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. Il trattamento di cardiache umane del campione <ol><li> Assicurarsi che tutti i fluidi, contenitori, strumenti, e la zona operativa dedicata sono sterili. </li><li> Posizionare il campione di tessuto cardiaco (ottenuta con la banca dei tessuti o all&#39;équipe chirurgica) nel supporto di memorizzazione composto medio refrigerata di Dulbecco modificato Eagle (DMEM) contenente 20% di siero fetale bovino (FBS) e 1% di penicillina-streptomicina (P / S) sul ghiaccio per il trasporto 3. </li><li> Rimuovere il...

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Una crescente evidenza supporta una limitata capacità rigenerativa del cuore umano adulto dopo l&#39;infortunio. Identificazione e caratterizzazione di cellule precursori native responsabili di tali risposte rigenerativa nei cuori feriti sono fondamentali sia per la comprensione dei meccanismi associati e vie di segnalazione e lo sviluppo di approcci di utilizzare queste cellule terapeuticamente. Protocolli precedenti hanno descritto l&#39;isolamento di sottoinsiemi di cellule precursori pe...

Il cuore è stato a lungo considerato un organo post-mitotico. Tuttavia, studi recenti hanno dimostrato la presenza di fatturato cardiomiociti limitata nel cuore degli uomini adulti 1. Cellule staminali / progenitrici nativa con cardiomiociti potenziale di differenziazione sono stati individuati all&#39;interno del miocardio in roditori adulti e cuori umani, tra cui Sca-1 +, c-kit +, Cardiosphere di formazione, e, più recentemente, le cellule precursori perivascolari 2,3. Queste cellule rappresentano candidati interessanti per le terapie volte a migliorare cardiaco riparazione / rigenerazione attraverso il trapianto di cellule o la stimolazione della proliferazione in situ. Staminali mesenchimali / cellule stromali (MSC) sono stati isolati da quasi tutti i tessuti umani 4,5 Studi clinici delle applicazioni terapeutiche di MSC sono state effettuate per molteplici condizioni patologiche quali la riparazione cardiovascolare 6, graft-versus-host-disease 7 </sup&gt;, E cirrosi epatica 8. Effetti benefici sono stati attribuiti alla capacità delle cellule staminali mesenchimali per: a casa per i siti di infiammazione 9; differenziarsi in diversi tipi cellulari 10; secernono molecole pro-riparativi 11; e modulare risposta immunitaria 12. L&#39;isolamento di cellule staminali mesenchimali ha tradizionalmente fatto affidamento su loro adesione preferenziale ai substrati plastici. Tuttavia, la popolazione di cellule risultante è tipicamente marcatamente eterogenea 13. Usando fluorescenza delle cellule attivate (FACS) con una combinazione di marcatori chiave di cellule perivascolari, siamo stati in grado di isolare e purificare un multipotenti MSC-come precursore della popolazione (CD146 + / CD31 - / CD34 - / CD45 - / CD56 -) da molteplici tessuti umani, tra cui il muscolo scheletrico adulto e grasso bianco 14. popolazioni di cellule perivascolari in vari tessuti non-cardiaci hanno dimostrato di avere proprietà staminali / progenitrici a cellulariND sono indagati per uso clinico in ambito cardiovascolare. Periciti, uno dei più noti sottopopolazioni di cellule perivascolari, sono una popolazione eterogenea che svolgono diversi ruoli fisiopatologici compresa nello sviluppo di nuovi vasi 15, la regolazione della pressione sanguigna 16, e il mantenimento dell&#39;integrità vascolare 17,18. Come mostrato in diversi tessuti, specifici sottoinsiemi di periciti nativamente esprimono antigeni MSC e sostenere le loro fenotipi MSC-come in coltura primaria dopo FACS purificazione 14. Inoltre, queste cellule mantengono stabilmente i loro fenotipi a lungo termine all&#39;interno della cultura e mostrano multi-lignaggio potenziale di differenziazione, simile a MSC 19,20. Questi risultati suggeriscono che periciti sono una delle origini della MSC sfuggente 14. Il potenziale terapeutico dei periciti è stata dimostrata con una riduzione cicatrici miocardica e migliorata funzione cardiaca dopo trapianto in ischemically feriticuori 21. Recentemente, abbiamo purificati con successo periciti dal miocardio umano e dimostrato la loro fenotipi MSC-simili e multipotenza (adipogenesi, Condrogenesi e osteogenesi) con l&#39;assenza di miogenesi scheletrico 3. Inoltre, periciti miocardio esposti differenziali capacità potenziali e angiogenici cardiomyogenic se confrontato con le controparti purificati da altri organi. Una seconda popolazione di cellule staminali / progenitrici multipotenti perivascolari, la cellula avventiziale, è stato isolato da safene umane sulla base di espressione CD34 positivo 22. Cellule avventiziale venose hanno dimostrato di avere un potenziale clonogenico, la capacità di differenziazione mesoderma e potenziale proangiogenico in vitro. Il trapianto di queste cellule nei cuori ischemically feriti di topi ha comportato una riduzione della fibrosi interstiziale, un aumento angiogenesi e flusso miocardico, ridotta dil ventricolarezione, e l&#39;aumento di eiezione cardiaca frazione 23. È interessante notare che le cellule adipose avventiziale hanno dimostrato di perdere l&#39;espressione di CD34 e CD146 upregulate espressione di cultura in risposta al trattamento angiopoietin II, suggerendo l&#39;adozione di un fenotipo pericyte con la stimolazione 24. All&#39;interno del cuore, tuttavia, la popolazione di cellule avventiziale non è ancora stato prospetticamente purificato mediante FACS e / o ben caratterizzato. Utilizzando le procedure di isolamento delle cellule descritte nelle sezioni seguenti, stiamo caratterizzando cellule miocardiche avventiziale e indagare il loro potenziale per applicazioni rigenerative. Qui si descrive un metodo per isolare e purificare due sottopopolazioni di cellule staminali / progenitrici perivascolari da miocardio fetale o umano adulto. Questo metodo di isolamento delle cellule prospettico permetterà ai ricercatori di ottenere isogenici sottoinsiemi di cellule staminali / progenitrici perivascolari da biopsie cardiache umane per gli studi comparativi e further esplorare il loro potenziale terapeutico in varie condizioni patologiche cardiache.

