Name: isolation of exosome-enriched extracellular vesicles carrying granulocyte-macrophage colony-stimulating factor from embryonic stem cells
Uploaded: 2021-11-11T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells at JoVE.com

We are grateful to Mr. Arkadiusz Slusarczyk and Kentucky Biomedical Research Infrastructure Network (KBRIN, P20GM103436) for acquiring transmission electron microscope images. This work was supported in part by grants from NIH AA018016-01 (J.W.E.), Commonwealth of Kentucky Research Challenge Trust Fund (J.W.E.), NIH CA106599 and CA175003 (C.L.), NIH CA198249 (K.Y.), and Free to Breathe Research Grant (K.Y.).

1. Culturing ES-D3 cells
<ol>
	<li>To generate exosome-free fetal bovine serum (FBS), load FBS into an ultracentrifuge and centrifuge at 100,000 x g for 16 h at 4 &#176;C. Following centrifugation, collect serum supernatant as exosome-free FBS for culturing the murine ESC cell line ES-D3 and acquiring exosome-enriched EVs.</li>
	<li>Before plating the ES-D3 cells, coat 15 cm tissue culture dishes using gelatin (0.1%) at room temperature for 30 min.</li>
	<li>Following a previously described protocol<sup class=...

<ol>
	<li>Thomson, J. A., 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=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282 (5391), 1145-1147 (1998).</li><li>Sakthiswary, R., Raymond, A. A. <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+therapy+in+neurodegenerative+diseases:+From+principles+to+practice.">Stem cell therapy in neurodegenerative diseases: From principles to practice.</a> Neural Regeneration Research. 7 (23), 1822-1831 (2012).</li><li>Liu, Y. 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This study shows a highly efficient method of producing exosome-enriched EVs carrying the immune-stimulatory protein GM-CSF, which can be employed to study the immune-modulatory effects of exosome-enriched EVs. Several studies suggest that exosomes exhibit immune-regulatory and anti-tumor functions22. Thus, exosomes from ESCs expressing GM-CSF might also possess biological activities that regulate the immune response. In this protocol, exogenous murine GM-CSF was stably overexpressed in murine ES-...

