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Biologia do Desenvolvimento

Epikardial Udvækst Kultur Assay og<em> Ex vivo</em> Vurdering af Epikardial-afledt cellemigration

Published: March 18, 2016
doi:
10.3791/53750
, ,
1Aab Cardiovascular Research Institute,University of Rochester School of Medicine and Dentistry, 2Department of Medicine,University of Rochester School of Medicine and Dentistry, 3Department of Pharmacology and Physiology,University of Rochester School of Medicine and Dentistry

Summary

Here, we describe methods for isolating primary mouse epicardial cells by an outgrowth culture assay and assessing the functional migration of epicardial-derived cells (EPDC) using an ex vivo heart culture system. These protocols are suitable for identifying genetic and chemical modulators of epicardial epithelial-to-mesenchymal transition (EMT) and motility.

Abstract

Et enkelt lag af epikardiale cellelinier hjertet, hvilket giver parakrine faktorer, der stimulerer cardiomyocyte proliferation og bidrager direkte kardiovaskulære progenitorer under udviklingen og sygdom. Mens en række faktorer har været impliceret i epikardiet-afledt celle (EPDC) mobilisering, er de mekanismer, der styrer deres efterfølgende migrering og differentiering dårligt forstået. Her præsenterer vi in vitro og ex vivo strategier til at studere EPDC motilitet og differentiering. Først beskriver vi en metode til at opnå primære epikardielle celler ved udvækst kultur fra den embryonale mus hjerte. Vi indfører også en detaljeret protokol til at vurdere tredimensionale migration af mærket EPDC i et organ kultursystem. Vi leverer beviser ved hjælp af disse teknikker, som genetisk sletning af myocardin-relaterede transkriptionsfaktorer i epikardiet dæmper EPDC migration. Denne fremgangsmåde fungerer som en platform til at vurdere kandidat modifikatorer af EPDCbiologi og kunne anvendes til at udvikle genetiske eller kemiske skærme for at identificere hidtil ukendte regulatorer af EPDC mobilisering der kan være nyttige til hjerte- reparation.

Introduction

Epicardiet er et enkelt lag af mesotelceller at linjer hjerte og påvirkninger cardiac udvikling, modning og reparation. Gennem en meget koordineret udveksling af parakrine signaler, den intime dialog mellem myocardium og epicardium er uundværlig for cardiac vækst og dannelse af ikke-myocyt kardiale afstamninger 1. Epikardielle-afledte celler (EPDC) frem fra en undergruppe af epikardiale celler gennem en epitelial-til-mesenchymal overgang (EMT) 2, invaderer subepicardial rum og underliggende myocardium, og differentiere i vid udstrækning i koronare vaskulære mural celler og kardiale fibroblaster, og til en mindre omfang, endotelceller og cardiomyocytter 3-9.

De mekanismer, der regulerer epikardial EMT og EPDC invasion er blevet tilskrevet den koordineret indsats af forskellige molekyler, herunder udskilles ligander 10-13, celleoverfladereceptorer og adhæsionsmolekyler 14,15, regulatorer af apikale-basal polaritet 16,17, små GTPaser 18,19, og transkriptionsfaktorer 2,20,21. Mens mange af de molekylære effektorer af EPDC migration er blevet defineret, at en bedre forståelse af de fysiologiske signaler, der stimulerer EPDC mobilisering i fosteret kan fremskynde udviklingen af ​​strategier manipulere denne proces i den voksne for forbedret hjertefunktion reparation.

Undersøgelser til formål at identificere nye regulatorer af EPDC mobilisering stole på rensning af denne celle population eller opsporing migrationen af ​​individuelle epikardielle celler. En eller en kombination af markørgener, herunder WT1, Tcf21, Tbx18, og / eller Aldh1a2, er almindeligt anvendt til at identificere fostrets epicardium 1. Men anvendelsen af ​​disse markører spore migrerende EPDC ikke er optimal som udtryk for epikardiale markører aftager i løbet af hjerte udvikling og er ofte tabt, når epikardiale celler undergår transdifrentiering i mesenchymale celler.

