细胞培养袋隔离

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

巨噬细胞集落刺激因子分化的人单核细胞衍生的巨噬细胞的培养

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

葡聚糖增强小鼠和人原发性 NK 细胞的慢转导效率

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

Megakaryocyte Differentiation and Platelet Formation from Human Cord Blood-derived CD34+ Cells

人脐血巨分化与血小板形成的 CD34+单元格

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

人类干细胞衍生的伊拉尔肠细胞单层中M细胞样细胞的诱导分化

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Isolation, Transfection, and Culture of Primary Human Monocytes

原人类单核细胞的隔离、转染和文化

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. ...