Name: Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells (Video) | JoVE Journal
Uploaded: 2021-11-11T13:00:00.000+00:00
Duration: 12 min 2 s
Description: Learn to isolate exosome-enriched extracellular vesicles carrying GM-CSF from embryonic stem cells using ultracentrifugation and characterization techniques. This protocol enables production of immune-modulatory vesicles for cell-free immunotherapy research.

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5 minutes

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 protocol16, culture the ES-D3 cells without feeder layer cells in the gelatin-coated 15 cm tissue culture dishes. The ES-D3 cell culture medium is composed of DMEM, exosome-free FBS (15%), nonessential amino acids (0.1 mM), L-glutamine (2 mM), &#946;-mercaptoethanol (0.1 mM), penicillin (50 units/mL), streptomycin (50 &#181;g/mL), and leukemia inhibitory factor (LIF; 100 units/mL). Culture ES-D3 cells at 37 &#176;C in a 5% CO2 humidified incubator.</li>
	<li>Once the ES-D3 cells reach around 90% confluency in 15 cm tissue culture dishes, remove the medium by aspiration. Wash the cells using trypsin (5 mL; 0.05%). Add trypsin (5 mL) to the dishes and incubate at 37 &#176;C for 5 min. Collect the cells from the dishes.</li>
	<li>Add fresh culture medium (5 mL) to the collected cells to inactivate the trypsin. Centrifuge the cells at 390 x g for 5 min. Resuspend the cells in fresh medium and determine the cell number using a hemocytometer.</li>
	<li>For passaging cells, plate the ES-D3 cells (5 x 106) in a gelatin-coated (0.1%) 15 cm tissue culture dish with fresh medium (15 mL) and culture for 3 days before subculturing the cells.</li>
	<li>To collect the cell culture supernatant for isolation of exosome-enriched EVs, plate the ES-D3 cells (1 x 107) in a gelatin-coated (0.1%) 15 cm tissue culture dish with fresh medium (15 mL) for 3 days prior to collecting cell culture supernatant.</li>
</ol>
2. Generation of GM-CSF expression plasmid
NOTE: Generate the transfection plasmid pEF1&#945;-mGM-CSF-IRES-hrGFP to overexpress GM-CSF in ES-D3 cells. In this plasmid, expression of murine GM-CSF cDNA along with the marker protein humanized Renilla reniformis GFP (hrGFP) is driven by the human polypeptide chain elongation factor 1&#945; (EF1&#945;) promoter17,18.
<ol>
	<li>Generate the vector backbone.
	<ol>
		<li>Digest the plasmid pEF1&#945;-FD3ER-IRES-hrGFP (20 &#181;g of DNA) using the restriction enzyme EcoRI (100 units) at 37 &#176;C for 2 h to generate two DNA fragments: the vector backbone (6.0 kb) and FD3ER insert (2.5 kb).</li>
		<li>Transfer 50% of digested plasmid DNA (10 &#181;g) into one 1.5 mL microcentrifuge tube and treat with alkaline phosphatase (20 units) at 37 &#176;C for 1 h. Resolve both untreated and dephosphorylated DNA using agarose gel electrophoresis (2%).</li>
		<li>Purify the vector backbone DNA fragments (6.0 kb) using a DNA gel extraction kit . While the untreated vector backbone will serve as the empty vector (pEF1&#945;-IRES-hrGFP), the dephosphorylated vector backbone will be used to generate GM-CSF-expressing plasmid (pEF1&#945;-mGM-CSF-IRES-hrGFP).</li>
	</ol>
	</li>
	<li>Generate the GM-CSF cDNA insert.
	<ol>
		<li>Digest the plasmid pMSCV-mGM-CSF-IRES-EGFP19 (20 &#181;g) using the restriction enzyme EcoRI (100 units) at 37 &#176;C for 2 h to produce two DNA fragments: the vector backbone (6.5 kb) and the murine GM-CSF cDNA insert (474 bp).</li>
		<li>Resolve the digested DNA through agarose gel electrophoresis (2%). Purify the murine GM-CSF cDNA fragment (474 bp) using a DNA gel extraction kit.</li>
	</ol>
	</li>
	<li>Generate the expression plasmids.
	<ol>
		<li>Set up 2 ligation reactions (10 &#956;L total volume) using a DNA ligation kit.
		<ol>
			<li>To generate the empty vector pEF1&#945;-IRES-hrGFP, ligate the untreated vector backbone from step 2.1.3 (6.0 kb; 500 ng).</li>
			<li>To generate pEF1&#945;-mGM-CSF-IRES-hrGFP, ligate the following DNA fragments: (1) the dephosphorylated vector backbone from step 2.1.3 (6.0 kb; 500 ng) and (2) the mGM-CSF cDNA fragment generated in step 2.2.2 (474 bp; 200 ng).</li>
		</ol>
		</li>
		<li>Ligate at 25 &#176;C for 5 min. Transform ligated DNA into DH5&#945; competent E. coli cells. Plate the transformed E. coli cells on LB-agar plates containing carbenicillin (50 &#956;g/mL).</li>
		<li>Purify plasmids from single E. coli colonies using a DNA isolation kit. Validate the identities of the plasmids by DNA sequencing.</li>
	</ol>
	</li>
</ol>
3. Generation of ES-D3 cells overexpressing GM-CSF
