Name: transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes
Uploaded: 2017-06-03T14:00:00
Description: Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Dit werk werd ondersteund door subsidies van de National Health Research Institutes (05A1-CSPP16-014, HJL) en van het Ministerie van Wetenschap en Technologie (MOST 103-2320-B-400-015-MY3, HJL).

Alle onderzoeken waarbij dieren betrokken zijn, voldoen aan protocollen die zijn goedgekeurd door het Institutioneel Comité voor dierenwelzijn. 1. Extracellulaire Vesikel / Exosome Isolatie en Validatie <ol><li> Cultuur mammale epitheliale basale cellen, NAMECs 4 , met vers, serumvrij medium gemaakt van 500 ml MCDB 170, pH 7,4 + 500 ml DMEM / F12 met natriumbicarbonaat (0,2438%); EGF (5 ng / ml); Hydrocoritison (0,5 μg / ml); Insuline (5 μg / ml); Boviene hypofys...

<ol>
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D., 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=Mammary+stem+cells+have+myoepithelial+cell+properties.">Mammary stem cells have myoepithelial cell properties.</a> Nat Cell Biol. 16 (10), 942-950 (2014).</li><li>Stingl, J., 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=Purification+and+unique+properties+of+mammary+epithelial+stem+cells.">Purification and unique properties of mammary epithelial stem cells.</a> Nature. 439 (7079), 993-997 (2006).</li><li>Gardiner, C., Ferreira, Y. J., Dragovic, R. A., Redman, C. W., Sargent, I. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem Biophys Res Commun. 28 (5), 815-820 (1967).</li><li>Riches, A., Campbell, E., Borger, E., Powis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+exosome+release+from+mammary+epithelial+and+breast+cancer+cells+-+a+new+regulatory+pathway.">Regulation of exosome release from mammary epithelial and breast cancer cells - a new regulatory pathway.</a> Eur J Cancer. 50 (5), 1025-1034 (2014).</li><li>Witwer, K. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+nano-sized+vesicles+released+by+cells+and+subsequent+quantitative+and+qualitative+analysis+by+high-resolution+flow+cytometry.">Fluorescent labeling of nano-sized vesicles released by cells and subsequent quantitative and qualitative analysis by high-resolution flow cytometry.</a> Nat Protoc. 7 (7), 1311-1326 (2012).</li><li>Kowal, J., 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=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proc Natl Acad Sci U S A. 113 (8), E968-E977 (2016).</li><li>Li, D., 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=ADVANCED+IMAGING.+Extended-resolution+structured+illumination+imaging+of+endocytic+and+cytoskeletal+dynamics.">ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.</a> Science. 349 (6251), (2015).</li><li>Outzen, H. C., Custer, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+human+normal+and+neoplastic+mammary+tissues+in+the+cleared+mammary+fat+pad+of+the+nude+mouse.">Growth of human normal and neoplastic mammary tissues in the cleared mammary fat pad of the nude mouse.</a> J Natl Cancer Inst. 55 (6), 1461-1466 (1975).</li><li>Sheffield, L. G., Welsch, C. 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Exosomen dragen vaak eigenschappen van de cellen die hen vrijgegeven, en de hoeveelheid vrijgegeven exosomen kan door stimuli 4 worden geïnduceerd. Het kweekmedium van cellen kan worden verzameld en onderworpen aan differentiële ultracentrifugatie voor EV / exosoom-collectie ( Figuur 1 ). Er is momenteel geen algemene overeenstemming over een ideale methode om EV&#39;s / exosomen te isoleren. De hier gebruikte optimale methode is bepaald door de downstream applicat...

