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
<ol>
	<li>Preparation of reagents.
	<ol>
		<li>Prepare EGM by adding endothelial basal medium (EBM) to 10% fetal bovine serum (FBS) supplemented with 20 ng/mL of insulin-like growth factor (IGF), 1 &#181;g/mL ascorbic acid, 5 ng/mL recombinant human epidermal growth factor (rhEGF), 22.5 &#181;g/mL heparin, and 0.5 ng/mL vascular endothelial growth factor (VEGF), along with 10 ng/mL of recombinant human Fibroblast growth factor B (rhFGF-B), 0.2 &#181;g/mL hydrocortisone, and 2% penicillin-streptomycin-glutamine. Sterilize the culture medium using a 0.2 &#181;m polyethersulfone (PES) membrane vacuum filter.</li>
		<li>Prepare rat tail I collagen solution at a concentration of 50 &#181;g/mL using 0.02 N acetic acid. Sterilize this solution using a 0.2 &#181;m syringe filter.</li>
		<li>Pre-coat 6-well plates with 2 mL of prepared rat tail I collagen solution for each well. Place them in an incubator maintained at 5% CO2 and 37 &#176;C for at least 1 h before seeding.</li>
		<li>Dilute approximately 25 mL of cord blood with Hanks Balanced Salt Solution (HBSS) at a 1:1 concentration.</li>
		<li>Prepare radio-immuno precipitation assay (RIPA) Buffer using 150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM &#946;-glycerophosphate, 0.1% SDS, and 50 mM Tris (pH 8.0).</li>
	</ol>
	</li>
	<li>Isolation of endothelial progenitor cells.
	<ol>
		<li>Warm all prepared reagents in a water bath maintained at 37 &#176;C prior to isolation.</li>
		<li>Add 20 mL of density gradient medium reagent to a 50 mL centrifuge tube.</li>
		<li>Carefully layer 20 mL of diluted cord blood in the centrifuge tube containing density gradient medium without breaking this layer by aiming the pipette towards the side walls of the tube.</li>
		<li>Separate the components of blood based on their densities (Figure 1) by centrifuging at 800 x g for 30 min at room temperature, with the brakes of the centrifuge rotor off. Do not disturb the different layers.</li>
		<li>Remove the plasma layer by carefully pipetting it out of the tube without disturbing other cell layers until the pipette tip is able to reach the MNC layer (see Figure 1). Discard the removed plasma. 
		NOTE: While using pipette tips to remove the plasma, leave a small layer of plasma above the MNC layer (Figure 1) to reduce disturbances.</li>
		<li>Collect the buffy coat that contains the MNCs using a syringe fitted to an 18-gauge needle and transfer it to a fresh centrifuge tube.</li>
		<li>Add an equal volume of EBM to the collected MNCs. Centrifuge these tubes at 500 x g for 10 min at 4 &#176;C. Discard the supernatant. 
		NOTE: The cell pellet will appear red because of the presence of red blood cells/erythrocytes.</li>
		<li>Add 5 mL of ammonium chloride solution to lyse the red blood cells. Incubate this tube on ice for 5 - 10 min, with occasional shaking. 
		NOTE: Caution should be taken during this step to not exceed the time duration, as it can be harmful to the MNCs.</li>
		<li>Centrifuge the tubes at 500 x g for 10 min at 4 &#176;C and discard the supernatant. If erythrocytes persist, repeat steps 1.2.8 and 1.2.9 until no red color is observed in the pellet.</li>
		<li>Resuspend the pellet in prepared EGM and use the hemocytometer to count the MNCs.</li>
		<li>Aspirate the rat tail I collagen-coated 6-well plate (from step 1.1.3) and wash the wells three times with 1x phosphate-buffered saline (PBS).</li>
		<li>Seed the MNC pellet resuspended in EGM to the collagen plate at a concentration of 1 x 107 MNCs in each well. Place it in the 37 &#176;C, 5% CO2 incubator.</li>
		<li>After 24 h, aspirate the medium and wash the wells once with EGM. Add 3 mL of EGM medium to each well and place it back in the 37 &#176;C, 5% CO2 incubator.</li>
		<li>Replenish the plate with fresh EGM medium every day for 7 days. After 7 days, change the EGM medium every other day.</li>
		<li>Using a bright-field microscope, take images of the 6-well plate every day to track the progression of endothelial cell colonies. Mark the plate where the colonies arise to keep track of their growth. 