NOTE: Transfect the ES-D3 cells with the plasmid pEF1&#945;-mGM-CSF-IRES-hrGFP to overexpress GM-CS. Cotransfect the plasmid pBabe-Neo into ES-D3 cells to facilitate selection of stably transfected cells18,20.
<ol>
	<li>Transfect the plasmids into the ES-D3 cells.
	<ol>
		<li>Plate the ES-D3 cells (1.4 x 106) in a gelatin-coated (0.1%) 10 cm tissue culture dish with culture medium (10 mL) for transfection. Culture plated ES-D3 cells at 37 &#176;C for 24 h.</li>
		<li>Prepare two plasmid mixtures in 1.5 mL microcentrifuge tubes: (1) pEF1&#945;-IRES-hrGFP (28 &#181;g, vector control) with pBabe-Neo (4 &#181;g) and (2) pEF1&#945;-mGM-CSF-IRES-hrGFP (28 &#181;g, expressing GM-CSF) with pBabe-Neo (4 &#181;g). Carry out transfection using a transfection kit following the manufacturer&#39;s protocol.</li>
		<li>Add transfection medium (1 mL) and transfection reagent (64 &#181;L) to each tube containing the plasmid mixtures and incubate at room temperature for 5 min.</li>
		<li>Add the transfection mixtures to respective 10 cm dishes of ES-D3 cells. Incubate at 37 &#176;C for 5 h.</li>
		<li>Replace the medium in 10 cm dishes with fresh culture medium (10 mL). Incubate at 37 &#176;C for 24 h.</li>
	</ol>
	</li>
	<li>Generate bulk populations of stably transfected ES-D3 cells.
	<ol>
		<li>Remove the medium from transfected ES-D3 cells. Wash the cells with trypsin (2 mL; 0.05%). Add trypsin (2 mL) and incubate at 37 &#176;C for 5 min. Transfer the cells to a 15 mL centrifuge tube and add 2 mL of fresh culture medium to neutralize trypsin. Centrifuge at 390 x g for 5 min.</li>
		<li>Resuspend the transfected cells in fresh culture medium (10 mL). Evaluate the fluorescence intensity of GFP in transfected ES-D3 cells using fluorescence-activated cell sorting (FACS), following manufacturer&#39;s protocol.</li>
		<li>Transfer the transfected cells into two 10 cm dishes containing fresh culture medium (10 mL). Add neomycin (0.5 mg/mL) to eliminate untransfected cells.</li>
		<li>Continue culturing the transfected cells in culture medium containing neomycin (0.5 mg/mL). When the transfected ES-D3 reach 90% confluency, transfer cells to 15 cm tissue culture dishes again. Repeat the procedure for 2 weeks.</li>
	</ol>
	</li>
	<li>Generate clones of stably transfected ES-D3 cells.
	<ol>
		<li>Once bulk populations of stably transfected ES-D3 cells are generated, collect the cells as before. Determine the cell numbers using a hemocytometer. Centrifuge the cells at 390 x g for 5 min. Resuspend the cells (1 x 107 cells/mL) in fresh culture medium.</li>
		<li>Filter the cells through a sterile 40 &#181;m cell strainer. Purify GFP-positive ES-D3 cells using FACS, following manufacturer&#39;s protocol.</li>
		<li>Plate a single sorted ES-D3 cell into one well of a gelatin-coated (0.1%) 96-well tissue culture plate containing parental ES-D3 cells (1 x 103) in neomycin-free medium (200 &#181;L). Co-culturing transfected ES-D3 cells with their untransfected parental counterparts ensures that stably transfected single ES-D3 cells survive and proliferate as a single clone.</li>
		<li>Culture the cells for 48 h, and then add neomycin (0.5 mg/mL) to 96-well plates to eliminate untransfected parental ES-D3 cells.</li>
		<li>Continue culturing the GFP-positive ES-D3 cells in 96-well tissue culture plates with medium containing neomycin (0.5 mg/mL) for 1 week. Transfer clonal ES-D3 cell lines to gelatin-coated (0.1%) 6 cm tissue culture dishes with culture medium (5 mL) containing neomycin (0.5 mg/mL) for 1 week.</li>
		<li>Determine the intensity of GFP fluorescence in each of transfected ES-D3 cell clones using FACS, following manufacturer&#39;s protocol. Select the ES-D3 clones expressing either GM-CSF or the empty vector with high levels of green fluorescence.</li>
		<li>Determine the amounts of GM-CSF secreted by ES-D3 cells using a murine GM-CSF ELISA kit, following manufacturer&#39;s protocol.</li>
	</ol>
	</li>
</ol>
4. Isolation of exosome-enriched extracellular vesicles
<ol>
	<li>Culture the ES-D3 cells (1 x 107) in 15 cm tissue culture dishes for 72 h at 37 &#176;C. Collect the cell culture supernatant. Store collected supernatant at 4 &#176;C up to 1 week to maintain exosomal integrity.</li>
	<li>Centrifuge the cell culture supernatant at 5,000 x g for 60 min at 4 &#176;C using a centrifuge to sediment large cell fragments.</li>
	<li>Collect the supernatant and centrifuge at 100,000 x g for 90 min at 4 &#176;C using an ultracentrifuge.</li>
	<li>Remove the supernatant. Gently rinse each pellet twice with phosphate-buffered saline (PBS; 1 mL) to remove residual culture supernatant.</li>
	<li>Resuspend each pellet in PBS. Quantify exosome-enriched EVs by their protein content5.