ESCs are derived from the blastocyst stage of a preimplantation embryo1. As pluripotent stem cells, ESCs have the capability to self-renew and differentiate into any type of embryonic cell. Due to their remarkable developmental potential and long-term proliferative capacity, ESCs are extremely valuable for biomedical research1. Current research efforts have largely focused on the therapeutic potential of ESCs for a variety of major pathological disorders, including diabetes, heart disease, and neurodegenerative diseases2,3,4.
Mammalian cells, including ESCs, are known to release vesicles with variable sizes to the extracellular environment, and these EVs possess many physiological and pathological functions due to their role in intercellular communication5. Among different subtypes of EVs, exosomes are small membrane vesicles released from various cell types into the extracellular space upon fusion of intermediate endocytic compartments, multivesicular bodies (MVBs), with the plasma membrane6. Exosomes have been reported to mediate intercellular communication and are critically involved in many (patho)physiological processes7,8. Exosomes inherit some biological functions from their own parental cells, because exosomes contain biological materials acquired from the cytosol, including proteins and nucleic acids. Thus, the associated antigens or factors stimulating the immune response specific for a given disease are encapsulated in the exosomes from particular types of cells9. This paved the way for clinical trials exploring tumor-derived exosomes as an anti-cancer vaccine10.
GM-CSF is a cytokine secreted by different types of immune cells11. Emerging evidence demonstrates that GM-CSF activates and regulates the immune system and plays an essential role in the antigen-presenting process12. For instance, a clinical report suggests that GM-CSF stimulates the immune response to tumors as a vaccine adjuvant13. Several GM-CSF-based cancer immunotherapy strategies to exploit the potent immune-stimulatory activity of GM-CSF have been investigated in clinical trials14. Among these, a cancer vaccine composed of irradiated GM-CSF-secreting tumor cells has shown some promise in advanced melanoma patients by inducing cellular and humoral antitumor responses and subsequent necrosis in metastasized tumors15.
Because the exosomes derived from ESCs possess similar biological activities as the original ESCs, maybe GM-CSF-carrying exosomes from ESCs could function as cell-free vesicles to regulate the immune response. In this paper, a detailed method to produce high-quality exosome-enriched EVs from ESCs expressing GM-CSF is described. These exosome-enriched EVs have the potential to serve as immune-regulatory vesicles to modulate the immune response.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Alkaline phosphate, Calf Intestinal</td>
			<td>New England Biolabs</td>
			<td>M0290S</td>
			<td>Dephosphorylating DNA plasmid</td>
		</tr>
		<tr>
			<td>anti-Annexin V mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone H-3, sc-74438</td>
			<td>Western blot, RRID:AB_1118989</td>
		</tr>
		<tr>
			<td>anti-CD81 mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone B-11, sc-166029</td>
			<td>Western blot, RRID:AB_2275892</td>
		</tr>
		<tr>
			<td>anti-cytochrome c mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone A-8, sc-13156</td>
			<td>Western blot, RRID:AB_627385</td>
		</tr>
		<tr>
			<td>anti-Flotillin-1 mAb</td>
			<td>Santa Cruz Biotechnology</td>
			<td>clone C-2; sc-74566</td>
			<td>Western blot, RRID:AB_2106563</td>
		</tr>
		<tr>
			<td>anti-GAPDH pAb</td>
			<td>Rockland</td>
			<td>600-401-A33S</td>
			<td>Western blot, RRID:AB_11182910</td>
		</tr>
		<tr>
			<td>anti-mouse IgG, goat, peroxidase-conjugated</td>
			<td>Thermo Fisher</td>
			<td>31430</td>
			<td>Western blot, RRID:AB_228307</td>
		</tr>
		<tr>
			<td>anti-Oxphos COX IV-subunit IV mAb</td>
			<td>Thermo Fisher</td>
			<td>clone 20E8C12 A21348</td>
			<td>Western blot, RRID:AB_221509</td>
		</tr>
		<tr>
			<td>anti-protein disulfide isomerase (PDI) pAb</td>
			<td>Enzo</td>
			<td>ADI-SPA-890</td>
			<td>Western blot, RRID:AB_10616242</td>
		</tr>
		<tr>
			<td>anti-rabbit IgG, goat, peroxidase-conjugated</td>
			<td>Thermo Fisher</td>
			<td>31460</td>
			<td>Western blot, RRID:AB_228341</td>
		</tr>
		<tr>
			<td>BCA (bicinchoninic acid) assay</td>
			<td>Thermo Fisher</td>
			<td>23223</td>
			<td>Determining protein concentrations</td>
		</tr>
		<tr>
