Name: a novel mammary fat pad transplantation technique to visualize the vessel generation of vascular endothelial stem cells
Uploaded: 2017-08-03T13:00:00
Description: Watch this Scientific Journal Video about A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells at JoVE.com

Dette arbejde blev støttet af National Natural Science Foundation of China (31530045 og 31371500 til YAZ, 31401245 til QCY), Kina&#39;s Ministerium for Videnskab og Teknologi (2014CB964800), Det kinesiske videnskabsakademi (XDB19000000 til YAZ) og den kinesiske Society of Cell Biology (Early Career Fellowship til QCY).

Eksperimentelle procedurer blev godkendt af dyrepleje- og brugskomitéen for Shanghai Institute of Biochemistry and Cell Biology, Det Kinesiske Videnskabsakademi under den godkendte protokol SIBCB-NAF-15-002-S335-003. 1. Isolering af VESC&#39;er fra Mouse Mammary Gland <ol><li> Mammary gland høst og væv fordøjelse. <ol><li> Brug 8 uger gamle Actin-GFP modne jomfru reportermus med vægt fra 20 til 25 g som vævsdonorer. Bemærk: Celler, der stammer fra do...

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+identification+of+bipotent+stem+cells+in+the+mammary+gland.">In situ identification of bipotent stem cells in the mammary gland.</a> Nature. 506, 322-327 (2014).</li><li>Barker, N., 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=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li><li>Wang, D. 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Angiogenese assays repræsenterer en god eksperimentel tilgang til at studere vaskulær dynamik. Mus retinal vaskulatur, som udvikler sig postnatalt, har vist sig at være en attraktiv model til at studere angiogenese 12 . På trods af at den er relativt tilgængelig, er den egentlige manipulation inden for nethinden vaskulær bed temmelig vanskelig. Hidtil har den bedst beskrevne in vivo- transplantation været plug-assayet, som omslutter celler inde i en kældermembranmatrixmasse og ki...

