Name: isolation of endothelial progenitor cells from human umbilical cord blood
Uploaded: 2017-09-14T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

This material is based upon work supported by the National Science Foundation under Grant No. CMMI-1452943 and by the University of Arkansas Honors College. We would also like to acknowledge the Arkansas Cord Blood Bank for providing us with cord blood units.

This research was carried out with the approval of the University of Arkansas Institutional Review Board (Approval number 16-04-722). Umbilical cord blood units were collected in citrate phosphate dextrose (CPD) solution at the Arkansas Cord Blood Bank, and units that did not meet the requirement for storage were donated for research. Cord blood units were couriered to the lab within 24 h of collection at ambient temperatures.
1. Isolation of Endothelial Progenitor Cells from Cord Blood
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As mentioned earlier, adherent EPCs possess a cobblestone morphology. Our isolated MNCs progressed from a spindle-shaped cell colony (Figure 2A-2D) in the early stages to a cobblestone colony (Figure 2E-2F) over a period of ten days in culture. EPCs have been labeled differently by different research groups, namely as late endothelial progenitor cells10, endothelial colony forming cells5...

Several investigators have studied the characteristics and potential of human EPCs5,10,11,12,13. EPCs can be described as circulating cells that have the ability to adhere to endothelial tissue in sites of hypoxia, ischemia, injury, or tumor formation and contribute to the formation of new vascular structures4,14. Their observed involvement in neovascularization, in the form of postnatal vasculogenesis, has led to an understanding of the pathophysiology of these cells and their use in therapeutic applications4,15,16. The number of EPCs in an individual has been shown to be correlated with cardiovascular pathology9,15,16,17,18,19,20. Other studies have also differentiated EPCs into a valve fibroblast-like phenotype and proposed that these cells could be used for tissue-engineering heart valves7,21.
The particular cell surface molecules needed to isolate EPCs have not been clearly identified due to discrepancies between investigations4. The adhesion of MNCs to a certain matrix, with exposure to a variety of culture conditions, has been performed by several groups1,17,22,23, suggesting that putative EPCs may display different phenotypic properties. These properties include a lack of phagocytotic ability, tube formation in Matrigel, and the uptake of Dil-acetylated low-density lipoproteins. The high clonogenic and proliferative potential are two properties with which EPCs can be hierarchized5. EPCs can also form in vitro tubules when cocultured with human fetal lung fibroblasts4. These cells are known to express endothelial cell surface markers and to share some of the hematopoietic markers13,24,25. The positively expressed markers that are widely accepted for phenotyping EPCs are CD31, CD34, vascular endothelial growth factor receptor 2 (VEGFR2), von Willebrand Factor (vWF), CD133, c-Kit, and vascular endothelial cadherin (VE-cadherin)4,18. Cells that co-express CD90, CD45, CD14, CD115, or alpha-smooth muscle actin (&#945;-SMA) are not considered to be EPCs because of their limited proliferative potential, ability to phagocytose bacteria, and inability to form de novo human vessels in vivo4,7. This article outlines a modified protocol for the isolation of endothelial progenitor cells from human umbilical cord blood without the need for any cell sorting protocols. For this article, we used CD31, CD34, and VEGFR2 as the positive markers, with &#945;-SMA as the negative indicator.
In this article, we propose a method of isolating and culturing endothelial progenitor cells from umbilical cord blood without cell sorting using specialized endothelial growth medium supplemented with growth factors (EGM). This EGM contains vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), which enhance the survival, proliferation, and migration of endothelial cells26. It also includes ascorbic acid, which is responsible for maintaining the cobblestone morphology of cells; insulin-like growth factor-1 (IGF-1), which provides angiogenic and migratory function; and heparin, which causes improved long-term stability of growth factors in the medium26. Other growth factors added to the endothelial cell culture medium includes supplementation with epidermal growth factor (EGF), which helps in stimulating cell proliferation and differentiation, and hydrocortisone, which sensitizes the cells to EGF26. We show that the use of this specific growth medium yields higher numbers of EPCs compared to endothelial basal medium (EBM) or Dulbecco&#39;s Modified Eagle Medium (DMEM).

