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

We are grateful to Mr. Poddar and Mr. Ludwig for generous funding support via Hackensack UMC Foundation. We appreciate Laura Zhao for English editing.

The protocol follows the guidelines of institutional human research ethics committee at Center for Discovery and Innovation, Hackensack Meridian Health. Human buffy coat blood units were purchased from the New York Blood Center (New York, NY). Human umbilical cord blood units were collected from healthy donors and purchased from Cryo-Cell International blood bank (Oldsmar, FL). Both New York Blood Center and Cryo-Cell have received all accreditations for blood collections and distributions, with IRB approval and signed C...

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	<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. 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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>

Application of exosomes is an emerging field for clinical diagnosis, drug developments and regenerative medicine. Here, we present a detailed protocol regarding the preparation of CB-SC-derived exosomes and the functional study of exosomes on the differentiation of human monocytes. The current protocol demonstrated that functional CB-SC-derived exosomes are isolated by sequential centrifugation and ultracentrifugation with high purity and exhibiting the immune modulation on monocytes.
As compa...

Cord blood stem cells (CB-SC) are unique type of stem cells identified from human cord blood and are distinguished from other known types of stem cells such as mesenchymal stem cells (MSC) and hematopoietic stem cells (HSC)1. Based on their unique properties of immune modulation and their ability to tightly adhere to the surface of Petri dishes, we developed a new technology designated as Stem Cell Educator (SCE) therapy in clinical trials2,3. During SCE therapy, a patient&#8217;s peripheral blood mononuclear cells (PBMC) are collected and circulated through a cell separator and co-cultured with adherent CB-SC in vitro. These &#8220;educated&#8221; cells (CB-SC-treated PBMC) are then returned to the patient&#8217;s circulation in a closed-loop system. Clinical trials have already demonstrated the clinical safety and efficacy of SCE therapy for the treatment of autoimmune diseases including type 1 diabetes (T1D)2,4 and alopecia areata (AA)5.
Exosomes are a family of nanoparticles with diameters ranging 30&#8210;150 nm and exist in all biofluid and cell culture media6. Exosomes are enriched with many bioactive molecules including lipids, mRNAs, proteins, and microRNAs (miRNA), and play an important role in cell-to-cell communications. Of late, exosomes have become more attractive for researchers and pharmaceutical companies due to their therapeutic potentials in clinics7,8,9. Recently, our mechanistic studies demonstrated that CB-SC-released exosomes contribute to the immune modulation of SCE therapy10.
Here, we describe the protocol to explore the mechanism of SCE therapy targeting monocytes by CB-SC-released exosomes. First, CB-SC-released exosomes were isolated from CB-SC-derived conditioned media using ultracentrifugation methods and validated by flow cytometry, western blot (WB) and dynamic light scattering (DLS). Second, CB-SC-derived exosomes were labeled with a green fluorescent lipophilic dye: Dio. Third, they were co-cultured with PBMC to examine the positive percentages of Dio-labeled CB-SC-derived exosomes at the different subpopulations of PBMC by flow cytometry. This protocol provides guidance to study the action of exosomes underlying the immune modulation of stem cells.

<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

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient&#8217;s blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient&#8217;s blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these &#8220;educated&#8221; immune cells to the patient&#8217;s blood. Exosomes are nano-sized extracellular vesicles between 30&#8210;150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Initially, the phenotype and purity of CB-SC were examined by flow cytometry with CB-SC-associated markers such as leukocyte common antigen CD45, ES cell-specific transcription factors OCT3/4, and SOX2. CB-SC display high levels of CD45, OCT3/4, SOX2, CD270, and galectin 9 expression, but no expression of CD34 (Figure 1A). Flow cytometry analysis confirmed the expression of exosome-specific markers including CD9, CD81, and CD63 were on CB-SC-derived exosomes (Figure 1B</...

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

Exosome application is an emerging tool for drug development and regenerative medicine. We establish an exosome isolation protocol with high purity to isolate exosomes from novel identified stem cells called CB-SC for mechanistic studies. We also coculture CB-SC-derived exosomes with human monocytes, leading to their differentiation into phenotypically distinct macrophages.