Under udvikling og homeostase finder vaskulær vækst og ombygning trofast sted i overensstemmelse med organvækst og reparation. Angiogenese beskriver dannelsen af ​​nye skibe fra eksisterende blodkar og anses for at være en vigtig kraft, der formidler disse dynamiske vaskulære ændringer. Hvert blodkar er inderforet med et lag af endotelceller (EC&#39;er), og de synes at være fundamentet for fartøjsarkitekturen. I lang tid forbliver den mekanisme, gennem hvilken EF-puljen genopfyldes under homeostasis, uklar, og argumenter blev rejst over, om vaskulær omsætning er resultatet af moden EC proliferation eller er bidraget fra vaskulær stamme / stamcelleraktivitet. På grund af manglen på direkte fysiologiske beviser forblev eksistensen og den cellulære identitet af vaskulære endotelceller (VESC&#39;er) også kontroversielle. En af de mest almindelige metoder til bekæmpelse af stamcellens adfærd er gennem transplanenTation af formodede stamceller i modtagende mus. Denne metode måler stammen potentialet af kandidat stamceller in vivo. Transplantation blev først anvendt til undersøgelsen af ​​knoglemarvstamceller 1 , som bidrog til etablering af de hierarkiske egenskaber ved det hæmatopoietiske system 2 . I endotelfeltet er en basementmembranmatrix ( f.eks. Matrigel) -plug, der er indsat subkutant under flankehuden, blevet et standard in vivo- angiogenese-assay anvendt til at adressere fartøjsdannelsesevnen af ​​transplanterede EC&#39;er. Flere eksperimentelle metoder, herunder kolonidannelse i 3D-kultursystemer og transplantation, har foreslået potentielle EC-progenitor / VESC-populationer 3 , 4 , 5 , 6 . Men da EC&#39;er indlejret i kælder membranmatrix er forholdsvis adskilt fraDet omgivende væv giver dette ikke det optimale nichemiljø, der kræves for fuldt ud at udforske det angiogene potentiale af transplanterede celler. Som et resultat er skibe dannet inden i matrixplugten overvejende kapillærlignende og funktionelt umærkelige. Brystkirtlen udvikler sig postnatalt, hvor den mest robuste vækst forekommer under pubertet og graviditet. I pubertetstadiet gennemgår brystepitelet hurtig ekspansion for at optage hele brystfedtpuden, ledsaget af en effektiv omformning af de omgivende vaskulære strukturer. Således giver brystkirtlen en fremragende model til undersøgelsen af ​​angiogenese. Det giver plads, matrix og rige angiogene stimuli fra det voksende brystepithel og er derfor et ideelt podningssted til vurdering af eksogencellernes angiogene potentiale. Derudover tillader brystfedtpuden at de dannede eksogene beholdere integreres med værtscirkulationssystemet, hvilket muliggør yderligere fUnctional evaluering og repræsenterer en fordel i forhold til subkutan transplantation. Selvom in vitro- dyrkning og transplantationsassays er som effektiv måde at undersøge regenereringsegenskaberne for en cellepopulation, er det kendt, at sådanne analyser kan stimulere plasticitet, da celler tages væk fra deres native habitater, og ændringer kan induceres, når cellerne afbrydes fra Deres fysiologiske omgivelser 7 . Derfor er opnåelse af direkte in vivo beviser for celleforebyggelse nøglen til at fremme den nuværende forståelse af adfærden hos endotelgrupper. Genetisk skæbneplanlægning ( dvs. in vivo lineage-sporing) er afgørende for identifikation af VESC&#39;er og til undersøgelse af deres egenskaber i kroppens system, da det kan afsløre in vivo stamcelleadfærd i sin fysiologiske kontekst, og den egentlige stilhed kan være vurderet. Lineage traciNg giver direkte bevis for den langsigtede persistens ( dvs. selvfornyelse) af kandidat VESC&#39;er og deres evne til at fremstille celletyper til oprindelsesvævet ( dvs. differentieringspotentiale). Denne protokol beskriver en ny brystfedtpude-transplantationsteknik og en linjesporingsmetode til observering af VESC&#39;ernes vådegenerationsevne. Disse teknikker overvinder mangler ved aktuelt tilgængelige analyser og tilvejebringer en ny måde at optimalt evaluere stamcelleegenskaber af VESC&#39;er. Disse tilgange er effektive værktøjer, der kan anvendes til at vurdere endotelpopulationers adfærd og fartøjdannende egenskaber såvel som at bestemme ændring af vaskulær cellepotentiale i et patologisk miljø.

