Name: phenotypic and functional characterization of endothelial colony forming cells derived from human umbilical cord blood
Uploaded: 2012-04-13T13:00:00
Description: Watch this Scientific Journal Video about Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood at JoVE.com

Dr. Yoder is consultant to EndGenitor Technologies, Inc. and a member of the board of Rimedion Technologies, Inc.

Reagents and Solutions
EMG-2 media (Lonza, Cat. No. cc-3162 containing EBM-2 basal medium and EGM-2 SingleQuot kit Supplements, &amp; growth factors)
EBM-2 (Lonza, Cat. No. cc-3156) supplemented with the entire SingleQuot kit supplements &amp; growth factors (Lonza, Cat. No. cc-4176), 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin (10,000 U/ml)/streptomycin (10,000 &mu;g/ml)/amphotericin (25 &mu;g/ml). Store up to 1 month at 4 &deg;C. Recommended EGM-2 volume...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vessel+wall-derived+endothelial+cells+rapidly+proliferate+because+they+contain+a+complete+hierarchy+of+endothelial+progenitor+cells.">Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells.</a> Blood. 105, 2783-276 (2005).</li><li>Ingram, D. A. <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+a+novel+hierarchy+of+endothelial+progenitor+cells+using+human+peripheral+and+umbilical+cord+blood.">Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood.</a> Blood. 104, 2752-2760 (2004).</li><li>Yoder, M. 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(32), e1525 (2009).</li><li>Au, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+in+vivo+potential+of+endothelial+progenitor+cells+from+human+umbilical+cord+blood+and+adult+peripheral+blood+to+form+functional+long-lasting+vessels.">Differential in vivo potential of endothelial progenitor cells from human umbilical cord blood and adult peripheral blood to form functional long-lasting vessels.</a> Blood. 111, 1302-135 (2008).</li><li>Critser, P. J., Kreger, S. T., Voytik-Harbin, S. L., Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+matrix+physical+properties+modulate+endothelial+colony+forming+cell-derived+vessels+in+vivo.">Collagen matrix physical properties modulate endothelial colony forming cell-derived vessels in vivo.</a> Microvasc. Res. 80, 23-30 (2010).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+robust+and+functional+vascular+networks+in+vivo+with+human+adult+and+cord+blood-derived+progenitor+cells.">Engineering robust and functional vascular networks in vivo with human adult and cord blood-derived progenitor cells.</a> Circ. Res. 103, 194-202 (2008).</li><li>Melero-Martin, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+vasculogenic+potential+of+human+blood-derived+endothelial+progenitor+cells.">In vivo vasculogenic potential of human blood-derived endothelial progenitor cells.</a> Blood. 109, 4761-4768 (2007).</li><li>Hofmann, N. A., Reinisch, A., Strunk, D. <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+Large+Scale+Expansion+of+Adult+Human+Endothelial+Colony+Forming+Progenitor+Cells.">Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells.</a> J. Vis. Exp. (32), e1524 (2009).</li><li>Lin, Y., Weisdorf, D. J., Solovey, A., Hebbel, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+circulating+endothelial+cells+and+endothelial+outgrowth+from+blood.">Origins of circulating endothelial cells and endothelial outgrowth from blood.</a> J. Clin. Invest. 105, 71-77 (2000).</li><li>Witting, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Large+Volume+Lentiviral+Vector+Production+Using+Flow+Electroporation.">Efficient Large Volume Lentiviral Vector Production Using Flow Electroporation.</a> Hum. Gene. Ther. , (2011).</li><li>Yoon, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+neovascularization+by+mixed+transplantation+of+early+endothelial+progenitor+cells+and+late+outgrowth+endothelial+cells:+the+role+of+angiogenic+cytokines+and+matrix+metalloproteinases.">Synergistic neovascularization by mixed transplantation of early endothelial progenitor cells and late outgrowth endothelial cells: the role of angiogenic cytokines and matrix metalloproteinases.</a> Circulation. , 112-1618 (2005).</li><li>Dubois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+progenitor+cell+populations+on+left+ventricular+remodeling+and+myocardial+neovascularization+after+myocardial+infarction.">Differential effects of progenitor cell populations on left ventricular remodeling and myocardial neovascularization after myocardial infarction.</a> J. Am. Coll. Cardiol. 55, 2232-2243 (2010).</li><li>Medina, R. J., O'Neill, C. L., Humphreys, M. W., Gardiner, T. A., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outgrowth+endothelial+cells:+characterization+and+their+potential+for+reversing+ischemic+retinopathy.">Outgrowth endothelial cells: characterization and their potential for reversing ischemic retinopathy.</a> Invest. Ophthalmol. Vis. Sci. 51, 5906-5913 (2010).</li></ol>

