Name: isolation of perivascular multipotent precursor cell populations from human cardiac tissue
Uploaded: 2016-10-08T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue at JoVE.com

The authors wish to thank Shonna Johnston, Claire Cryer, Fiona Rossi and Will Ramsay at the University of Edinburgh and Alison Logar and Megan Blanchard at the University of Pittsburgh for their expert assistance with flow cytometry. We also wish to thank Anne Saunderson and Lindsay Mock for their help with obtaining human tissues. Human adult and fetal heart tissue samples were procured with full ethics permission of the NHS Scotland Tayside Committee on Medical Research Ethics and the NHS Lothian Research Ethics Committee (REC08/S1101/1) respectively. This work was supported by grants from the Medical Research Council (BP), British Heart Foundation (BP), Commonwealth of Pennsylvania (BP), Children's Hospital of Pittsburgh (BP), National Institute of Health R01AR49684 (JH) and R21HL083057 (BP), and the Henry J. Mankin Endowed Chair at University of Pittsburgh (JH). JEB was supported by a British Heart Foundation Centre of Research Excellence doctoral training award (RE/08/001/23904). WC was supported in part by an American Heart Association predoctoral fellowship (11PRE7490001).

1. Processing of Human Cardiac Sample <ol><li> Påse at alle væsker, containere, instrumenter, og den dedikerte operasjonsområdet er sterile. </li><li> Plasser hjertevevet prøven (innhentes av vev bank eller kirurgisk lag) i lagringsmediet består av kjølt Dulbeccos modifiserte Eagle medium (DMEM) inneholdende 20% føtalt bovint serum (FBS) og 1% penicillin-streptomycin (P / S) på is for transport 3. </li><li> Fjern det kardiale prøven fra lagringsmediet og vaskes med et vaskemedium bestående av fosf...

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+contribution+of+pericytes+to+angiogenic+sprouting+and+tube+formation.">Early contribution of pericytes to angiogenic sprouting and tube formation.</a> Angiogenesis. 6 (3), 241-249 (2003).</li><li>Rucker, H. K., Wynder, H. J., Thomas, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+CNS+pericytes.">Cellular mechanisms of CNS pericytes.</a> Brain research bulletin. 51 (5), 363-369 (2000).</li><li>Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insight+into+the+physiological+functions+of+PDGF+through+genetic+studies+in+mice.">Insight into the physiological functions of PDGF through genetic studies in mice.</a> Cytokine &amp; Growth Factor Reviews. 15 (4), 215-228 (2004).</li><li>Gerhardt, H., Betsholtz, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-pericyte+interactions+in+angiogenesis.">Endothelial-pericyte interactions in angiogenesis.</a> Cell and tissue research. 314 (1), 15-23 (2003).</li><li>Crisan, M., Chen, C. W., Corselli, M., Andriolo, G., Lazzari, L., P&#233;ault, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+multipotent+progenitor+cells+in+human+organs.">Perivascular multipotent progenitor cells in human organs.</a> Annals of the New York Academy of Sciences. 1176, 118-123 (2009).</li><li>Kang, S. G., 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=Isolation+and+perivascular+localization+of+mesenchymal+stem+cells+from+mouse+brain.">Isolation and perivascular localization of mesenchymal stem cells from mouse brain.</a> Neurosurgery. 67 (3), 711-720 (2010).</li><li>Chen, C. W., 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=Human+pericytes+for+ischemic+heart+repair.">Human pericytes for ischemic heart repair.</a> Stem cells (Dayton, Ohio). 31 (2), 305-316 (2013).</li><li>Campagnolo, P., 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=Human+adult+vena+saphena+contains+perivascular+progenitor+cells+endowed+with+clonogenic+and+proangiogenic+potential.">Human adult vena saphena contains perivascular progenitor cells endowed with clonogenic and proangiogenic potential.</a> Circulation. 121 (15), 1735-1745 (2010).</li><li>Katare, R., 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=Transplantation+of+human+pericyte+progenitor+cells+improves+the+repair+of+infarcted+heart+through+activation+of+an+angiogenic+program+involving+micro-RNA-132.">Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132.</a> Circulation research. 109 (8), 894-906 (2011).</li><li>Corselli, M., Chen, C. W., Sun, B., Yap, S., Rubin, J. P., P&#233;ault, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tunica+Adventitia+of+Human+Arteries+and+Veins+As+a+Source+of+Mesenchymal+Stem+Cells.">The Tunica Adventitia of Human Arteries and Veins As a Source of Mesenchymal Stem Cells.</a> Stem Cells and Development. 21 (8), 1299-1308 (2012).</li><li>Crisan, M., 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+long-term+culture+of+multipotent+progenitor+cells+affiliated+with+the+walls+of+human+blood+vessels:+myoendothelial+cells+and+pericytes.">Purification and long-term culture of multipotent progenitor cells affiliated with the walls of human blood vessels: myoendothelial cells and pericytes.</a> Methods in cell biology. 86, 295-309 (2008).</li></ol>