Anvendelsen af Cre / loxP-system, sammen med slægt-tracing journalister, har været nyttig i permanent mærkning af epicardium og dens efterkommere under hjerte udvikling og efter iskæmisk skade i den voksne 7,22,23. Adskillige epikardielle-begrænset Cre linjer er blevet genereret og er almindeligt anvendt til at mærke EPDC og for betinget gendeletion strategier 1. Disse undersøgelser har ført til karakteriseringen af ​​forskellige epikardielle slægter, og identifikationen af ​​kritiske formidlere af EPDC motilitet og differentiering. Men akkumulere tyder også, epicardiet er en heterogen population af progenitorceller 4,24,25. Således vil kun en delmængde af epikardiale celler målrettes under anvendelse af en given Cre driver.

For at mærke hele epicardium uanset Cre specificitet, har en række laboratorier udnyttet udvækst cultidige systemer eller ex vivo organ dyrkningsmetoder til trofast isolere eller mærke epikardielle celler uden afhængigt af ekspressionen af et genetisk afstamning markører 26-28. For ex vivo studier migration, er embryonale hjerter isoleret før epikardial EMT og dyrket i medium suppleret med et grønt fluorescerende protein (GFP) udtrykkende adenovirus (Ad / GFP) 9,18. Denne fremgangsmåde giver mulighed for effektiv mærkning af hele epicardium snarere end en undergruppe af celler ved Cre-medieret rekombination. Heart kulturer efterfølgende eksponering for kendte induktorer af EMT at stimulere mobilisering af EPDC 28,29. Ex vivo og in vivo analyser suppleres af in vitro epikardielle eksplantatkulturer, som er en særlig nyttig metode til at udforske detaljerede mekanismer kørsel EPDC migration.

Her beskriver vi metoder til isolering primære epikardielle celler til in vitro-undersøgelser af epicardial EMT, samt et organ dyrkningssystem til ex vivo-analyser af EPDC motilitet. Vi har for nylig demonstreret nytten og robustheden af denne metode ved genetisk modulere myocardin-relaterede transskription faktor (MRTF) / serum respons faktor (SRF) signalering akse til at manipulere actin-baserede migration af EPDC 9. Mens vores resultater fremhæve en signalvej nødvendig til regulering EPDC migration, disse metoder er egnede til optrævling de mekanismer, der kollektivt orkestrerer EPDC migration og differentiering. Endvidere kunne eksplantatet og ex vivo dyrkningssystemer implementeres i funktionelle skærme at identificere nye regulatorer af EPDC mobilisering til terapeutiske anvendelser i hjerte-regenerering.

Protocol

BEMÆRK: Alle eksperimenter med mus blev godkendt af University udvalg for Animal Resources ved University of Rochester. 1. Epikardial Udvækst Culture Assay (figur 1A) Forberedelser Forbered 5 ml medium A til primær epikardial celleisolering ved supplering Medium 199 (M199) med 5% føtalt bovint serum (FBS) og 1% penicillin / streptomycin (Pen / Strep). Pre-varm medium A og 100 ml Hanks 'Balanced Salt Solution (HBSS) i en 37 ° C vandbad. Tillad collagencoatede kamme…

Representative Results

Den epicardium effektivt kan isoleres ved hjælp af en udvækst kultur assay ved at udnytte dens ydre placering og udnytte den udviklingsmæssige plasticitet og iboende vandrende adfærd EPDC. Murine embryonale hjerte isoleres ved E11.5 før epikardial EMT og dyrkes dorsale side ned på collagen-coatede kammerskiver 26 (figur 1A, B). Eksplanterede hjerter vil fortsætte med at trække sig sammen; dog vil epicardiet holde sig til collagen matrix og migrerer ud …

Discussion

Her har vi skitsere detaljerede metoder til isolering primære epikardial celle ved udvækst og spore EPDC migration i ex vivo hjerte kulturer. For udvækst kulturer, et passende tidspunkt til at fjerne epikardiet-udtømte hjerte varierer lidt mellem eksperimenter. Periodisk monitorering af eksplantaterne efter inkubering natten anbefales at måle omfanget af epicardiale udvækst og at uddrage hjertet før fibroblaster vises. For at sikre renhed, bør hjerter fjernes inden 24 timer af eksplantering at forhindre…

Declarações

The authors have nothing to disclose.