		NOTE: It usually takes between 5 and 9 days for the colonies to appear in culture.</li>
	</ol>
	</li>
	<li>Expansion of endothelial progenitor cells.
	<ol>
		<li>Coat the T-25 culture flask with rat tail I collagen (50 &#181;g/mL) and place it in the 37 &#176;C, 5% CO2 incubator for at least 60 min. Aspirate and wash the T-25 cell culture flask three times with 1x PBS prior to seeding the cells.</li>
		<li>Trypsinize the endothelial cell colonies when they reach about 3 mm in size using 150 &#181;L of 0.05% Trypsin-EDTA solution in each well. Place the 6-well plate back into the 37 &#176;C, 5% CO2 incubator for 2 - 3 min.</li>
		<li>Gently tap the 6-well plate to dislodge the attached cells. Immediately add 2 mL of EGM and recover the cells in a 15 mL centrifuge tube.</li>
		<li>Count the cells using a hemocytometer and calculate the initial seeding density.</li>
		<li>Spin the 15 mL centrifuge tubes at 400 x g for 5 min. Resuspend the cell pellet in EGM and seed the T-25 flask with ~500,000 cells. Mark this flask as Passage 1 and place the T-25 flask back in the 37 &#176;C, 5% CO2 incubator. 
		NOTE: Cells can be plated in EBM and DMEM to compare the growth with EGM.</li>
		<li>For further passages, when the flask reaches 90% confluence, follow steps 1.3.1 - 1.3.5 and reseed the cells at 104 cells/cm2 on T-75 or T-175 cell culture flasks. Proceed to the characterization of the isolated cells (steps 2 and 3).</li>
	</ol>
	</li>
</ol>
2. Characterization of Isolated Cells Using Western Blotting
<ol>
	<li>Add 50 - 100 &#181;L of lysis buffer at every passage when following the expansion steps to characterize the isolated cells. Collect proteins from approximately 500,000 or more cells.</li>
	<li>Pipette up and down vigorously to rupture the cells and release the proteins. Collect and transfer the buffer to a microcentrifuge tube.</li>
	<li>Spin the microcentrifuge tube at 500 x g and carefully transfer the supernatant to a new microcentrifuge tube. Label and store the tubes at -80 &#176;C for long-term storage.</li>
	<li>Quantify the amount of proteins in each tube using either Bradford&#39;s or BCA assay27,28,29.</li>
	<li>Carry out Western blotting using standard procedures27,28,29. Analyze 5 &#181;g of total protein for the expression of CD31, CD34, VEGFR2, and &#945;-SMA. Use &#945;-tubulin for normalizing the data.</li>
</ol>
3. Indirect Immunofluorescence
<ol>
	<li>Sonicate 18-mm glass coverslips in 50% ethanol and dry them. Leave the clean coverslips in a 12-well plate overnight in the biosafety hood with a UV light on to sterilize them.</li>
	<li>Coat the coverslips with 50 &#181;g/mL of rat tail I collagen and transfer the plate to a 37 &#176;C, 5% CO2 incubator for at least 60 min.</li>
	<li>Aspirate the collagen and add 1 mL of 1x PBS to the 12-well plate to wash the coverslips. Aspirate the 1x PBS and repeat twice.</li>
	<li>Seed 250,000 cultured EPCs in each well and place them back in the 37 &#176;C, 5% CO2 incubator for at least 3 days before carrying out immunostaining.</li>
	<li>Wash the plate 3 times with 1x PBS. Add 1 mL of ice-cold methanol to each well and incubate at room temperature for 15 min to fix the cells.</li>
	<li>Carry out immunostaining using a standard protocol27,28,29. Use the antibodies at their appropriate dilutions (e.g., CD31 (1:20), CD34 (1:50), &#945;-SMA (1:100), and VEGFR2 (1:50)).</li>
	<li>Take images of the immunostained coverslips using an epifluorescence microscope.</li>
</ol>

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The authors have nothing to disclose.