	<ol>
		<li>Measure the protein concentration of exosome-enriched EVs with a bicinchoninic acid (BCA) assay. The expected yield of exosome-enriched EVs from ES-D3 cells is approximately 4 &#181;g protein/mL of cell culture supernatant. Resuspend the exosome-enriched EVs in PBS (protein concentration: ~6 &#181;g/&#181;L). Store the exosome-enriched EVs at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
5. Characterization of exosome-enriched extracellular vesicles by transmission electron microscopy
NOTE: Investigate the composition and the structure of the exosome-enriched EVs isolated from ESCs using transmission electron microscopy (TEM)5.
<ol>
	<li>Fix the exosome-enriched EVs (3-5 &#181;g/&#181;L) with a final concentration of 2% EM grade paraformaldehyde at room temperature for 2 h.</li>
	<li>Load fixed samples (10 &#181;L) onto copper grids with carbon support film. Incubate the samples with copper grids for 1 min, and then drain the grids with filter paper.</li>
	<li>Stain the grids with a staining solution following manufacturer&#39;s protocol.</li>
	<li>Transfer the grids to a piece of filter paper using tweezers. Allow the grids to dry overnight at room temperature.</li>
	<li>Acquire electron microscopy images using a transmission electron microscope (50,000x magnification), following manufacturer&#39;s protocol.</li>
</ol>
6. Evaluation of exosome-enriched extracellular vesicles by western blot analysis
<ol>
	<li>Prepare whole cell extracts.
	<ol>
		<li>Remove the medium from ES-D3 cells cultured in 15 cm dishes. Wash the cells with trypsin (5 mL; 0.05%). Add trypsin (5 mL) to the cells. Incubate at 37 &#176;C for 5 min. Collect the cells and add fresh culture medium (5 mL) to neutralize trypsin. Centrifuge the cells at 390 x g for 5 min. Resuspend the cells in PBS.</li>
		<li>Determine cell numbers using a hemocytometer. Centrifuge the cells again at 390 x g for 5 min. Resuspend the cells in SDS-PAGE loading buffer containing 0.5% SDS (5,000 cells/&#181;L).</li>
		<li>Sonicate the samples for 10 s using a sonicator with 10% amplitude (wattage: 500 W; ultrasonic frequency: 20 kHz). Heat the samples at 100 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Prepare the lysates of exosome-enriched EVs.
	<ol>
		<li>Resuspend the exosome-enriched EVs in SDS-PAGE loading buffer containing 0.5% SDS at a concentration of 1.2 &#181;g/&#181;L.</li>
		<li>Sonicate the samples for 10 s using a sonicator with 10% amplitude (wattage: 500 W; ultrasonic frequency: 20 kHz). Heat the samples at 100 &#176;C for 5 min.</li>
	</ol>
	</li>
	<li>Detect proteins by Western blot.
	<ol>
		<li>Load whole cell extracts (10 &#181;L; 5,000 cells/&#181;L) and exosome-enriched EV lysates (10 &#181;L; 1.2 &#181;g/&#181;L) into each well of a Bis-Tris PAGE gel (4-20%). Transfer proteins onto polyvinylidene fluoride (PVDF) membranes.</li>
		<li>Incubate membranes with appropriate primary and secondary antibodies. Dilute antibodies (at the concentrations indicated below) in blotting buffer containing PBS, Tween-20 (0.2%), and nonfat dry milk (10% w/v).
		<ol>
			<li>Use the following primary antibodies: anti-Annexin V (200 ng/mL), anti-CD81 (50 ng/mL), anti-Flotillin-1 (200 ng/mL), anti-cytochrome c (100 ng/mL), anti-protein disulfide isomerase (200 ng/mL), anti-GAPDH (33 ng/mL), and anti-Oxphos COX IV-subunit IV (600 ng/mL).</li>
			<li>Use the following secondary antibodies: peroxidase-conjugated goat anti-rabbit IgG (20 ng/mL) and peroxidase-conjugated goat anti-mouse IgG (20 ng/mL).</li>
		</ol>
		</li>
		<li>Detect proteins using an enhanced chemiluminescence detection kit.</li>
	</ol>
	</li>
</ol>
7. Determining GM-CSF concentrations in exosome-enriched extracellular vesicles by ELISA
NOTE: Evaluate the amounts of GM-CSF in exosome-enriched EVs by ELISA using a kit for murine GM-CSF, following manufacturer&#39;s protocol with some modifications.
<ol>
	<li>Coat the ELISA plate with capture antibody. Treat exosome-enriched EVs (0.6 &#181;g) in PBS alone or PBS + 0.05% Tween-20 (100 &#181;L) at room temperature for 30 min. Add treated samples to the coated ELISA plate and incubate at room temperature for 1 h. Wash the plate with PBS alone or PBS + 0.05% Tween-20.</li>
	<li>Add detection antibody to the samples. Incubate at room temperature for 1 h. Wash the plate with PBS alone or PBS + 0.05% Tween-20. Add Avidin-HRP to the samples. Incubate at room temperature for 30 min. Wash the plate with PBS alone or PBS + 0.05% Tween-20.</li>
	<li>Determine the concentrations of GM-CSF in exosome-enriched EVs by measuring the absorbance at 450 nm on a microplate reader.</li>
</ol>