Økende bevis støtter en begrenset evne til reproduksjon av den voksne menneskehjertet etter skade. Identifisering og karakterisering av innfødte forloperceller ansvarlige for slike regenerativ respons i skadde hjerter er avgjørende for både forståelsen av tilhørende mekanismer og signalveier og utvikling av metoder for å utnytte disse cellene terapeutisk. Tidligere protokoller er beskrevet isoleringen av perivaskulære forløper celleundergrupper fra human skjelettmuskel 25.</su...

Hjertet har lenge vært betraktet som en post-mitotisk organ. Imidlertid har nyere studier vist tilstedeværelse av begrenset kardiomyocytt omsetningen hos voksne menneskers hjerter 1. Native stilk / stamceller med kardiomyocytt differensiering potensial har også blitt identifisert innen hjertemuskelen i voksen gnager og menneskelige hjerter, inkludert Sca-1 +, c-kit +, cardiosphere dannende, og sist, perivaskulær forloperceller 2,3. Disse cellene representerer attraktive kandidater for behandling med sikte på å styrke hjerte reparasjon / regenerering gjennom celletransplantasjon eller stimulering av in-situ spredning. Mesenchymale stilk / stromaceller (MSC) har blitt isolert fra nesten alle humant vev 4,5 Kliniske studier av de terapeutiske anvendelser av MSC har blitt utført for flere patologiske tilstander slik som kardiovaskulær reparasjon 6, graft-versus-host-sykdom 7 </sup&gt;, Og levercirrhose 8. Gunstige effekter har blitt tilskrevet evnen til MSC til: hjem til områder av betennelse 9; differensiere til forskjellige celletyper 10; utsondre pro-reparerende molekyler 11; og modulere vert immunresponser 12. Isolasjon av MSC har tradisjonelt støttet seg på sin fortrinnsrett tilslutning til plast underlag. Men den resulterende populasjon av celler er typisk markert heterogen 13. Ved å bruke fluorescerende aktivert celle sortering (FACS) med en kombinasjon av sentrale perivaskulær celle markører, har vi vært i stand til å isolere og rense et multipotent MSC-lignende forløper befolkningen (CD146 + / CD31 - / CD34 - / CD45 - / CD56 -) fra flere menneskelig vev, inkludert voksen skjelettmuskulatur og hvitt fett til 14. Perivaskulær celle populasjoner i ulike ikke-kardiale vev har vist seg å ha stilk / stamcelleegenskaper ennd blir undersøkt for klinisk bruk i hjerte-innstillingen. Pericytes, en av de mest kjente perivaskulær celle undergrupper, er en heterogen befolkning som spiller flere patofysiologiske roller inkludert i utviklingen av nye skip 15, regulering av blodtrykk 16, og vedlikehold av vaskulær integritet 17,18. Som vist i flere vev, bestemte undergrupper av pericytes opprinnelig uttrykke MSC antigener og opprettholde sine MSC-lignende fenotyper i primærkultur etter FACS rensing 14. Videre disse cellene stabilt opprettholde sine langsiktige fenotyper innen kultur og vise multi-avstamning differensiering potensial, i likhet med MSC 19,20. Disse resultatene tyder på at pericytes er en av opprinnelsen til den unnvikende MSC 14. Det terapeutiske potensialet for pericytes er vist med en reduksjon i myokardialt arrdannelse og forbedret hjertefunksjon etter transplantasjon inn i ischemisk skadehjerter 21. Nylig har vi lykkes renset pericytes fra menneskehjertemuskelen og demonstrerte sin MSC-lignende fenotyper og multipotency (adipogenesen, chondrogenesis og Osteogensesis) med fravær av skjelett myogenesen tre. I tillegg hjerteinfarkt pericytes utstilt differensial cardiomyogenic potensielle og angiogene kapasitet sammenlignet med kolleger renset fra andre organer. En andre populasjon av multipotente perivaskulære stilk / stamceller, den adventitia-cellen, har blitt isolert fra humane saphenous vener på grunnlag av positiv CD34 ekspresjon 22. Venøs adventitia-celler har vist seg å ha klonogene potensial, mesodermal differensiering kapasitet og proangiogenic potensial in vitro. Transplantasjon av disse cellene inn i ischemisk skadede hjerter mus resulterte i en reduksjon i interstitiell fibrose, en økning i angiogenese og myokardial blodstrøm, redusert ventrikulær dilasjon, og økt hjerte ejeksjonsfraksjon 23. Interessant, har adipose adventitia celler er vist å miste CD34 uttrykk og oppregulere CD146 uttrykk i kulturen som svar på Angiopoietin II behandling, noe som tyder på at innføringen av en pericyte fenotype med stimulering 24. I hjertet, men de adventitia cellepopulasjon har enda