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, or endothelial progenitor cells12. It should be noted that these cells are functionally the same and express similar cell surface markers. The number of cells observed during the culture period (Figure 3) increased to almost 108 cells, analogous to previous publications and typical of the high proliferative potential of EPCs3,5. Indeed, it was found that EPCs obtained from human umbilical cord blood showed a high proliferative potential and demonstrated several population doublings before undergoing senescence compared to EPCs isolated from adult human peripheral blood5,10.
Our Western blotting results (Figure 4A) showed the presence of CD31, CD34, and VEGFR2 on the cell lysates obtained from EPCs compared to the positive control, HUVEC. HUVECs express CD31, CD34, and VEGFR2 and activated VIC express &#945;-sSMA. Similar to previous reports10,18, the expression of CD31 decreased with higher passages. The expression pattern was also similar in the case of CD34 and VEGFR2, where both the markers were expressed strongly in the first passage and in relatively lower but equal amounts in later passages. This data shows a trend that is in agreement with previous groups&#39; results, in which immunosorting-based protocols were used for isolating cord blood-derived EPCs12. Generally, EPCs do not express mesenchymal markers and, to verify that the isolated cells were not mesenchymal in nature, we immunolabeled our Western blot membranes with &#945;-SMA. Our results clearly show that there was no &#945;-SMA expression in the cultured EPCs when compared with positive control cell lysates collected from VICs, which exhibit strong expression of &#945;-SMA antibody.
To demonstrate that EGM allows for increased cell proliferation compared to other culture media used for EPCs, we cultured the cells in three different types of media: DMEM, EBM, and EGM (i.e., EBM supplemented with growth factors, as outlined earlier). All were supplemented with 10% FBS. We seeded cells of different passages in each well of a 12-well plate. Supplemental Figure 1 shows the number of cells per unit area in each well over time in culture in the different media formulations. We note that the EPCs cultured in EGM showed a steady increase in cell number, and for cells in EBM, there was a slower rate of increase in cell number during the first three days, which subsequently plateaued. This suggests that the EPCs could still survive in EBM, but the absence of growth factors hindered proliferation. We also note that EPCs cultured in DMEM decline in number, suggesting that this particular medium formulation is not suitable for their survival or proliferation. Our immunostaining data shows that the EPCs cultured in EGM (Figure 5) expressed CD31 more when compared to EPCs cultured in EBM (Supplemental Figure 2) and DMEM (Supplemental Figure 3). Comparing Figure 5 with Supplemental Figure 2 and Supplemental Figure 3, it can be seen that the cells were not contaminated with other cell types. Also, when comparing the immunostained EPCs (Figure 5), we can qualitatively state that the expression of CD31 and CD34 decreased over higher passages, supporting our Western blot data (Figure 4).
To reiterate, the primary goal of our proposed protocol is to demonstrate that one can isolate endothelial progenitor cells by culturing blood MNCs in specialized selective media. This is to show that EPCs can be isolated without sophisticated equipment and procedures, unlike other methods that involve the use of flow cytometry - either immediately after MNC isolation1,3,20,30 or after the colonies have grown into a monolayer - followed by replating4,10,12,13,23. The EPC colonies usually appear between days 5 and 9. Our method is thus a faster and more cost-efficient method for isolating EPCs. The primary limitation of this technique is that the expression of CD31 and CD34 decreases with subsequent passages, which could suggest that the cells are losing their progenitor phenotype. Thus, it is recommended that one should use cells obtained from passage 5 or lower to conduct experiments if using this protocol.