Exosomen kunnen cellulaire communicatie bemiddelen door membraan- en cytosolische eiwitten, lipiden en RNA&#39;s tussen cellen 1 over te brengen . Exosoomgemedieerde communicatie is aangetoond dat zij betrokken zijn bij veel fysiologische en pathologische processen ( dwz antigeenpresentatie, ontwikkeling van tolerantie 2 en tumorprogressie 3 ). Exosomen hebben vaak dezelfde inhoud als die van de broncellen die ze vrijmaken. Zo kunnen de exosomen specifieke celeigenschappen van de broncellen dragen en deze eigenschappen overbrengen naar de cellen die ze innemen 4 . Exosomen zijn 50- tot 150 nm dubbellaagse membraan vesicles en aanwezige specifieke markers ( bijv. CD9, CD81, CD63, HSP70, Alix en TSG101). Zo moeten exosomen worden gekenmerkt door verschillende methoden voor verschillende aspecten. Transmissie elektronenmicroscopie kan worden gebruikt om membraan vesicles te visualiserenZoals exosomen 4 , 5 . Nanoparticle tracking analyse (NTA) en dynamische lichtverspreiding analyse (DLS) worden gebruikt voor het meten van de grootte en het aantal gezuiverde exosomen 4 . Het lipidemembraangehalte van exosomen kan door dichtheidsgradiënt worden gecontroleerd. Exosomale markers, zoals CD9, CD81, CD63, HSP70, Alix en TSG101 6 , 7 kunnen worden gemeten door Western blotting. Mammary basale cellen hebben de mogelijkheid om mammekirtels te genereren wanneer ze in vetpads geïmplanteerd worden, terwijl luminale cellen niet kunnen 8 , 9 , 10 . Zo worden ook basaalcellen van mammoet ook aangeduid als mammografische repopulerende eenheden. Door gebruik te maken van het model van basale en luminale cellen van de mammoet kan het vermogen van EV&#39;s / exosomen om celkenmerken tussen verschillende celpopulaties over te dragen, worden onderzocht. Dit werkDemonstreert de werkwijze voor het overbrengen van kliervormend vermogen van basale epitheelcellen naar mammary luminale epitheelcellen door gebruik te maken van EV&#39;s / exosomen afgeleid van mammale basale epitheelcellen. Luminale mammary epithelialcellen verworven basale cel eigenschappen na de inname van EV&#39;s / exosomen afgescheiden van basale cellen en kunnen dan mammary klieren 4 vormen .

