Name: isolation of endothelial progenitor cells from healthy volunteers and their migratory potential influenced by serum samples after cardiac surgery
Uploaded: 2017-02-14T14:00:00
Description: Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery at JoVE.com

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Blood for the isolation of EPCs was obtained from healthy volunteers after informed consent in accordance with the local ethics committee. Serum samples used in the migration assays were obtained from patients that underwent conventional cardiac surgery with the use of cardiopulmonary bypass (CPB). Exclusion criteria were emergency operations, known or suspected pregnancy, patient`s age less than 18 years, and failure to obtain informed consent. Serum samples were drawn in addition to clinical routine measurements (immed...

The first part of this study included the isolation of human EPCs from the peripheral blood of healthy volunteers to enable a comprehensive evaluation of the blood of cardiac surgery patients. Therefore, a density gradient centrifugation was performed to separate the PBMC fraction from plasma, granulocytes and erythrocytes. To remove most of the contaminating platelets, this cell fraction was subjected to short and slow washing steps29,30. Next, CD34+ ...

Endothelial progenitor cells (EPCs) are circulating in the human blood and have the ability to differentiate into endothelial cells2. They participate in vasculogenesis and are capable of minimizing the damage caused by inflammation and ischemia/reperfusion (I/R) injuries in various ways3,4. For example, EPCs show elevated levels of intracellular antioxidant enzymes like catalase, glutathione peroxidase or manganese superoxide dismutases (MnSOD)5. The elevated resistance against oxidative stress allows EPCs to function in microenvironments with elevated reactive oxygen species (ROS) after ischemic injury6. Previous studies also indicated that the number of EPCs might be correlated to vascular repair and that a reduced number of circulating EPCs predicts the occurrence of cardiovascular events7,8. However, a clear definition of an EPC has not been found yet. Up to now, there is no specific cell surface marker or consistent phenotype for EPCs and these cells are very rare in the peripheral blood9. A human EPC should be considered as a circulating cell with the ability to contribute to the reconstruction of the injured endothelium and new vascular structures.
One way of isolating and characterizing EPCs is through adhesion to fibronectin. Thereby, the capacity of these cells is used to show a superior adhesion to fibronectin coated dishes compared to type 1 collagen, for example3,10,11. However, others found that plating mononuclear cells on fibronectin-coated dishes without any previous or further purification step leads to colonies including myeloid progenitor cells, monocytes, and T lymphocytes12,13,14. Moreover, in this case, platelets might contaminate the mononuclear cell (MNC) fraction and thereby transfer plasma membrane proteins to any adherent cells15.
Besides characterization through in vitro adhesion assays, a combination of different cell surface markers is used to describe a cell type considered as an EPC. In this case, after fibronectin-mediated adhesion, the cells are analyzed concerning their endothelial-like attributes. In this process, the two endothelial cell-associated markers, acetylated-low density lipoprotein (acLDL) and vascular endothelial growth factor receptor 2 (VEGFR-2, KDR), play a role. Endothelial cells and macrophages have been shown to specifically take up acLDL in a process called &#34;scavenger cell pathway&#34;16. Another marker protein is KDR as the main VEGF receptor on endothelial cells17. However, as EPCs in general are cultured in media supplemented with endothelial growth factors and fetal calf serum, it is possible that macrophages, which might also have been mistakenly isolated, exhibit an endothelial-like marker profile. As previously shown, if cultured in an endothelial-conditioned medium, macrophages express &#34;endothelial-specific&#34; proteins18.
In general, there are two categories of EPCs within more subtypes, which can be found in the blood or be cultured in vitro. Late-outgrowth EPCs (late-EPCs) appear after 2-3 weeks of culture. These cells are integrated faster into a monolayer of human umbilical vein endothelial cells and can form capillary tubes19. Besides, so-called &#34;early-EPCs&#34; circulate in the blood for about one week and act in a more passive way through delivering angiogenic molecules, such as vascular endothelial growth factor (VEGF), or CXCL819. Patients with coronary artery disease (CAD) showed significantly lower amounts of early-EPCs compared to a control group without CAD20. Interestingly, the same group showed higher amounts of late-EPCs compared to a control group. Another study showed that early-EPCs protect differentiated EPCs from apoptosis under oxidative conditions in a paracrine manner6. Therefore, early EPCs might provide relevant protective effects through the migration of other cells in an auto- or paracrine manner within the peripheral blood.
This protocol describes a method to purify early-EPCs by first isolating the PBMC-fraction from human peripheral blood and subsequently isolating CD34+ cells from the PBMC-fraction to clear this cell suspension from unwanted cells. CD34 is a marker, which is used for the isolation of human hematopoietic stem cells9. Afterwards, CD34+ cells are cultured on fibronectin-coated tissue culture surfaces. After three days, the medium is changed, thereby losing all non-adherent cells. Finally, isolated EPCs are stained to verify the uptake of acLDL and the presence of KDR as endothelial cell-marker by using fluorescence-activated cell sorting (FACS). As an additional marker, we analyzed platelet endothelial cell adhesion molecule (PECAM-1, CD31), which also occurs on endothelial cells.
Restoration of damaged or infarcted myocardial tissue by enhanced recruitment of EPCs belongs to the intensively investigated treatment strategies in cardiovascular diseases. However, the translation of experimental results into clinical practice is still challenging, given the complex cellular interplay in the human body during various pathophysiological conditions. Furthermore, the myocardial I/R injuries trigger an excessive secretion of various cytokines, hormones and growth factors, which control the homing of EPCs to regions of blood vessel formation13. As already shown, CXCL8, stromal cell-derived factor 1&#945; (SDF-1&#945;, CXCL12), VEGF and macrophage migration inhibitory factor (MIF) are significantly increased in serum samples following myocardial I/R injury1. Among these factors, MIF is a pleiotropic chemokine-like cytokine with predominantly pro-inflammatory characteristics. In contrast to its historic name, MIF has pro-migratory functions, acting as a true chemokine on various cell types1,21,22. MIF-mediated cell recruitment processes have been linked to the chemokine receptors CXCR2 and CXCR4, which MIF binds to and activates in a non-cognate manner21. Of note, EPCs express both of these receptors on their surface, which additionally become up-regulated under hypoxic conditions23,24. Moreover, accumulating evidence suggests that MIF has an overall cardio-protective effect during I/R injury of the heart22,25,26. In this context, it has been further shown that MIF may support the neovascularization during hypoxic stress that is of particular relevance, when considering the limited recovery mechanisms of the injured myocardium27. Previous in vitro studies and experiments in pre-clinical mouse models provided first evidence about the role of MIF in EPCs recruitment4. Of note, MIF also is a prominent cargo protein of EPCs that may be released during EPCs recruitment within ischemic sites28. However, studies in clinical settings in particular in comparison with other (angiogenic) serum cytokines remain elusive.

