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

Summary

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

Abstract

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.

Introduction

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 diseases^2,3,4.

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 processes^7,8. 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.

Protocol

1. Culturing ES-D3 cells

1. 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 °C. Following centrifugation, collect serum supernatant as exosome-free FBS for culturing the murine ESC cell line ES-D3 and acquiring exosome-enriched EVs.
2. Before plating the ES-D3 cells, coat 15 cm tissue culture dishes using gelatin (0.1%) at room temperature for 30 min.
3. 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), β-mercaptoethanol (0.1 mM), penicillin (50 units/mL), streptomycin (50 µg/mL), and leukemia inhibitory factor (LIF; 100 units/mL). Culture ES-D3 cells at 37 °C in a 5% CO₂ humidified incubator.
4. 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 °C for 5 min. Collect the cells from the dishes.
5. 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.
6. 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.
7. 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.

2. Generation of GM-CSF expression plasmid

NOTE: Generate the transfection plasmid pEF1α-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 1α (EF1α) promoter^17,18.

1. Generate the vector backbone.
   1. Digest the plasmid pEF1α-FD3ER-IRES-hrGFP (20 µg of DNA) using the restriction enzyme EcoRI (100 units) at 37 °C for 2 h to generate two DNA fragments: the vector backbone (6.0 kb) and FD3ER insert (2.5 kb).
   2. Transfer 50% of digested plasmid DNA (10 µg) into one 1.5 mL microcentrifuge tube and treat with alkaline phosphatase (20 units) at 37 °C for 1 h. Resolve both untreated and dephosphorylated DNA using agarose gel electrophoresis (2%).
   3. 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 (pEF1α-IRES-hrGFP), the dephosphorylated vector backbone will be used to generate GM-CSF-expressing plasmid (pEF1α-mGM-CSF-IRES-hrGFP).
2. Generate the GM-CSF cDNA insert.
   1. Digest the plasmid pMSCV-mGM-CSF-IRES-EGFP¹⁹ (20 µg) using the restriction enzyme EcoRI (100 units) at 37 °C for 2 h to produce two DNA fragments: the vector backbone (6.5 kb) and the murine GM-CSF cDNA insert (474 bp).
   2. Resolve the digested DNA through agarose gel electrophoresis (2%). Purify the murine GM-CSF cDNA fragment (474 bp) using a DNA gel extraction kit.
3. Generate the expression plasmids.
   1. Set up 2 ligation reactions (10 μL total volume) using a DNA ligation kit.
      1. To generate the empty vector pEF1α-IRES-hrGFP, ligate the untreated vector backbone from step 2.1.3 (6.0 kb; 500 ng).
      2. To generate pEF1α-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).
   2. Ligate at 25 °C for 5 min. Transform ligated DNA into DH5α competent E. coli cells. Plate the transformed E. coli cells on LB-agar plates containing carbenicillin (50 μg/mL).
   3. Purify plasmids from single E. coli colonies using a DNA isolation kit. Validate the identities of the plasmids by DNA sequencing.

3. Generation of ES-D3 cells overexpressing GM-CSF

NOTE: Transfect the ES-D3 cells with the plasmid pEF1α-mGM-CSF-IRES-hrGFP to overexpress GM-CS. Cotransfect the plasmid pBabe-Neo into ES-D3 cells to facilitate selection of stably transfected cells^18,20.

1. Transfect the plasmids into the ES-D3 cells.
   1. 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 °C for 24 h.
   2. Prepare two plasmid mixtures in 1.5 mL microcentrifuge tubes: (1) pEF1α-IRES-hrGFP (28 µg, vector control) with pBabe-Neo (4 µg) and (2) pEF1α-mGM-CSF-IRES-hrGFP (28 µg, expressing GM-CSF) with pBabe-Neo (4 µg). Carry out transfection using a transfection kit following the manufacturer's protocol.
   3. Add transfection medium (1 mL) and transfection reagent (64 µL) to each tube containing the plasmid mixtures and incubate at room temperature for 5 min.
   4. Add the transfection mixtures to respective 10 cm dishes of ES-D3 cells. Incubate at 37 °C for 5 h.
   5. Replace the medium in 10 cm dishes with fresh culture medium (10 mL). Incubate at 37 °C for 24 h.
2. Generate bulk populations of stably transfected ES-D3 cells.
   1. 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 °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.
   2. 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's protocol.
   3. 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.
   4. 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.
3. Generate clones of stably transfected ES-D3 cells.
   1. 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.
   2. Filter the cells through a sterile 40 µm cell strainer. Purify GFP-positive ES-D3 cells using FACS, following manufacturer's protocol.
   3. 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 µL). 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.
   4. 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.
   5. 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.
   6. Determine the intensity of GFP fluorescence in each of transfected ES-D3 cell clones using FACS, following manufacturer's protocol. Select the ES-D3 clones expressing either GM-CSF or the empty vector with high levels of green fluorescence.
   7. Determine the amounts of GM-CSF secreted by ES-D3 cells using a murine GM-CSF ELISA kit, following manufacturer's protocol.