			<td>Bis-Tris PAGE Gel, ExpressPlus, 4-20%</td>
			<td>Genscript</td>
			<td>M42015</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Carbenicillin, Disodium Salt</td>
			<td>Thermo Fisher</td>
			<td>10177012</td>
			<td>Selecting E. coli colonies</td>
		</tr>
		<tr>
			<td>Centrifuge, Avanti J-26 XPI</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Centrifuge rotor, JA-10</td>
			<td>Beckman Coulter</td>
			<td>09U1597</td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Centrifuge bottle, Nalgene PPCO</td>
			<td>Thermo Fisher</td>
			<td>3120-0500PK</td>
			<td>Low speed centrifugation</td>
		</tr>
		<tr>
			<td>Cu grids with carbon support film</td>
			<td>Electron Microscopy Sciences</td>
			<td>FF200-Cu</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>EcoRI</td>
			<td>New England Biolabs</td>
			<td>R0101</td>
			<td>Digesting DNA plasmid</td>
		</tr>
		<tr>
			<td>Enhanced chemiluminescence detection system</td>
			<td>Thermo Fisher</td>
			<td>32106</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>FACScalibur flow cytometer</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>Examining GFP levels of ES-D3 cells</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>ATCC</td>
			<td>SCRR-30-2020</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Fisherbrand Sterile Cell Strainers; Mesh Size: 40&#956;m</td>
			<td>Thermo Fisher</td>
			<td>22-363-547</td>
			<td>Filtering ES-D3 cells for FACS sorting</td>
		</tr>
		<tr>
			<td>Gelatin (0.1%)</td>
			<td>Thermo Fisher</td>
			<td>ES006B</td>
			<td>Culturing ES-D3 cells</td>
		</tr>
		<tr>
			<td>GM-CSF ELISA kit</td>
			<td>Thermo Fisher</td>
			<td>88733422</td>
			<td>Determining GM-CSF concentrations</td>
		</tr>
		<tr>
			<td>KnockOut Dulbecco&#8217;s Modified Eagle&#8217;s Medium</td>
			<td>Thermo Fisher</td>
			<td>10-829-018</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Leukemia Inhibitory Factor</td>
			<td>Thermo Fisher</td>
			<td>ESG1106</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>VWR</td>
			<td>VWRL0131-0100</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 transfection reagent</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td>Transfecting ES-D3 cells</td>
		</tr>
		<tr>
			<td>Microplate reader, PowerWave XS</td>
			<td>BioTek</td>
			<td></td>
			<td>Determining GM-CSF concentrations</td>
		</tr>
		<tr>
			<td>MoFlo XDP high-speed cell sorter</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>Isolating single ES-D3 cell clones</td>
		</tr>
		<tr>
			<td>NEB 5-alpha Competent E. coli</td>
			<td>New England Biolabs</td>
			<td>C2988J</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Neomycin</td>
			<td>Thermo Fisher</td>
			<td>10-131-035</td>
			<td>Selecting ES-D3 clones</td>
		</tr>
		<tr>
			<td>Non-essential amino acids</td>
			<td>Thermo Fisher</td>
			<td>SH3023801</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Non-fat dry milk</td>
			<td>Thermo Fisher</td>
			<td>NC9022655</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Thermo Fisher</td>
			<td>31985062</td>
			<td>Transfecting ES-D3 cells</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>VWR</td>
			<td>sc45000-652</td>
			<td>Medium for ES-D3 cells</td>
		</tr>
		<tr>
			<td>Plasmid pEF1a-FD3ER-IRES-hrGFP</td>
			<td>Addgene</td>
			<td>37270</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>PVDF membranes</td>
			<td>Millipore EMD</td>
			<td>IPVH00010</td>
			<td>Western blot</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit (250)</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit (50)</td>
			<td>QIAGEN</td>
			<td>28704</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Quick Ligation Kit</td>
			<td>New England Biolabs</td>
			<td>M2200S</td>
			<td>Generating GM-CSF expression plasmid</td>
		</tr>
		<tr>
			<td>Transmission electron microscope</td>
			<td>Hitachi</td>
			<td>HT7700</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>VWR</td>
			<td>45000-660</td>
			<td>Culturing ES-D3 cells</td>
		</tr>
		<tr>
			<td>Ultracentrifuge, OptimaTM L-100 XP</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge rotor, 45Ti</td>
			<td>Beckman Coulter</td>
			<td>09U4454</td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>Ultracentrifuge polycarbonate bottle</td>
			<td>Beckman Coulter</td>
			<td>355622</td>
			<td>High speed centrifugation</td>
		</tr>
		<tr>
			<td>UranyLess staining solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>22409</td>
			<td>Acquiring electron microscopy images</td>
		</tr>
	</tbody>
</table>