<table border="0" cellpadding="0" cellspacing="0" width="1265">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:407px;">0.05% Trypsin-Ethylene Diamine Tetraacetic Acid(EDTA) (1X)</td>
			<td style="width:263px;">Gibco (Life Technologies)</td>
			<td style="width:208px;">25300-062</td>
			<td style="width:387px;">0.05% Trypsin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22 &#181;m Filter Unit</td>
			<td>Merck Millipore Ltd.</td>
			<td>SLGP033RB</td>
			<td>0.22 &#181;m Filter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methylbutanol-2, ReagentPlus</td>
			<td>Sigma-Aldrich</td>
			<td>24, 048-6</td>
			<td>Tert Amyl Alcohol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">222, Tribromethanol</td>
			<td>Sigma-Aldrich</td>
			<td>T48402-25g</td>
			<td>222, Tribromethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4&#39;,6-diamidino-2-phenylindole(DAPI)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m Cell Strainer</td>
			<td>BD FAL</td>
			<td>352350</td>
			<td>70 &#181;m Cell Strainer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adhesion Microscope Slides</td>
			<td>CITOGLAS</td>
			<td>188105</td>
			<td>Glass slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD105 APC</td>
			<td>eBioscience</td>
			<td>17-1051-82, clone MJ7/18</td>
			<td>for FACS analusis use at 1 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD201 Biotin</td>
			<td>eBioscience</td>
			<td>13-2012-82</td>
			<td>for FACS analusis use at 2.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse CD31 PE-Cyanine 7</td>
			<td>eBioscience</td>
			<td>25-0311-82, clone 390</td>
			<td>for FACS analusis use at 1 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD FACSJazz Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>655489</td>
			<td>FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>100190332</td>
			<td>BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Centrifuge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type 3</td>
			<td>Worthington-Biochem</td>
			<td>#LS004183</td>
			<td>Collagenase III</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscopy</td>
			<td>Leica</td>
			<td>sp8</td>
			<td>Confocal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deoxyribonuclease I from bovine pancreas</td>
			<td>Sigma</td>
			<td>D2463-5VL</td>
			<td>Dnase I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey anti-Rat Cy3</td>
			<td>Jackson ImmunResearch</td>
			<td>712-165-150</td>
			<td>Secondary antibody, use at 0.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps</td>
			<td>WPI</td>
			<td>500342</td>
			<td>Froceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Forceps - Micro-Blunted Tips</td>
			<td>FST</td>
			<td>11253-20</td>
			<td style="width:387px;">Forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FITC Ms CD3/Gr1/CD11b/CD45R(B220)/Ter-119</td>
			<td>BioLegend</td>
			<td>78022</td>
			<td>Blood lineage cocktail for FACS analysis, use at 25 &#181;l per test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma</td>
			<td>G6279</td>
			<td>Glycerol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscov&#39;s Modified Dulbecco&#39;s Medium</td>
			<td>Gibco (Life Technologies)</td>
			<td>12440-053</td>
			<td>IMDM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isolectin GS-IB4 from Griffonia Simplicifolia, Alexa Fluora&#160; 647</td>
			<td>Invitrogen</td>
			<td>Z32450</td>
			<td>Isolectin-647</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel&#160; Matrix (growth factor reduced)</td>
			<td>BD</td>
			<td>356231</td>
			<td>Matrigel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain (Actin-GFP)</td>
			<td>Jax Laboratories</td>
			<td>3773</td>
			<td>Actin-GFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain (C57BL/6)</td>
			<td>SLAC</td>
			<td>C57BL/6</td>
			<td>C57BL/6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse strain&#160; (BALB/c nude)</td>
			<td>SLAC</td>
			<td>BALB/c nude</td>
			<td>Nude mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>PFA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicilin Streptomycin</td>
			<td>Gibco (Life Technologies)</td>
			<td>5140-122</td>
			<td>Pen/Strep</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (pH7.2) 1X</td>
			<td>Gibco (Life Technologies)</td>
			<td>c20012500BT</td>
			<td>PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PRIM1640</td>
			<td>Gibco (Life Technologies)</td>
			<td>c11875500CP</td>
			<td>PRIM1640</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold antifade reagent</td>
			<td>Life Technologies</td>
			<td>P36934</td>
			<td>Mounting Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti CD144</td>
			<td>BD Biosciences</td>
			<td>550548</td>
			<td>for whole-mount analysis, anti-VE-Cadherin / Cdh5 antibdy for endothelial tight junction, use at 2.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti CD31-biotin</td>
			<td>BD Biosciences</td>
			<td>553371</td>
			<td>for whole-mount analusis, anti-CD31/PECAM1 antibody for endothelial surface adhesion molecule, use at 10 ug/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Red Cell Lysis Buffer</td>
			<td>Sigma</td>
			<td>R7767-100ML</td>
			<td>Red blood cell lysing buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Straight/Sharp-Blunt/10cm</td>
			<td>FST</td>
			<td>14028-10</td>
			<td>Fine Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin eFluor 450</td>
			<td>eBioscience</td>
			<td>48-4317-82</td>
			<td>for FACS analysis use at 0.5 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tamoxifen</td>
			<td>Sigma</td>
			<td>101551374</td>
			<td>TAM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787-250ML</td>
			<td>TritonX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wax Coated Braided Silk (Size 5-0 USP (1 Metric), 18 inches (45 cm) BLACK on C-1 Needle)</td>
			<td>COVIDIEN</td>
			<td>S1173</td>
			<td>Suture&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Disposable Scaplels</td>
			<td>Swann Morton</td>
			<td>#10</td>
			<td>Scalpel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Betadine</td>
			<td>Yifeng Medical</td>
			<td align="right">20160101</td>
			<td>Antiseptic solution</td>
		</tr>
	</tbody>
</table>

a novel mammary fat pad transplantation technique to visualize the vessel generation of vascular endothelial stem cells