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

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

The Stem Cell Educator therapy was developed to treat type I diabetes and the other autoimmune disease. This method was developed to explore the molecule mechanism underlying immune moderation of Stem Cell Educator therapy through CB-SC released exosomes. This protocol provide guidance on how to purify exosomes from a human cord blood stem cells cultures and determine their immune modulation of monocytes.Demonstrating the procedure will be Wei Hu, a PhD student, from my lab. To begin, centrifuge the CB-SC-derived conditioned medium three times as described in the text manuscript, collecting the supernatant into a new 50 milliliter conical tube after each centrifugation. After the third centrifugation, filter the obtained supernatant with a 0.22 microliter filter and transfer 15 milliliters of media to each 10 kilodalton centrifugal filter unit.Centrifuges at 4, 000 x g for 30 minutes to isolate the concentrated exosome media. Transfer the concentrated exosomes to an ultracentrifuge tube. Then pellet the exosomes at 100, 000 x g for 80 minutes at four degrees Celsius.Then discard the supernatant and resuspend the pelleted exosomes in 10 milliliters of PBS. Perform a final ultracentrifugation to collect the exome pellet and resuspend it in 200 microliters of PBS. Add 30 million human PBMCs to a 15 milliliter tube and centrifuge at 300 x g for 10 minutes at four degrees Celsius.Resuspend the cells in 300 microliters of cold PBS and add 60 microliters of CD14 microbeads. Mix well and incubate the cells on ice for 15 minutes. Add six more milliliters of cold PBS and centrifuge at 300 x g for 10 minutes at four degrees Celsius.Then resuspend the pelleted cells in 500 microliters of cold running buffer. Place the separation column in the magnet separator and wash it three times with two milliliters of cold running buffer. Transfer the resuspended cells into the separation column and let them pass.Again, wash the column three times with two milliliters of running buffer per wash. Then lift the column from the magnet separator and place it over a 15 milliliter centrifuge tube on ice. Elute the CD14 positive cells with two milliliters of cold running buffer and centrifuged at 300 x g for 10 minutes at four degrees Celsius to pellet the cells.Resuspend the pellet in two milliliters of cold chemical defined serum medium and transfer 50 microliters of it into a 1.5 milliliter tube. Stain the obtained cells with 10 microliters of Krome Orange-conjugated anti-human CD14 monoclonal antibodies for 20 minutes. Add one milliliter of PBS and centrifuge at 300 x g for 10 minutes to pellet the cells.Resuspend the cell pellet in 200 microliters of PBS and transfer it into a five milliliter tube. Determine the purity of CD14+monocytes by flow cytometry. Seed one million of the obtained purified monocytes in a tissue culture treated six-well plate and incubate for two hours at 37 degrees Celsius under 5%carbon dioxide.After incubation discard the supernatant and gently add two milliliters of 37 degree pre-warmed chemical defined serum-free culture medium, then add 80 micrograms of the CB-SC-derived exosomes to the monocyte culture in the six-well plate with a total volume of two milliliters. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for three to four days. Then image the cell morphology using an inverted microscope at 200X magnification.Add one milliliter of PBS-based cell dissociation buffer to detach the cells by pipetting up and down and harvest the remaining cells with a cell scraper. Collect the cells by centrifuging at 1, 690 x g for five minutes and resuspend them in 200 microliters of PBS. Add five microliters of FC blocker to block the non-specific binding, then add the antibodies.Incubate the cells at room temperature for 30 minutes. Add one milliliter of PBS to the incubated cells and centrifuge them at 300 x g for 10 minutes. Resuspend the pellet in 200 microliters of PBS and add five microliters of propidium iodide per sample.Transfer the cells to a new five milliliter flow tube and perform flow cytometry to evaluate the levels of CD14, CD80, CD86, CD163, CD206, and CD209 expression. The phenotype and purity of CB-SCs were examined by flow cytometry with CB-SC associated markers. The analysis showed high levels of CD45, OCT3/4, SOX2, CD270 and galectin 9 expression but no expression of CD34 was observed.Flow cytometry analysis also confirm the expression of exome specific markers, CD9, CD 81, and CD63 on CB-SC-derived exosomes. The size morphology of exosomes was characterized by TEM and the size distribution was determined by DLS. Western blot further prove the expression of the exome associated marker Alix without expression of the ER associated marker, calnexin.The direct interaction of Dio-labeled CB-SC-Exo with PBMC was observed using fluorescence microscopy. To better define which cell population interacted with the dial labeled CB-SC-Exo, different cell compartments were gated with cell specific markers, such as CD3 for T cells, CD11C for myeloid dendritic cells, CD14 for monocytes, CD19 for B cells, and CD56 for NK cells. Monocytes were primarily targeted by the CB-SC-derived exosomes as they exhibited higher median fluorescence intensities than those of other immune cells.The exosome treated monocytes successfully differentiated into spindle-like morphologies. There was an upregulation in the level of M2 associated markers, such as CD163, CD206, and CD209 after the treatment with CB-SC-derived exosomes. Phenotypic comparison between conventional M2 macrophages and the CB-SC exome-induced M2 macrophages showed no significant differences.The key step in this protocol is adding CB-SC-derived exosomes to the monocyte of culture in six-well plate and incubate it for three to four days, which needed to the cell's differentiation to other type 2 macrophages with spindle-like morphology. Next, we are going to explore the molecule components inside of CB-SC-derived exosomes that contributed to the induction of differentiation of monocyte into type 2 macrophages.