Precautions should be taken after the density centrifugation to not disturb the different layers when taking the centrifuged tubes back into the hood and also when removing the plasma. Another critical step pertains to erythrocyte lysis, where a longer incubation time could potentially damage the MNC. It is thus recommended not to repeat this step more than twice. Indeed, since the medium aspiration is carried out 24 h after plating, trace amounts of erythrocytes are removed, as they are non-adherent. Alternative substrates, like fibronectin or gelatin, while not tested by us, can also be used according to the specific protocols for creating cell culture coatings. By employing minor modification in the initial steps, researchers may isolate non-adherent EPCs30.
In conclusion, we have reported a method of isolating EPCs from umbilical cord blood. It is important to note that the EPCs do not express markers like &#945;-SMA, suggesting that they are not mesenchymal-like cells. Indeed, &#945;-SMA serves as an important marker when trying to study the endothelial-mesenchymal transition (endo-MT) of EPCs. Endo-MT is a cellular transdifferentiation process during which endothelial cells are known to lose their specific markers and to transform to a mesenchymal-like cell phenotype. It has been shown that EPCs can undergo this transition in the presence of transforming growth factor-&#946;7 and can release extracellular membrane proteins on their tissue-engineered scaffolds8. All these observations indicate the promise of EPCs for use as an autologous cell source to create a heart valve or other cardiovascular tissue-engineered constructs.

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).

<table><tbody><tr><td>&lt;strong&gt;A) For isolation and culturing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EGM-2 BulletKit</td><td>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><td>Fetal Bovine Serum</td><td>Thermofisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Pencillin-Streptomycin-Glutamine (100x)</td><td>Thermofisher Scientific</td><td>10378016</td><td></td></tr><tr><td>Ficoll-Paque</td><td>GE Heatlhcare</td><td>17-1440-02</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution</td><td>Thermofisher Scientific</td><td>14170-112</td><td></td></tr><tr><td>Ammonium Chloride</td><td>Stem Cell Technologies</td><td>7850</td><td></td></tr><tr><td>1x Phosphate Buffer Saline</td><td>Thermofisher Scientific</td><td>14190250</td><td></td></tr><tr><td>Rat Tail I Collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Amresco</td><td>0714-500ML</td><td></td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Thermofisher Scientific</td><td>25300054</td><td></td></tr><tr><td>HEPES buffer</td><td>Thermofisher Scientific</td><td>15630080</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Thermofisher Scientific</td><td>10566-016</td><td></td></tr><tr><td>&lt;strong&gt;B) Antibodies and cell lysates&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD31&amp;nbsp;</td><td>Abcam</td><td>ab28364</td><td>1:250 dilution&amp;nbsp; for Western blotting</td></tr><tr><td>CD34</td><td>Santa Cruz Biotechnology</td><td>sc-7045</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-SMA</td><td>abcam</td><td>ab5694</td><td>1:100 dilution for Western blotting</td></tr><tr><td>&amp;alpha;-tubulin</td><td>abcam</td><td>ab7291</td><td>1:2,500 dilution for Western blotting</td></tr><tr><td>VEGFR2</td><td>abcam</td><td>sc504</td><td>1:100 dilution for Western blotting</td></tr><tr><td>Human umbilical vein endothelial cell lysate</td><td>Santa Cruz Biotechnology</td><td>sc24709&amp;nbsp;</td><td></td></tr><tr><td>Valve interstitial cell lysate</td><td></td><td></td><td>Primary cell line cultured from own lab and lysed with RIPA buffer</td></tr><tr><td>&lt;strong&gt;C) Western blotting and immunostaining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x Tris/Glycine/SDS buffer</td><td>Biorad</td><td>161-0772</td><td>Used as running buffer</td></tr><tr><td>10x Tris/Glycine buffer</td><td>Biorad</td><td>161-0771</td><td>Used as transfer