Endotelceller (EC&#39;er) er de grundlæggende byggesten i den vaskulære arkitektur og formidler vaskulær vækst og remodeling for at sikre ordentlig fartøjsudvikling og homeostase. Undersøgelser om endotel-lineær hierarki forbliver imidlertid ubehagelige på grund af manglende værktøjer til at få adgang såvel som direkte at evaluere deres adfærd in vivo . For at afhjælpe denne mangel er der udviklet en ny vævsmodel til undersøgelse af angiogenese ved anvendelse af brystfedtpuden. Brystkirtlen udvikler sig hovedsageligt i postnatale stadier, herunder pubertet og graviditet, hvorigennem robust epithelsproliferation ledsages af omfattende vaskulær remodeling. Mammedriktspads giver plads, matrix og rige angiogene stimuli fra det voksende brystepitel. Desuden er brystfedtpuder placeret uden for peritonealhulrummet, hvilket gør dem til et let tilgængeligt podningssted til vurdering af eksogene cellers angiogene potentiale. Dette arbejde beskriver også en effektivitetNt-sporingstilgang ved anvendelse af fluorescerende reportermus for specifikt at mærke den målrettede population af vaskulære endotelceller (VESC&#39;er) in vivo . Denne linjesporingsmetode kombineret med efterfølgende fuldmikroskopi af væv muliggør direkte visualisering af målrettede celler og deres efterkommere, hvorved proliferationskapaciteten kan kvantificeres, og differentieringsforpligtelsen kan være skæbneplaneret. Ved anvendelse af disse metoder er en population af bipotent protein C-receptor (Procr), der udtrykker VESC&#39;er, for nylig blevet identificeret i flere vaskulære systemer. Procr + VESC&#39;er, der giver anledning til både nye EC&#39;er og pericytter, bidrager aktivt til angiogenese under udvikling, homeostase og skadereparation. Samlet set beskriver dette manuskript en ny brystfedtpudstransplantation og in vivo lineage-sporingsteknikker, der kan bruges til at evaluere stamcelleegenskaber af VESC&#39;er.

Mammary VESCs Isolation: For at isolere endotel-populationen for den deraf følgende transplantationsassay blev modne (8 uger gamle) høstede mavekirtler høstet som donormateriale. Endotelceller blev isoleret under anvendelse af en antistofbaseret cellesorteringsteknik. Repræsentative plots af FACS-analysen af ​​VESC-populationen i 8-ugers gamle C57BL / 6-brystkirtlen EC&#39;er er vist i Figur 1</strong...

Watch this Scientific Journal Video about A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells at JoVE.com

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Dette arbejde demonstrerer en ny tilgang til vurdering af proliferation, differentiering og fartøjsdannende potentiale af vaskulære endotelceller (VESC&#39;er) gennem brystfedtpudstransplantation efterfulgt af helbårne vævsblandinger til mikroskopisk observation. En linjesporingstrategi til undersøgelse af VESCs adfærd in vivo præsenteres også.

En ny mammedisk fedtpudstransplantationsteknik til visualisering af fartøjsgenerering af vaskulære endotelceller

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Research

JoVE Journal

Developmental Biology

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An Efficient Method to Obtain Dedifferentiated Fat Cells

En effektiv metode til at opnå dedifferentierede fedtceller

Tissue engineering and cell therapy hold great promise clinically. In this regard, multipotent cells, such as mesenchymal stem cells (MSCs), may be used ...

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

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Generation af 3D hud Organoid fra ledningen blod-afledte induceret pluripotente stamceller

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The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

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