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<li>Bunggulawa, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advancements+in+the+use+of+exosomes+as+drug+delivery+systems%5BTitle%5D)+AND+%22Journal+of+Nanobiotechnology%22%5BJournal%5D)">Recent advancements in the use of exosomes as drug delivery systems.</a> Journal of Nanobiotechnology. 16 (1), 81(2018).</li>
<li>Schlesinger, S., Lee, A. H., Wang, G. Z., Green, L., Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Proviral+silencing+in+embryonic+cells+is+regulated+by+Yin+Yang+1%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Proviral silencing in embryonic cells is regulated by Yin Yang 1.</a> Cell Reports. 4 (1), 50-58 (2013).</li>
<li>Dranoff, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((GM-CSF-based+cancer+vaccines%5BTitle%5D)+AND+%22Immunological+Reviews%22%5BJournal%5D)">GM-CSF-based cancer vaccines.</a> Immunological Reviews. 188, 147-154 (2002).</li>
<li>Park, Y. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Feeder+Cell+Types+on+Culture+of+Mouse+Embryonic+Stem+Cell+In+Vitro%5BTitle%5D)+AND+%22Development+and+Reproduction%22%5BJournal%5D)">Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro.</a> Development and Reproduction. 19 (3), 119-126 (2015).</li>
<li>Lin, S., Talbot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+culturing+mouse+and+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Methods for culturing mouse and human embryonic stem cells.</a> Methods in Molecular Biology. 690, 31-56 (2011).</li>
<li>Yaddanapudi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exosomes+from+GM-CSF+expressing+embryonic+stem+cells+are+an+effective+prophylactic+vaccine+for+cancer+prevention%5BTitle%5D)+AND+%22OncoImmunology%22%5BJournal%5D)">Exosomes from GM-CSF expressing embryonic stem cells are an effective prophylactic vaccine for cancer prevention.</a> OncoImmunology. 8 (3), 1561119(2019).</li>
</ol>