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	<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>
</table>

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.

singole cellule si distinguevano da detriti e doppiette sulla base delle distribuzioni in avanti e scatter laterale. cellule vive sono stati identificati per la loro incapacità di prendere il colorante DAPI. La strategia di gating è stato scelto sulla base di etichettatura controllo isotipico di questo live, dissociazione whole-cell cardiaca (Figura 1). Dalle cellule vive, cellule CD45 + sono stati gated, seguito da CD56 + cellule. Cellule endotel...

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

tessuto cardiaco umano alberga popolazioni di cellule precursore perivascolari multipotenti che possono essere adatti per la rigenerazione del miocardio. La tecnica qui descritta permette l&#39;isolamento e la purificazione simultanea di due popolazioni di cellule stromali multipotenti associati con i vasi sanguigni nativi, cioè CD146 + CD34 - periciti e CD34 + CD146 - cellule avventiziale, dal miocardio umano.

Isolamento di perivascolare Multipotent precursori delle cellule popolazioni dal cardiache umane 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

Isolamento e quantitativa immunocitochimica caratterizzazione di cellule miogeniche primarie e fibroblasti da muscolo scheletrico umano

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

Ricerca

Biologia dello sviluppo

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Saggio popolazioni cellulari del sangue<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

Istituzione di Genome-cura pluripotenti umane Linee di cellule staminali: Da Targeting per isolamento

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

Avvio di differenziazione in cellule multipotenti immortalizzate Otic progenitrici

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

Isolamento e l&#39;espansione delle staminali mesenchimali stromali derivate / cellule da placenta umana 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

L&#39;espansione e l&#39;adipogenesi dell&#39;induzione di progenitori adipociti da tessuto adiposo perivascolare isolati da cellule magnetiche attivate

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

Analisi quantitativa intero-monta immunofluorescenza delle popolazioni di cellule progenitrici cardiache in embrioni di topo

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

Rilevamento rapido dei fenotipi di Neurodevelopmental in cellule precursori neurali umane (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

Un metodo semplificato ed efficiente per isolare i Keratinocytes umani primari dal tessuto pelle adulta

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

Isolamento di popolazioni di neuroni specifici da nematode Caenorhabditis elegans

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