ikke blitt prospektivt renset ved FACS og / eller godt karakterisert. Utnytte celleisolasjonsprosedyrer som er beskrevet i de neste avsnittene, er vi for tiden karakter hjerteinfarkt adventitia celler og undersøke deres potensial for fornybare applikasjoner. Heri vi beskriver en metode for å isolere og rense to subpopulasjoner av perivaskulære stammen / stamceller fra human fetal eller voksen myokard. Denne prospektive celle isolasjonsmetoden gjør at forskerne å få isogene perivaskulær stilk / stamcelle undergrupper fra menneskelige hjerte biopsier for komparative studier og further utforske deres terapeutiske potensiale i forskjellige hjerte patologiske tilstander.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AbC Anti-mouse Bead Kit</td>
			<td>Molecular Probes</td>
			<td>A-10344</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase I</td>
			<td>Gibco</td>
			<td>17100-017</td>
			<td rowspan="3">Reconstitute powder as required and filter sterilise</td>
		</tr>
		<tr>
			<td>Collagenase II</td>
			<td>Gibco</td>
			<td>17101-015</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104-019</td>
		</tr>
		<tr>
			<td>anti-human CD34-PE</td>
			<td>BD Pharmingen</td>
			<td>555822</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD45-APC-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557833</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD56-PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557747</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD144-PerCP-Cy5.5</td>
			<td>BD Pharmingen</td>
			<td>561566</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>anti-human CD146-AF647</td>
			<td>AbD Serotec</td>
			<td>MCA2141A647</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>EGM2-BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td>For collection of cells and culture until adhered</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX without sodium pyruvate</td>
			<td>ThermoFischer Scientific</td>
			<td>10566-016</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFischer Scientific</td>
			<td>10500-064</td>
			<td>Freeze in aliquots and keep sterile</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma Aldrich</td>
			<td>G1393</td>
			<td>Dilute with sterile water</td>
		</tr>
		<tr>
			<td>IgG1k-PE</td>
			<td>BD Pharmingen</td>
			<td>559320</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-APC-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557873</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-PE-Cy7</td>
			<td>BD Pharmingen</td>
			<td>557872</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-PerCP-Cy5.5</td>
			<td>BD Pharmingen</td>
			<td>561566</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>IgG1k-647</td>
			<td>AbD Serotec</td>
			<td>MCA1209A647</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Mouse serum</td>
			<td>Sigma Aldrich</td>
			<td>M5905</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Paraffin Film - Parafilm M</td>
			<td>Sigma Aldrich</td>
			<td>P7793</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15979-063</td>
			<td>Freeze in aliquots and keep sterile</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline pH 7.4</td>
			<td>ThermoFischer Scientific</td>
			<td>10010-023</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysing Buffer Hybri-Max</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution</td>
			<td>Sigma Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA 0.5%(10X)</td>
			<td>Invitrogen</td>
			<td>15400-054</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;FACSARIA FUSION</td>
			<td>BD Pharmingen</td>
			<td></td>
			<td>Fluorescence Activated Cell Sorter</td>
		</tr>
	</tbody>
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isolation of perivascular multipotent precursor cell populations from human cardiac tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Enkeltceller ble skilles fra rusk og dubletter på grunnlag av forover og side scatter distribusjoner. Levende celler, ble identifisert ved deres manglende evne til å ta opp fargestoff DAPI. Den gating strategien ble valgt på grunnlag av isotypekontrollantistoff merking av dette live, hel-hjerte celledissosiasjon (figur 1). Fra levende celler, ble CD45 + -celler første silt ut, etterfulgt av CD56 + celler. CD144 + endotelceller ble der...