Acknowledgements

EMS blev støttet af tilskud fra National Institutes of Health (NIH) [tilskud nummer R01HL120919]; American Heart Association [tilskud nummer 10SDG4350046] den; University of Rochester CTSA pris fra NIH [tilskud nummer UL1 TR000042]; og start midler fra Aab CVRI. MAT blev delvist understøttet af et tilskud til University of Rochester School of Medicine og Dentistry fra Howard Hughes Medical Institute Med-Ind-Grad initiativet; og en Institutional Ruth L. Kirschstein National Research Service Award fra NIH [tilskud nummer GM068411].

Materials

Dulbecco's Modified Eagle Medium Hyclone  SH3002201 DMEM
Hanks' Balanced Salt Solution Hyclone SH3003102 HBSS
Medium 199 Hyclone SH3025301 M199
Dulbecco’s Phosphate-Buffered Saline  Hyclone SH3002802 DPBS
Fetal Bovine Serum  Gemini  100-106 FBS
Penicillin/Streptomycin Solution Hyclone SV30010
Corning Biocoat Collagen I, 4-Well Culture Slides Corning 354557
Multiwell 24-well plates Falcon 08-772-1
Dissecting microscope Leica M50 Equipped with Leica KL300 LED might source
Confocal miscroscope Olympus 1X81 Equipped with muli-argon laser 405/488/515, FV10-MCPSU laser 405/440/635, 559 laser, and mercury lamp
Bioruptor Diagenode  UCD-200
Transforming growth factor beta 1 R&D Systems 100-B-001 TGF-β1
Platelet-derived growth factor BB R&D Systems 220-BB-010 PDGF-BB
Trizol Reagent Applied Biosystems 15596-026 Caution, hazardous material
TURBO DNA-free Kit Life Technologies AM1907 DNaseI
iScript cDNA Synthesis Kit BioRad 1708891
UltraPure Glycogen Life Technologies 10814-010
SYBR Green BioRad 170-8880
Protease inhibtor cocktail tablets Roche 11836170001
Alexa Goat-anti-rabbit 488 Life Technologies A11008 Secondary antibody
Alexa Goat anti-rabbit 594 Life Technologies A-11012 Secondary antibody
Alexa Goat anti-mouse 594 Life Technologies A-11005 Secondary antibody
Mouse anti-smooth muscle alpha actin, Cy3-conjugated  Sigma-Aldrich C6198 monoclonal antibody, clone 1A4
Mouse anti-Wilms tumor 1 (WT1) Life Technologies MA1-46028 monoclonal antibody, clone 6F-H2
Rabbit anti-ZO1 Life Technologies 40-2200 polyclonal antibody
Rabbit anti-Collagen Type 4 Millipore AB756P polyclonal antibody
4',6-Diamidino-2-Phenylindole, Dihydrochloride  Life Technologies D1306 DAPI
Fluorescent mounting medium  DAKO S3023
16% Paraformaldehyde solution Electron Microscopy Sciences 15710 PFA diluted to 4% in DPBS. Caution, hazardous material
Sucrose Sigma-Aldrich S0389
Tissue Freezing Medium Triangle Biomedical Sciences TFM-B
Pap pen DAKO S2002
Disposable mictrotome blades  Sakura Finetek 4689
Microscope slides Globe Scientific 1358W positively charged, 25 x 75 x 1mm
Cryostat  Leica CM1950
Forceps Fine Science Tools 11295-00
Surgical Scissors Fine Science Tools 91460-11
Disposable base molds Fisher Scientific 22-363-553
Ketamine Hospira NDC 0409-2051-05
Xylazine Akorn NADA 139-236
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Tags

Epicardial OutgrowthEpicardial-derived Cell MigrationEx Vivo AssessmentPrimary Epicardial Cell CultureEpithelial-to-mesenchymal TransitionCardiac Regenerative MedicineCollagen-coated Chamber SlidesMouse EmbryoExplant MediumMesothelial CellsMesenchymal Cell ContaminationTrizol

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Trembley, M. A., Velasquez, L. S., Small, E. M. Epicardial Outgrowth Culture Assay and Ex Vivo Assessment of Epicardial-derived Cell Migration. J. Vis. Exp. (109), e53750, doi:10.3791/53750 (2016).
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