<table>
	<tbody>
		<tr>
			<td>MCDB 170&#160;</td>
			<td>USBiological</td>
			<td>M2162</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Thermo</td>
			<td>1250062</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-100K ultracentrifuge</td>
			<td>Beckman</td>
			<td>393253</td>
			<td></td>
		</tr>
		<tr>
			<td>SW28 Ti Rotor</td>
			<td>Beckman</td>
			<td>342204</td>
			<td></td>
		</tr>
		<tr>
			<td>SW41 Rotor</td>
			<td>Beckman</td>
			<td>331306</td>
			<td></td>
		</tr>
		<tr>
			<td>NANOSIGHT LM10</td>
			<td>Malvern</td>
			<td>NANOSIGHT LM10</td>
			<td>for nanoparticle tracking analysis (NTA)</td>
		</tr>
		<tr>
			<td>Optiprep&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D1556</td>
			<td>60% (w/v) solution of iodixanol in water (sterile).</td>
		</tr>
		<tr>
			<td>CD81 antibody</td>
			<td>GeneTex</td>
			<td>GTX101766</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CD9 antibody</td>
			<td>GeneTex</td>
			<td>GTX100912</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CD63 antibody</td>
			<td>Abcam</td>
			<td>Ab59479</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>TSG101 antibody</td>
			<td>GeneTex</td>
			<td>GTX118736</td>
			<td>1:1000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>GeneTex</td>
			<td>GTX100118</td>
			<td>1:6000 in 5% w/v nonfat dry milk, 1X TBS, 0.1% Tween 20 at 4&#176;C, overnight&#160;</td>
		</tr>
		<tr>
			<td>CFSE (carboxyfluorescein succinimidyl diacetate ester)</td>
			<td>Thermo</td>
			<td>V12883</td>
			<td></td>
		</tr>
		<tr>
			<td>FACSCalibur</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>fluorescence cell analyzer</td>
		</tr>
		<tr>
			<td>collagenase Type IV&#160;</td>
			<td>Thermo</td>
			<td>17104019</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin</td>
			<td>Thermo</td>
			<td>27250018</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;ITS</td>
			<td>Sigma-Aldrich</td>
			<td>I3146</td>
			<td>a mixture of recombinant human insulin, human transferrin, and sodium selenite</td>
		</tr>
		<tr>
			<td>accutase</td>
			<td>ebioscience</td>
			<td>00-4555-56</td>
			<td>a natural enzyme mixture with proteolytic and collagenolytic enzyme activity</td>
		</tr>
		<tr>
			<td>dispase&#160;</td>
			<td>STEMCELL</td>
			<td>7913</td>
			<td>5 mg/ml = 5 U/ml</td>
		</tr>
		<tr>
			<td>anti-CD49f antibody</td>
			<td>Biolegend</td>
			<td>313611</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>anti-EpCAM antibody</td>
			<td>Biolegend</td>
			<td>118213</td>
			<td>1:200</td>
		</tr>
		<tr>
			<td>FACSAria</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>cell sorter</td>
		</tr>
		<tr>
			<td>carmine alum</td>
			<td>Sigma-Aldrich</td>
			<td>C1022</td>
			<td></td>
		</tr>
		<tr>
			<td>human mammary epithelial cells (HMLE cells, NAMECs)</td>
			<td></td>
			<td></td>
			<td>gifts from Dr. Robert Weinberg</td>
		</tr>
		<tr>
			<td>permount</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Zymeset&#160;</td>
			<td>BSB101</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>AF-100-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocoritisone</td>
			<td>Sigma-Aldrich</td>
			<td>SI-H0888</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SI-I9278</td>
			<td></td>
		</tr>
		<tr>
			<td>BPE (bovine pituitary extract)</td>
			<td>Hammod Cell Tech&#160;</td>
			<td>1078-NZ</td>
			<td></td>
		</tr>
		<tr>
			<td>GW627368X&#160;</td>
			<td>Cayman</td>
			<td>10009162</td>
			<td></td>
		</tr>
		<tr>
			<td>15-cm culture dish</td>
			<td>Falcon&#160;</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>table-top centrifuge</td>
			<td>Eppendrof&#160;</td>
			<td>Centrifuge 3415R</td>
			<td></td>
		</tr>
		<tr>
			<td>ultracentrifuge tube</td>
			<td>Beckman</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate-buffered saline)&#160;</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>23228</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy</td>
			<td>Hitachi</td>
			<td>HT7700</td>
			<td></td>
		</tr>
		<tr>
			<td>gelatin&#160;</td>
			<td>STEMCELL</td>
			<td>7903</td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm culture dish</td>
			<td>Falcon&#160;</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture dish</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>female C57BL/6 mice</td>
			<td>NLAC (National Laboratory Animal Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS (Fetal Bovine Serum)</td>
			<td>BioWest&#160;</td>
			<td>S01520</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamycin</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>15710072</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Corning</td>
			<td>30-002-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>5PRIMER</td>
			<td>2500120</td>
			<td></td>
		</tr>
		<tr>
			<td>isofluorane&#160;</td>
			<td>Halocarbon</td>
			<td>NPC12164-002-25</td>
			<td></td>
		</tr>
		<tr>
			<td>formaldehyde</td>
			<td>MACRON</td>
			<td>H121-08</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH (Ethanol)</td>
			<td>J.T. Baker</td>
			<td>800605</td>
			<td></td>
		</tr>
		<tr>
			<td>glacial acetic acid</td>
			<td>Panreac</td>
			<td>131008.1611</td>
			<td></td>
		</tr>
		<tr>
			<td>aluminum potassium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>12625</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene&#160;</td>
			<td>Leica</td>
			<td>3803665</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m membranes</td>
			<td>Merck Millipore</td>
			<td>Millex-GP</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clips, 9 mm</td>
			<td>BD Biosciences</td>
			<td>427631</td>
			<td></td>
		</tr>
		<tr>
			<td>AUTOCLIP Wound Clip Applier</td>
			<td>BD Biosciences</td>
			<td>427630</td>
			<td></td>
		</tr>
		<tr>
			<td>CellMask&#8482; Deep Red</td>
			<td>Thermo Fisher Scientific&#160;</td>
			<td>C10046</td>
			<td>plasma membrane stain</td>
		</tr>
	</tbody>
</table>

transfer of mammary gland-forming ability between mammary basal epithelial cells and mammary luminal cells via extracellular vesicles/exosomes