Phenotypic and functional characterization of putative endothelial progenitor cells is important to identify the bona fide ECFCs that are capable of clonally and serially re-plating in culture and give rise to durable and functional implantable blood vessels in vivo. Human umbilical cord blood is enriched with ECFCs and the concentration of these circulating cells declines with aging or disease10. Recent studies suggest that ECFC may play important roles in vascular repair or regeneration in ...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Heparin Sodium Injection, USP</td>
			<td>APP Pharmaceuticals</td>
			<td>504031</td>
		</tr>
		<tr>
			<td>Ficoll-Pague</td>
			<td>Amersham Biosciences</td>
			<td>17-1440-03</td>
		</tr>
		<tr>
			<td>Mixing cannula</td>
			<td>Maersk Medical</td>
			<td>500.11.012</td>
		</tr>
		<tr>
			<td>EGM-2</td>
			<td>Lonza</td>
			<td>CC-3162</td>
		</tr>
		<tr>
			<td>Defined FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
		</tr>
		<tr>
			<td>TrypLE express</td>
			<td>Gibco</td>
			<td>12605</td>
		</tr>
		<tr>
			<td>Rat type I collagen</td>
			<td>BD Biosciences</td>
			<td>354236</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>356234</td>
		</tr>
		<tr>
			<td>FcR Block</td>
			<td>Miltenyi Biotech</td>
			<td>130-059-901</td>
		</tr>
		<tr>
			<td>hCD31, FITC conjugated</td>
			<td>BD Pharmingen</td>
			<td>555445</td>
		</tr>
		<tr>
			<td>hCD45, FITC conjugated</td>
			<td>BD Pharmingen</td>
			<td>555482</td>
		</tr>
		<tr>
			<td>hCD14, FITC conjugated</td>
			<td>BD Pharmingen</td>
			<td>555397</td>
		</tr>
		<tr>
			<td>hCD144, PE conjugated</td>
			<td>eBioscience</td>
			<td>12-1449-80</td>
		</tr>
		<tr>
			<td>hCD146, PE conjugated</td>
			<td>BD Pharmingen</td>
			<td>550315</td>
		</tr>
		<tr>
			<td>hCD105, PE conjugated</td>
			<td>Invitrogen</td>
			<td>MHCD10504</td>
		</tr>
		<tr>
			<td>Ms IgG1,k antibody, FITC conjugated</td>
			<td>BD Pharmingen</td>
			<td>555748</td>
		</tr>
		<tr>
			<td>Ms IgG1,k antibody, PE conjugated</td>
			<td>BD Pharmingen</td>
			<td>559320</td>
		</tr>
		<tr>
			<td>Ms IgG2a,k antibody, FITC conjugated</td>
			<td>BD Pharmingen</td>
			<td>555573</td>
		</tr>
		<tr>
			<td>Anti-human CD31</td>
			<td>Dako</td>
			<td>clone JC70/A</td>
		</tr>
		<tr>
			<td>Anti-mouse CD31</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
		</tr>
		<tr>
			<td>0.22-&mu;m vacuum filtration system</td>
			<td>Millipore</td>
			<td>SCGPU05RE</td>
		</tr>
		<tr>
			<td>Glacial acetic acid, 17.4N</td>
			<td>Fisher</td>
			<td>A38-500</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Invitrogen</td>
			<td>15240-062</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
		</tr>
		<tr>
			<td>IHC Zinc Fixative</td>
			<td>BD Biosciences</td>
			<td>550523</td>
		</tr>
		<tr>
			<td>Sytox green reagent</td>
			<td>Invitrogen</td>
			<td>S33025</td>
		</tr>
		<tr>
			<td>Cloning cylinders, sterile</td>
			<td>Fisher Scientific</td>
			<td>07-907-10</td>
		</tr>
	</tbody>
</table>

phenotypic and functional characterization of endothelial colony forming cells derived from human umbilical cord blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Watch this Scientific Journal Video about Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood at JoVE.com

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

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Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

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Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...