isolation of exosome-enriched extracellular vesicles carrying granulocyte-macrophage colony-stimulating factor from embryonic stem cells

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell types, ESCs release small membrane vesicles, such as exosomes, to the extracellular environment. Exosomes serve as essential mediators of intercellular communication and play a basic role in many (patho)physiological processes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) functions as a cytokine to modulate the immune response. The presence of GM-CSF in exosomes has the potential to boost their immune-regulatory function. Here, GM-CSF was stably overexpressed in the murine ESC cell line ES-D3. A protocol was developed to isolate high-quality exosome-enriched extracellular vesicles (EVs) from ES-D3 cells overexpressing GM-CSF. Isolated exosome-enriched EVs were characterized by a variety of experimental approaches. Importantly, significant amounts of GM-CSF were found to be present in exosome-enriched EVs. Overall, GM-CSF-bearing exosome-enriched EVs from ESCs might function as cell-free vesicles to exert their immune-regulatory activities.

GM-CSF is overexpressed in murine ESCs. 
To stably overexpress GM-CSF in ES-D3 cells, murine GM-CSF cDNA was cloned into a transfection vector to generate the expression vector pEF1&#945;-mGM-CSF-IRES-hrGFP (Figure 1A). GM-CSF was overexpressed in ES-D3 cells by transfection, and about 20% of transiently transfected ES-D3 cells were GFP-positive. Cell clones stably overexpressing GM-CSF or the empty vector control were a...

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Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells

This study describes a method to isolate exosome-enriched extracellular vesicles carrying immune-stimulatory granulocyte macrophage colony-stimulating factors from embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent stem cells capable of self-renewal and differentiation into all types of embryonic cells. Like many other cell ...