Kavitha Yaddanapudi, Chi Li, and John W. Eaton submitted a US patent application "Compositions comprising engineered embryonic stem cell-derived exosomes and methods of use thereof."

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><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 &lt;em&gt;E. coli&lt;/em&gt; 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&amp;mu;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&amp;rsquo;s Modified Eagle&amp;rsquo;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

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 pEF1α-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 acquired...

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 ...

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Research

JoVE Journal

Cancer Research

This video describes a method based on size-exclusion chromatography (SEC) to isolate extracellular vesicles or EVs derived from the brain immune cells―the microglia. The EVs act as molecular cargo to deliver mediators, which regulate an immune response. Therefore, the isolated EVs can be used as an in vitro model to study the microglia-neuron crosstalk during a neuronal immune response.

SEC-based Isolation of Microglia-derived Extracellular Vesicles: A Technique to Isolate Extracellular Vesicles from Microglia Conditioned Culture Media

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

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

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells' small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Here, we describe a magnetic separation-assisted high-speed homogenization method for large-scale production of endosome-derived nanovesicles as a new type of exosome mimics (EMs) that share the same biological origin and similar structure, morphology, and protein composition of native extracellular vesicles (EVs).

A Magnetic Separation-Assisted High-Speed Homogenization Method for Large-Scale Production of Endosome-Derived Vesicles

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

Learn to isolate exosome-enriched extracellular vesicles carrying GM-CSF from embryonic stem cells using ultracentrifugation and characterization techniques. This protocol enables production of immune-modulatory vesicles for cell-free immunotherapy research.

Isolation of Exosome-Enriched Extracellular Vesicles Carrying Granulocyte-Macrophage Colony-Stimulating Factor from Embryonic Stem Cells (Video) | JoVE Journal

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

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