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			<td height="20" style="width:204px;height:20px;">A) For isolation and culturing</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:807px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">EGM-2 BulletKit</td>
			<td style="width:127px;">Lonza</td>
			<td>CC-3162</td>
			<td>This product comes with all the growth factors needed to make the Endothelial Growth Medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Fetal Bovine Serum</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">26140079</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Pencillin-Streptomycin-Glutamine (100X)</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">10378016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Ficoll-Paque</td>
			<td style="width:127px;">GE Heatlhcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Ammonium Chloride</td>
			<td style="width:127px;">Stem Cell Technologies</td>
			<td style="width:119px;">7850</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">1X Phosphate Buffer Saline</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Rat Tail I Collagen</td>
			<td style="width:127px;">Corning</td>
			<td style="width:119px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Glacial Acetic Acid</td>
			<td style="width:127px;">Amresco</td>
			<td style="width:119px;">0714-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">25300054</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">HEPES buffer</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">15630080</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">10566-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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			<td height="20" style="width:204px;height:20px;">B) Antibodies and cell lysates</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">CD31&#160;</td>
			<td style="width:127px;">Abcam</td>
			<td style="width:119px;">ab28364</td>
			<td>1:250 dilution&#160; for Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">CD34</td>
			<td style="width:127px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-7045</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">&#945;-SMA</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">ab5694</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">&#945;-tubulin</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">ab7291</td>
			<td>1:2500 dilution for Western blotting</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">VEGFR2</td>
			<td style="width:127px;">abcam</td>
			<td style="width:119px;">sc504</td>
			<td>1:100 dilution for Western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Human umbilical vein endothelial cell lysate</td>
			<td style="width:127px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc24709&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Valve interstitial cell lysate</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td style="width:807px;">Primary cell line cultured from own lab and lysed with RIPA buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;"></td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
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			<td height="40" style="width:204px;height:40px;">C) Western blotting and immunostaining</td>
			<td style="width:127px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10X Tris/Glycine/SDS buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0772</td>
			<td>Used as running buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10X Tris/Glycine buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0771</td>
			<td>Used as transfer buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Immobilon-FL transfer membrane</td>
			<td style="width:127px;">Merck Millipore</td>
			<td style="width:119px;">IPFL0010</td>
			<td>This is a PVDF transfer membrane that has 45 &#181;m pore size and is mentioned in the protocol as western blot membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">4X Laemmli sample buffer</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0747</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">2-mercaptoethanol</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">10% Criterion TGX precast gel</td>
			<td style="width:127px;">Biorad</td>
			<td style="width:119px;">5671033</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:204px;height:42px;">Prolong Gold antifade</td>
			<td style="width:127px;">Thermofisher Scientific</td>
			<td style="width:119px;">P36930</td>
			<td>Used for mounting immunostained coverslips for long term storage</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:204px;height:21px;">Methanol</td>
			<td style="width:127px;">VWR Analytical</td>
			<td style="width:119px;">BDH1135-4LP</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of endothelial progenitor cells from human umbilical cord blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

Isolation and Expansion of Endothelial Progenitor Cells: 
A schematic (Figure 1) is provided depicting the overall protocol. The different blood component layers were observed following density gradient centrifugation of human umbilical cord blood with density gradient medium. Upon seeding MNCs onto the collagen-treated plates, the outgrowth of colonies was first observed between Days 5 and 7 (Figure 2<stron...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood at JoVE.com

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Research

JoVE Journal

Developmental Biology

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

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Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

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Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

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Isolation and Characterization of Human Umbilical Cord-derived Mesenchymal Stem Cells from Preterm and Term Infants

Mesenchymal stem cells (MSCs) have considerable therapeutic potential and attract increasing interest in the biomedical field. MSCs are originally ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...