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>Ensure that all fluids, containers, instruments, and the dedicated operational area are sterile.</li>
	<li>Place the cardiac tissue sample (procured by the tissue bank or surgical team) in storage medium composed of chilled Dulbecco&#39;s modified Eagle medium (DMEM) containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S) on ice for transportation3.</li>
	<li>Remove the cardiac sample from the storage medium and wash with a washing medium c...

<ol>
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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=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>

Increasing evidence supports a limited regenerative capacity of the adult human heart after injury. Identification and characterization of native precursor cells responsible for such regenerative responses in injured hearts are critical for both the understanding of associated mechanisms and signalling pathways and the development of approaches to utilize these cells therapeutically.
Previous protocols have described the isolation of perivascular precursor cell subsets from human skeletal musc...

The heart has long been considered a post-mitotic organ. However, recent studies have demonstrated the presence of limited cardiomyocyte turnover in adult human hearts1. Native stem/progenitor cells with cardiomyocyte differentiation potential have also been identified within the myocardium in adult rodent and human hearts, including Sca-1+, c-kit+, cardiosphere-forming, and most recently, perivascular precursor cells2,3. These cells represent attractive candidates for therapies aimed at enhancing cardiac repair/regeneration through cell transplantation or stimulation of in-situ proliferation.
Mesenchymal stem/stromal cells (MSC) have been isolated from almost every human tissue4,5 Clinical trials of the therapeutic applications of MSC have been carried out for multiple pathological conditions such as cardiovascular repair6, graft-versus-host-disease7, and liver cirrhosis8. Beneficial effects have been attributed to the ability of MSCs to: home to sites of inflammation9; differentiate into different cell types10; secrete pro-reparative molecules11; and modulate host immune responses12. The isolation of MSCs has traditionally relied on their preferential adherence to plastic substrates. However, the resulting population of cells is typically markedly heterogenous13. By using fluorescent activated cell sorting (FACS) with a combination of key perivascular cell markers, we have been able to isolate and purify a multipotent MSC-like precursor population (CD146+/CD31-/CD34-/CD45-/CD56-) from multiple human tissues including adult skeletal muscle and white fat14.
Perivascular cell populations in various non-cardiac tissues have been shown to have stem/progenitor cell properties and are being investigated for clinical use in the cardiovascular setting. Pericytes, one of the most well-known perivascular cell subsets, are a heterogeneous population that play several pathophysiological roles including in the development of new vessels15, the regulation of blood pressure16, and maintenance of vascular integrity17,18. As shown in multiple tissues, specific subsets of pericytes natively express MSC antigens and sustain their MSC-like phenotypes in primary culture after FACS purification14. Moreover, these cells stably maintain their long-term phenotypes within culture and exhibit multi-lineage differentiation potential, similar to MSCs19,20. These results suggest that pericytes are one of the origins of the elusive MSC14. The therapeutic potential of pericytes has been demonstrated with a reduction in myocardial scarring and enhanced cardiac function following transplantation into ischemically injured hearts21. Recently, we successfully purified pericytes from the human myocardium and demonstrated their MSC-like phenotypes and multipotency (adipogenesis, chondrogenesis and osteogenesis) with the absence of skeletal myogenesis3. In addition, myocardial pericytes exhibited differential cardiomyogenic potential and angiogenic capacities when compared with counterparts purified from other organs.
A second population of multipotent perivascular stem/progenitor cells, the adventitial cell, has been isolated from human saphenous veins on the basis of positive CD34 expression22. Venous adventitial cells have been shown to have clonogenic potential, mesodermal differentiation capacity and proangiogenic potential in vitro. Transplantation of these cells into the ischemically injured hearts of mice resulted in a reduction in interstitial fibrosis, an increase in angiogenesis and myocardial blood flow, reduced ventricular dilation, and increased cardiac ejection fraction23. Interestingly, adipose adventitial cells have been shown to lose CD34 expression and upregulate CD146 expression in culture in response to angiopoietin II treatment, suggesting the adoption of a pericyte phenotype with stimulation24. Within the heart, however, the adventitial cell population has not yet been prospectively purified by FACS and/or well characterized. Utilizing the cell isolation procedures described in the following sections, we are currently characterizing myocardial adventitial cells and investigating their potential for regenerative applications.
Herein we describe a method to isolate and purify two subpopulations of perivascular stem/progenitor cells from human fetal or adult myocardium. This prospective cell isolation method will enable researchers to obtain isogenic perivascular stem/progenitor cell subsets from human heart biopsies for comparative studies and further explore their therapeutic potential in various cardiac pathological conditions.

<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>
</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.

Single cells were distinguished from debris and doublets on the basis of forward and side scatter distributions. Live cells were identified by their failure to take up the DAPI dye. The gating strategy was chosen on the basis of isotype control labelling of this live, whole-cardiac cell dissociation (Figure 1). From the live cells, CD45+ cells were first gated out, followed by CD56+ cells. CD144+ endothelial cells were then removed from th...

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

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

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

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