<table border="0" cellpadding="0" cellspacing="0" width="803">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Fibronectin</td>
			<td style="width:185px;">Biochrom AG</td>
			<td style="width:155px;">L7117</td>
			<td style="width:207px;">Coating of T-75 flasks</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Aqua ad iniectabilia</td>
			<td style="width:185px;">Fresenius Kabi</td>
			<td style="width:155px;"></td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Endothelial cell growth medium MV2</td>
			<td style="width:185px;">Promo Cell</td>
			<td style="width:155px;">C-22221</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Endothelial cell growth medium MV2 SupplementMix</td>
			<td style="width:185px;">Promo Cell</td>
			<td style="width:155px;">C-39226</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">Ficoll-Paque plus</td>
			<td style="width:185px;">GE Healthcare</td>
			<td style="width:155px;">17-1440-03</td>
			<td style="width:207px;">Density centrifugation</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">EasySep human CD34 positive Selection Kit</td>
			<td style="width:185px;">Stemcell Technologies</td>
			<td style="width:155px;">18056</td>
			<td style="width:207px;">Isolation of CD34+ cells</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">EasySep magnet</td>
			<td style="width:185px;">Stemcell Technologies</td>
			<td style="width:155px;">18000</td>
			<td style="width:207px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:257px;">Accutase</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:155px;">A6964-100ML</td>
			<td style="width:207px;">Detachment of cells</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Corning HTS transwell 96 well permeable supports</td>
			<td style="width:185px;">Sigma-Aldrich</td>
			<td style="width:155px;">CLS3387-8EA</td>
			<td style="width:207px;">Migration system</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:257px;">Hoechst solution</td>
			<td style="width:185px;">ThermoFisher</td>
			<td style="width:155px;">33342</td>
			<td style="width:207px;">Staining of migrated cells</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:257px;">ImageJ</td>
			<td style="width:185px;">National institutes of health</td>
			<td style="width:155px;">xxx</td>
			<td style="width:207px;">Counting of migrated cells</td>
		</tr>
	</tbody>
</table>

isolation of endothelial progenitor cells from healthy volunteers and their migratory potential influenced by serum samples after cardiac surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Characterization of Isolated Endothelial Progenitor Cells
First, the uptake of acLDL was verified, as well as the expression of KDR, and CD31 on the surface of the isolated cell population. As Figure 7a shows, 85.1% of the isolated EPCs showed an uptake of acLDL and expressed CD31. Figures 7b and 7c further demonstrate a homogenous distribution of both markers, although there seems...