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Research

JoVE Journal

Immunology and Infection

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

Virus infections after stem cell transplantation are among the most common causes of death, especially after cord blood (CB) transplantation (CBT) where the CB does not contain appreciable numbers of virus-experienced T cells which can protect the recipient from infection.1-4 We and others have shown that virus-specific CTL generated from seropositive donors and infused to the recipient are safe and protective.5-8 However, until recently, virus-specific T cells could not be generated from cord blood, likely due to the absence of virus-specific memory T cells. 
In an effort to better mimic the in vivo priming conditions of na&iuml;ve T cells, we established a method that used CB-derived dendritic cells (DC) transduced with an adenoviral vector (Ad5f35pp65) containing the immunodominant CMV antigen pp65, hence driving T cell specificity towards CMV and adenovirus.9 At initiation, we use these matured DCs as well as CB-derived T cells in the presence of the cytokines IL-7, IL-12, and IL-15.10 At the second stimulation we used EBV-transformed B cells, or EBV-LCL, which express both latent and lytic EBV antigens. Ad5f35pp65-transduced EBV-LCL are used to stimulate the T cells in the presence of IL-15 at the second stimulation. Subsequent stimulations use Ad5f35pp65-transduced EBV-LCL and IL-2. 
From 50x106 CB mononuclear cells we are able to generate upwards of 150 x 106 virus-specific T cells that lyse antigen-pulsed targets and release cytokines in response to antigenic stimulation.11 These cells were manufactured in a GMP-compliant manner using only the 20% fraction of a fractionated cord blood unit and have been translated for clinical use.

Here we describe the first good manufacturing practice (GMP)-compliant method of producing virus-specific cytotoxic T lymphocytes (CTL) from umbilical cord blood, a source of predominantly na&#238;ve T cells.

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

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

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 superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-&gamma; receptor 1) for the loading of monoclonal antibodies (mAb)12. In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR+ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of &gamma;-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-2113. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.

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

T cells expressing a CD19-specific chimeric antigen receptor (CAR) are infused as investigational treatment of B-cell malignancies in our first-in-human gene therapy trials. We describe genetic modification of T cells using the Sleeping Beauty (SB) system to introduce CD19-specific CAR and selective propagation on designer CD19+ artificial antigen presenting cells.

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

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

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

Human macrophages are involved in a plethora of pathologic processes ranging from infectious diseases to cancer. Thus they pose a valuable tool to understand the underlying mechanisms of these diseases. We therefore present a straightforward protocol for the isolation of human monocytes from buffy coats, followed by a differentiation procedure which results in high macrophage yields. The technique relies mostly on commonly available lab equipment and thus provides a cost and time effective way to obtain large quantities of human macrophages. Briefly, buffy coats from healthy blood donors are subjected to a double density gradient centrifugation to harvest monocytes from the peripheral blood. These monocytes are then cultured in fluorinated ethylene propylene (FEP) Teflon-coated cell culture bags in the presence of macrophage colony-stimulating factor (M-CSF). The differentiated macrophages can be easily harvested and used for subsequent studies and functional assays. Important methods for quality control and validation of the isolation and differentiation steps will be highlighted within the protocol. In summary, the protocol described here enables scientists to routinely and reproducibly isolate human macrophages without the need for cost intensive tools. Furthermore, disease models can be studied in a syngeneic human system circumventing the use of murine macrophages.

We present a simple and efficient protocol for the generation of human macrophages. Buffy coats are processed by double density gradient centrifugation and isolated monocytes are then differentiated to macrophages in Teflon-coated cell culture bags. This maximizes macrophage yields and facilitates cell harvesting for subsequent experiments.

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

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

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

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

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

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

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

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

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

Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.

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

A highly pure population of megakaryocytes can be obtained from cord blood-derived CD34+ cells. A method for CD34+ cell isolation and megakaryocyte differentiation is described here.

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

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 immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

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

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4+ T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14+ monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs.

Presented here is an optimized protocol for isolating, culturing, transfecting, and differentiating human primary monocytes from HIV-infected individuals and healthy controls.

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