Our protocol can be used to produce high quality exosome-enriched extracellular vesicles from embryonic stem cells that express the immune stimulatory factor, GM-CSF. Exosome enriched extracellular vesicles carrying GM-CSF, have the potential to serve a cell-free immune regulatory vesicles that can modulate the immune response. Researchers with the basic molecular and cellular biology training, Should be able to easily as could this protocol, but anyone performing this protocol for the first time should follow the directions closely.Since our protocol is complicated, visualizing the intricate details of each step will help other researchers To generate exosome free FBS, ultracentrifuge the desired volume of FBS and collect the exosome free supernatant. Before plating the ESD three cells, coat 15 centimeter tissue culture dishes with 0.1%Gelatin at room temperature for 30 minutes. Remove Gelatin by aspiration and culture the ESD three cells without feeder layer cells in ESD three cell culture medium at 37 degrees Celsius in 5%Carbon dioxide humidified incubator until the cells reach 90%confluency.Wash the almost confluent cultures with five milliliters of 0.05%trypsin per dish followed by a five minute incubation at 37 degrees Celsius in fresh trypsin. At the end of the incubation pull the detached cells in a centrifuge tube and inactivate the trypsin with five milliliters of fresh culture medium. Sediment the cells by centrifugation and re-suspend the pallets in fresh medium for counting For passaging, plate five times 10 to the sixth of the ESD three cells in 15 milliliters of cell culture medium onto new Gelatin coated plates for three days of culture before sub-culturing the cells To collect the cell culture supernatant for the isolation of exosome enriched extracellular vesicles plate one times 10 to the seventh ESD three cells in 15 milliliters of cell culture medium per new Gelatin coated plate for three days prior to collecting the cell culture supernatants For exosome enriched extra cellular vesicle isolation, First sediment the large cell fragments within the supernatants collected from 72 hour cultured ESD three cells by centrifugation.After collecting the supernatant, Ultracentrifuge the samples and discard the supernatants. Gently rinse each pallette two times with one milliliter of PBS per wash to remove any residual culture supernatant and quantify the exosome enriched extracellular vesicle protein content with a Bicinchoninic acid assay, according to the manufacturer's instruction. Then re-suspend the exosome enriched extracellular vesicles in PBS at a six micrograms per micro meter concentration for storage at minus 80 degrees Celsius.To visualize the exosome enriched extracellular vesicles by transmission electron microscopy fix three to five micrograms per milliliter of the extracellular vesicles with the final concentration of 2%electron microscope grid paraformaldehyde at room temperature for two hours. At the end of the incubation, load 10 microliters of the fixed samples onto Copper Grids with carbon support film for one minute before draining the grids with filter paper, stay in the grids with an appropriate staining solution, according to the manufacturer's protocol and use tweezers to transfer the grids to a piece of filter paper, then use the transmission electron microscope with a 50, 000 times magnification to acquire electron microscopy images, according to the standard protocols. To prepare Whole Cell Extracts, collect ESD three cells from culture as demonstrated, and re-suspend the harvested cells in PBS for counting.After a second centrifugation, re-suspend the cells out of five times 10 to the third cells per microliter of SDS page loading buffer containing 0.5%SDS concentration and Sonicate the samples for 10 seconds on a Sonicator with a 10%amplitude. Then heat the samples at 100 degrees Celsius for five minutes. To prepare lysates for the exosome enriched extracellular vesicles, Re-suspend the exosome enriched extracellular vesicles in SDS page loading buffer containing 0.5%SDS at a 1.2 micrograms per microliter concentration and Sonicate the samples for 10 seconds as demonstrated then heat the samples at 100 degrees Celsius for five minutes.For Western Blot Analysis, load 10 microliter wholesale extract and exosome enriched extracellular vesicle lysate samples into individual Wells of a best stress page gel. At the end of the run, transfer the proteins onto PVDF membranes and incubate the membranes with the appropriate primary and secondary antibodies of interest, then detect the protein expression using an enhanced chemiluminescence detection kit, According to the manufacturer's instructions. To evaluate the amount of GM-CSF within exosome enriched extracellular vesicles, use a kit Murine GM-CSF to coat an ELISA Plate with anti GM-CSF capture antibody next, treat 0.6 micrograms of exosome enriched extracellular vesicles samples with 100 microliters of PBS alone, or PBS plus 0.05%tween 20 at room temperature for 30 minutes.At the end of the incubation, add the treated samples to individual Wells of the prepared ELISA Plate for a one hour incubation at room temperature followed by washing of the appropriate corresponding Wells with PBS alone, or PBS plus 0.05%tween 20. After the wash, add the detection antibody to the samples for a one hour incubation at room temperature followed by a wash with PBS alone or PBS plus 0.05%tween 20 as demonstrated, Then add Avidin Horseradish Peroxidase to the samples for a 30 minute incubation at room temperature, followed by a wash and measuring the absorbance in each well on a Microplate Reader at 450 nanometers. The GFP fluorescence intensity of a GM-CSF expressing ESD three cell line or an ESD three cell line expressing an empty vector is much higher than that of their parental counterparts.ELISA reveals that ESD three cells expressing GM-CSF produce markedly higher levels of GMCSF in their cell culture supernatant than do empty vector control cells. Furthermore, the amount of GM-CSF generated by GM-CSF expressing ESD three cells is similar to that produced by STO fibroblasts expressing GM-CSF. Transmission electron microscopy of extracellular vesicles isolated from vector transduced and GM-CSF transduced cell cultures reveal vesicles of different sizes that fall within the expected 30 to 100 nanometer diameter range.The expression of exosomal markers, including CD81, Annexin V and flotillin-1 is markedly enhanced in extracellular vesicles, isolated from ESD three cells compared to corresponding wholesale extracts by Western blot analysis. Importantly, the presence of other sub-cellular compartment markers in ESD three derived extracellular vesicles was not detected including the endoplasmic reticulum marker, Protein Disulfide Isomerase, the mitochondrial markers, cytochrome C and Oxphos complex IV-subunit IV and the cytosolic marker GAPDH. After washing with 0.05%tween 20, the background GM-CSF levels detected in the control Extracellular vesicles was significantly reduced.In contrast, GM-CSF levels in the extracellular vesicles of GM-CSF expressing cells was significantly increased by tween 20. Acquiring high quality exosome enriched extracellular vesicles carrying GMCSF is the most important step for the success of this protocol. Researchers can perform additional experiments, such as studies in GM-CSF dependent cell lines and animals to determine whether exosome enriched extracellular vesicles caring GM-CSF can function as cell-free immune regulatory vesicles.These exosome enriched extracellular vesicles can then be used to explore how modulation of the immune response affects different disease conditions.

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