Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes
Summary
This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/exosomes.
Abstract
Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.
Introduction
Exosomes can mediate cellular communication by transferring membrane and cytosolic proteins, lipids, and RNAs between cells1. Exosome-mediated communication has been demonstrated to be involved in many physiological and pathological processes (i.e., antigen presentation, development of tolerance2, and tumor progression3). Exosomes often have contents similar to those of the source cells releasing them. Thus, the exosomes can carry specific cell properties from the source cells and transfer these properties to the cells ingesting them4.
Exosomes are 50- to 150-nm double-layer membrane vesicles and present specific markers (e.g., CD9, CD81, CD63, HSP70, Alix, and TSG101). Thus, exosomes must be characterized by various methods for different aspects. Transmission electron microscopy can be used to visualize membrane vesicles such as exosomes4,5. Nanoparticle tracking analysis (NTA) and dynamic light scattering analysis (DLS) are used for measuring the size and number of purified exosomes4. The lipid membrane content of exosomes can be verified by density gradient. Exosomal markers, such as CD9, CD81, CD63, HSP70, Alix, and TSG1016,7, can be measured by Western blotting.
Mammary basal cells have the ability to generate mammary glands when implanted into fat pads, while luminal cells cannot8,9,10. Thus, mammary basal cells are also referred to as mammary repopulating units. By using the model of mammary basal and luminal cells, the ability of EVs/exosomes to transfer cell characteristics between different cell populations can be examined. This work demonstrates the method of transferring gland-forming ability from mammary basal epithelial cells to mammary luminal epithelial cells by using EVs/exosomes derived from mammary basal epithelial cells. Luminal mammary epithelial cells acquired basal cell properties following the ingestion of EVs/exosomes secreted from basal cells and can then form mammary glands4.
Protocol
Representative Results
Discussion
Exosomes often carry characteristics of the cells that released them, and the amount of released exosomes can be induced by stimuli4. The culture medium of cells can be collected and subjected to differential ultracentrifugation for EV/exosome collection (Figure 1). There is currently no general agreement on an ideal method to isolate EVs/exosomes. The optimal method used here has been determined by the downstream application14. Ultracentrifuga…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from the National Health Research Institutes (05A1-CSPP16-014, H.J.L.) and from the Ministry of Science and Technology (MOST 103-2320-B-400-015-MY3, H.J.L).
Materials
| MCDB 170 | USBiological | M2162 | |
| DMEM/F12 | Thermo | 1250062 | |
| Optima L-100K ultracentrifuge | Beckman | 393253 | |
| SW28 Ti Rotor | Beckman | 342204 | |
| SW41 Rotor | Beckman | 331306 | |
| NANOSIGHT LM10 | Malvern | NANOSIGHT LM10 | for nanoparticle tracking analysis (NTA) |
| Optiprep | Sigma-Aldrich | D1556 | 60% (w/v) solution of iodixanol in water (sterile). |
| CD81 antibody | GeneTex | GTX101766 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| CD9 antibody | GeneTex | GTX100912 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| CD63 antibody | Abcam | Ab59479 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| TSG101 antibody | GeneTex | GTX118736 | 1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| GAPDH | GeneTex | GTX100118 | 1:6000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4°C, overnight |
| CFSE (carboxyfluorescein succinimidyl diacetate ester) | Thermo | V12883 | |
| FACSCalibur | BD Biosciences | fluorescence cell analyzer | |
| collagenase Type IV | Thermo | 17104019 | |
| trypsin | Thermo | 27250018 | |
| ITS | Sigma-Aldrich | I3146 | a mixture of recombinant human insulin, human transferrin, and sodium selenite |
| accutase | ebioscience | 00-4555-56 | a natural enzyme mixture with proteolytic and collagenolytic enzyme activity |
| dispase | STEMCELL | 7913 | 5 mg/ml = 5 U/ml |
| anti-CD49f antibody | Biolegend | 313611 | 1:50 |
| anti-EpCAM antibody | Biolegend | 118213 | 1:200 |
| FACSAria | BD Biosciences | cell sorter | |
| carmine alum | Sigma-Aldrich | C1022 | |
| human mammary epithelial cells (HMLE cells, NAMECs) | gifts from Dr. Robert Weinberg | ||
| permount | Thermo Fisher Scientific | SP15-500 | |
| sodium bicarbonate | Zymeset | BSB101 | |
| EGF | Peprotech | AF-100-015 | |
| Hydrocoritisone | Sigma-Aldrich | SI-H0888 | |
| Insulin | Sigma-Aldrich | SI-I9278 | |
| BPE (bovine pituitary extract) | Hammod Cell Tech | 1078-NZ | |
| GW627368X | Cayman | 10009162 | |
| 15-cm culture dish | Falcon | 353025 | |
| table-top centrifuge | Eppendrof | Centrifuge 3415R | |
| ultracentrifuge tube | Beckman | 344058 | |
| PBS (Phosphate-buffered saline) | Corning | 46-013-CM | |
| BCA Protein Assay | Thermo Fisher Scientific | 23228 | |
| Transmission Electron Microscopy | Hitachi | HT7700 | |
| gelatin | STEMCELL | 7903 | |
| 10-cm culture dish | Falcon | 353003 | |
| 6-well culture dish | Corning | 3516 | |
| female C57BL/6 mice | NLAC (National Laboratory Animal Center | ||
| FBS (Fetal Bovine Serum) | BioWest | S01520 | |
| gentamycin | Thermo Fisher Scientific | 15710072 | |
| Pen/Strep | Corning | 30-002-Cl | |
| DNase I | 5PRIMER | 2500120 | |
| isofluorane | Halocarbon | NPC12164-002-25 | |
| formaldehyde | MACRON | H121-08 | |
| EtOH (Ethanol) | J.T. Baker | 800605 | |
| glacial acetic acid | Panreac | 131008.1611 | |
| aluminum potassium sulfate | Sigma-Aldrich | 12625 | |
| Xylene | Leica | 3803665 | |
| 0.22 μm membranes | Merck Millipore | Millex-GP | |
| AUTOCLIP Wound Clips, 9 mm | BD Biosciences | 427631 | |
| AUTOCLIP Wound Clip Applier | BD Biosciences | 427630 | |
| CellMask™ Deep Red | Thermo Fisher Scientific | C10046 | plasma membrane stain |
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