Cellen kunnen communiceren via exosomen, ~ 100 nm extracellulaire vesicles (EV&#39;s) die proteïnen, lipiden en nucleïnezuren bevatten. Non-adherent / mesenchymale mammary epithelial cell (NAMEC) -gerelateerde extracellulaire vesicles kunnen worden geïsoleerd uit NAMEC medium via differentiële ultracentrifugatie. Op grond van hun dichtheid kunnen EV&#39;s worden gezuiverd via ultracentrifugatie bij 110.000 x g. De EV-bereiding uit ultracentrifugatie kan verder worden gescheiden door gebruik te maken van een continu dichtheidsgradiënt om verontreiniging met oplosbare eiwitten te voorkomen. De gezuiverde EV&#39;s kunnen vervolgens verder geëvalueerd worden met behulp van nanoparticle-tracking analyse, die de grootte en het aantal blaasjes in de bereiding meet. De extracellulaire blaasjes met een grootte van 50 tot 150 nm zijn exosomen. De NAMEC-afgeleide EV&#39;s / exosomen kunnen worden ingezet door mammale epitheelcellen, die kunnen worden gemeten door flowcytometrie en confocale microscopie. Sommige mammale stamcellen eigenschappen ( bijv. Membraanvormende vermogen) kunnenWorden overgebracht van de stamachtige NAMEC&#39;s ​​naar epitheliale cellen via de NAMEC-afgeleide EV&#39;s / exosomen. Geïsoleerde primaire EpCAM hi / CD49f lo luminale mammary epithelialcellen kunnen geen mammaklieren vormen nadat ze in muizenvetpads zijn getransplanteerd, terwijl EpCAM lo / CD49f hi basale mammary epitheelcellen na de transplantatie membraan vormen. Toepassing van NAMEC-afgeleide EV&#39;s / exosomen door EpCAM hi / CD49f lo luminale mammary epithelialcellen stelt hen in staat om melkklippen te genereren na transplantatie in vetpads. De EV&#39;s / exosomen afgeleid van stamachtige mammale epitheelcellen overdragen de kliervormende vermogen van EpCAM hi / CD49f lo luminale mammary epitheelcellen.

Aangezien aangetoond is dat het blokkeren van PGE 2 / EP 4 signalering triggers EV / exosome vrijgave van basale basale stamcellen 4, presenteert dit werk een werkwijze voor het isoleren van de geïnduceerde EV&#39;s / exosomen uit mammary epithelial basal cell (NAMEC) cultuur. Aangezien NAMEC&#39;s ​​worden gekweekt in serumvrij medium, zijn er geen bestaande EV&#39;s / exosomen afgeleid van serum 13 . Voor cellen die in serum...

Watch this Scientific Journal Video about Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes at JoVE.com

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Dit protocol beschrijft methoden voor het zuiveren, kwantificeren en karakteriseren van extracellulaire vesicles (EVs) / exosomen van niet-aanhechtende / mesenchymale mammary epitheelcellen en voor het gebruik ervan om het vermogen van de borstkanker te overdragen naar luminale mammary epitheelcellen. EV&#39;s / exosomen afgeleid van stamachtige mammale epitheelcellen kunnen deze celeigenschap overbrengen aan cellen die de EV&#39;s / exosomen innemen.

Overdracht van Mammary Gland-vormende Vermogen Tussen Mammary Basal Epithelial Cellen en Mammary Luminale Cellen via Extracellulaire Vesicles / Exosomen

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

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