Watch this Scientific Journal Video about Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery at JoVE.com

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

The overall goal of this cell isolation procedure and migration protocol is to show a reliable way of isolating endothelial progenitor cells and their migratory potential towards serum samples of cardiac surgical patients. This method can help to answer key questions in the regeneration of the endothelial lining and blood vessels, which is needed after cardiac surgery due to ischemia and re-perfusion related injuries. The main advantage of this technique is the relatively simple way of isolating progenitor cells, including the pre-isolation of CD-34 positive cells.In addition to deaths, major complications of the cardiac surgery remain too common. Underlining the need to identify the tie risk and protective mechanisms during cardiac surgery. Endothelial progenitor cells are crucially involved in the neovascularization of ischemic tissues and known to provide cardio-protective properties.To begin the experiment, mix blood, one-to-one, with calcium free and magnesium free PBS. Add 15 milliliters of density gradient solution to a 50 milliliter tube. And slowly layer the diluted blood on top of the density gradient solution.Next, centrifuge the samples. Using a sterile plastic pipette, carefully collect the buffy coat layer of every tube, and place it into another tube while avoiding the density gradient solution. Dilute the peripheral blood mononuclear cell, or PBMC fraction, with at least three volumes of PBS and mix the solution by pipetting.Centrifuge the mixture at room temperature for 15 minutes at 200 times G.Then, aspirate the supernatant, and resuspend the cell pellet in five milliliters of endothelial cell growth medium, MV-2. After resuspending the cells, add 100 microliters of human CD-34 anti-body per used buffy coat and rotate the cells. Add 50 microliters of Dextran coated magnetic beads per used buffy coat and rotate the cells.After incubation, transfer the suspension to fax tubes with a maximum of three milliliters to each tube. Next, insert the fax tubes into the magnets and wait for five minutes. Discard the super natant without pulling the fax tubes out of the magnet.Then, outside of the magnets, resuspend the cells in each fax tube with three milliliters of MV-2 medium. Transfer the cell suspension into pre-coated T-75 flasks. Add 17 milliliters of MV-2 medium to each flask.To prepare the migration assay, use a pipette to remove the medium from the endothelial progenitor cells or the EPCs, in the T-75 flask. Wash the cells with five milliliters of phosphate buffered saline or PBS, and carefully shake the flask. Next, remove the PBS and add five milliliters of commercial cell detachment solution.Then, wait until the cells are detached under a light microscope. Accelerate the detachment by carefully tapping the bottom of the flask. When the cells are detached, quickly add five milliliters of MV-2 complete medium, and transfer the cell suspension into another tube.Centrifuge the cells at 2, 000 x G for five minutes. After pelleting the cells, resuspend them in five to ten milliliters of PBS. And centrifuge them again.Resuspend the cells in five to ten milliliters of PBS again and follow with centrifugation. Resuspend the cell pellet in MV-2 starved medium. Next, dilute the serum sample one to five in MV-2 starved medium.Prepare the migration plate by adding 235 microliters of the serum sample into the lower chamber. Add the insert shortly before adding the cell solution. Then, add 75 microliters of the cell solution into the upper chamber.Allow the EPCs to migrate. Remove the upper chamber containing all of the non-migrated cells. Add 75 microliters of 3.6 percent paraformaldehyde solution including hoechst dye diluted at one to 1, 000.Centrifuge the plate shortly to get all of the cells into the same focal plane at 2, 000 x G for one to two minutes. To avoid serum auto-fluorescence artifacts, quantify the migrated cells by taking five pictures per well with 100x magnification under a microscope. Finally, count the migrated cells using the semi-automated software.Fax analysis of the EPCs verify the uptake of acLDL, as well as the expression of CD-31 on the surface of the isolated cell population. Isolating the analysis of acLDL and CD-31 reveals a homogenous distribution of each marker. Elyso to identify the influence of cardiac surgery following myocardial reperfusion injury on the concentrations of circulating serum levels of MIF, CXCL12, CXCL8, and VEGF.Serum samples were drawn pre and intraoperatively. Serum levels of MIF, CXCL12 and CXCL8 showed a significant intraoperative increase compared to baseline values. In contrast, VEGF concentrations did not show any significant changes.An ex vivo migration assay, using serum samples drawn prior to and during surgery was performed using EPCs isolated from healthy volunteers, and revealed a significantly increased migration rate toward the intraoperatively taken samples. Following this technique, peripheral blood mononuclear cells can easily be isolated to, in order to answer additional inflammatory related questions.

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