Watch this Scientific Journal Video about Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue at JoVE.com

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Menneskelig hjertevev havner multipotente perivaskulær forløper cellepopulasjoner som kan være egnet for hjerteinfarkt regenerering. Teknikken som er beskrevet her gjør det mulig for samtidig isolering og rensing av to multipotente stromale cellepopulasjoner forbundet med native blodkar, dvs. CD146 + CD34 - pericytes og CD34 + CD146 - adventitia-celler fra den humane myokardium.

Isolering av perivaskulær multipotente forløpercelle populasjoner fra humane kardiale Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Research

JoVE Journal

Developmental Biology

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Isolasjon og Kvantitativ immunocytokjemisk Karakterisering av Primære myogenic celler og fibroblaster fra Menneskelig Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Analyse av blodcellepopulasjoner i<em&gt; Drosophila melanogaster</em&gt; Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

Establishment of Genome-edited Human Pluripotent Stem Cell Lines: From Targeting to Isolation

Etablering av Genome-redigerte Menneskelig Pluripotent Stem Cell Lines: Fra Targeting å Isolering

Genome-editing of human pluripotent stem cells (hPSCs) provides a genetically controlled and clinically relevant platform from which to understand human ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Initiere Differensiering i udødeligmultipotente Otic stamceller

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolasjon og utvidelse av Mesenchymale Stem / stromal celler avledet fra human placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Expansion and Adipogenesis Induction of Adipocyte Progenitors from Perivascular Adipose Tissue Isolated by Magnetic Activated Cell Sorting

Ekspansjon og adipogeneseinduksjon av adipocytprogenitorer fra perivaskulær adiposevev isolert ved magnetisk aktivert cellesortering

Expansion of Perivascular Adipose Tissue (PVAT), a major regulator of vascular function through paracrine signaling, is directly related to the ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Kvantitativ hele-mount Immunofluorescence analyse av Cardiac stamfar populasjoner i Mouse embryoer

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs)

Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells NPCs

Rask påvisning av Neurodevelopmental fenotyper i menneskelig Neural Precursor celler (NPC)

Human brain development proceeds through a series of precisely orchestrated processes, with earlier stages distinguished by proliferation, migration, and ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

En forenklet og effektiv metode å isolere primære menneskelige keratinocytter fra voksen hud vev

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Isolation of Specific Neuron Populations from Roundworm Caenorhabditis elegans

Isolering av spesifikke Nevron populasjoner fra roundworm Caenorhabditis elegans

During the aging process, many cells accumulate high levels of damage leading to cellular dysfunction, which underlies many geriatric and pathological ...