4. Isolation of exosome-enriched extracellular vesicles

1. Culture the ES-D3 cells (1 x 10⁷) in 15 cm tissue culture dishes for 72 h at 37 °C. Collect the cell culture supernatant. Store collected supernatant at 4 °C up to 1 week to maintain exosomal integrity.
2. Centrifuge the cell culture supernatant at 5,000 x g for 60 min at 4 °C using a centrifuge to sediment large cell fragments.
3. Collect the supernatant and centrifuge at 100,000 x g for 90 min at 4 °C using an ultracentrifuge.
4. Remove the supernatant. Gently rinse each pellet twice with phosphate-buffered saline (PBS; 1 mL) to remove residual culture supernatant.
5. Resuspend each pellet in PBS. Quantify exosome-enriched EVs by their protein content⁵.
   1. 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 µg protein/mL of cell culture supernatant. Resuspend the exosome-enriched EVs in PBS (protein concentration: ~6 µg/µL). Store the exosome-enriched EVs at -80 °C.

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.

1. Fix the exosome-enriched EVs (3-5 µg/µL) with a final concentration of 2% EM grade paraformaldehyde at room temperature for 2 h.
2. Load fixed samples (10 µL) onto copper grids with carbon support film. Incubate the samples with copper grids for 1 min, and then drain the grids with filter paper.
3. Stain the grids with a staining solution following manufacturer's protocol.
4. Transfer the grids to a piece of filter paper using tweezers. Allow the grids to dry overnight at room temperature.
5. Acquire electron microscopy images using a transmission electron microscope (50,000x magnification), following manufacturer's protocol.

6. Evaluation of exosome-enriched extracellular vesicles by western blot analysis

1. Prepare whole cell extracts.
   1. 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 °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.
   2. 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/µL).
   3. Sonicate the samples for 10 s using a sonicator with 10% amplitude (wattage: 500 W; ultrasonic frequency: 20 kHz). Heat the samples at 100 °C for 5 min.
2. Prepare the lysates of exosome-enriched EVs.
   1. Resuspend the exosome-enriched EVs in SDS-PAGE loading buffer containing 0.5% SDS at a concentration of 1.2 µg/µL.
   2. Sonicate the samples for 10 s using a sonicator with 10% amplitude (wattage: 500 W; ultrasonic frequency: 20 kHz). Heat the samples at 100 °C for 5 min.
3. Detect proteins by Western blot.
   1. Load whole cell extracts (10 µL; 5,000 cells/µL) and exosome-enriched EV lysates (10 µL; 1.2 µg/µL) into each well of a Bis-Tris PAGE gel (4-20%). Transfer proteins onto polyvinylidene fluoride (PVDF) membranes.
   2. 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).
      1. 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).
      2. Use the following secondary antibodies: peroxidase-conjugated goat anti-rabbit IgG (20 ng/mL) and peroxidase-conjugated goat anti-mouse IgG (20 ng/mL).
   3. Detect proteins using an enhanced chemiluminescence detection kit.

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's protocol with some modifications.

1. Coat the ELISA plate with capture antibody. Treat exosome-enriched EVs (0.6 µg) in PBS alone or PBS + 0.05% Tween-20 (100 µL) 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.
2. 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.
3. Determine the concentrations of GM-CSF in exosome-enriched EVs by measuring the absorbance at 450 nm on a microplate reader.

Results

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...

Discussion

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-...

Materials

List of materials used in this article

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