buffer</td></tr><tr><td>Immobilon-FL transfer membrane</td><td>Merck Millipore</td><td>IPFL0010</td><td>This is a PVDF transfer membrane that has 45 &amp;micro;m pore size and is mentioned in the protocol as western blot membrane</td></tr><tr><td>4x Laemmli sample buffer</td><td>Biorad</td><td>161-0747</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Biorad</td><td>161-0710</td><td></td></tr><tr><td>10% Criterion TGX precast gel</td><td>Biorad</td><td>5671033</td><td></td></tr><tr><td>Prolong Gold antifade</td><td>Thermofisher Scientific</td><td>P36930</td><td>Used for mounting immunostained coverslips for long term storage</td></tr><tr><td>Methanol</td><td>VWR Analytical</td><td>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 2A). These colonies continued to grow and had a spindle-shaped cell morphology (Figure 2A-2D) in the early stages, which later progressed to a cobblestone-like morphology (Figure 2E-2F). Figure 3A shows the total number of cells versus time in culture, and each data point represents the cumulative number of cells harvested at each passage. Figure 3B depicts the average time taken for the first colony to arise in the collagen-treated 6-well plate after seeding the MNCs.
Characterization Using Western Blotting: 
Figure 4A shows the representative results of the Western blot membrane that was tested against the various antibodies. Our results suggest that the cells were positive for CD31 and CD34. The expression of CD31 (Figure 4B) and CD34 (Figure 4C) appeared to decrease over subsequent passages, whereas VEGFR2 (Figure 4D) was expressed equally in later passages, with the first passage having higher expression. We also observed that the EPCs did not express the mesenchymal cell marker &#945;-SMA (Figure 4E). Human umbilical vein endothelial cells (HUVEC) and valvular interstitial cells (VIC) cell lysates were used as positive and negative controls, respectively. HUVECs are known to express CD31, CD34, and VEGFR2, while VICs express &#945;-SMA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56021/56021fig1.jpg" /> 
Figure 1: Schematic of EPC Isolation. Start the isolation by diluting cord blood with HBSS and layering it carefully on top of the density gradient medium, as shown. Density gradient centrifugation of the layered blood is carried out to obtain distinct layers of the blood, which is comprised of MNCs (buffy coat), density gradient medium, granulocytes, and RBCs. The subset image provided shows these different layers. MNCs are collected and reseeded onto a collagen-treated cell culture plate. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56021/56021fig2.jpg" /> 
Figure 2. Progression of the Cell Colony. (A-E) Representative bright-field images of EPC colony progression over time. Note that the colony has spindle-shaped cells at the early stages, adopting the cobblestone morphology. (E) Representative image of EPC cultured in a T-75 flask at passage 3. Scale bar = 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56021/56021fig3.jpg" /> 
Figure 3: Growth Curves of EPCs. (A) The graph depicts the number of cells growing over a period of time, and each of the data point denotes the cell number harvested during each passage (P0 - P10) (n = 4). (B) Time taken for the first EPC colony to appear in the 6-well plate (n = 4). The error bars denote the standard error of the mean (SEM). No statistical significance was found using one-way ANOVA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56021/56021fig4.jpg" /> 
Figure 4: Western Blotting. (A) Western blot membrane stained with various antibodies. HUVEC lysate and VIC lysate were used as positive controls. (B) Average intensity of CD31 bands normalized with &#945;-tubulin. (C) Average intensity of CD34 bands normalized with &#945;-tubulin. (D) Average intensity of VEGFR2 bands normalized with &#945;-tubulin. (E) Average intensity of &#945;-SMA bands normalized with &#945;-tubulin (n = 3 - 6). The error bars denote the SEM. No statistical significance was found using one-way ANOVA. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56021/56021fig5.jpg" /> 
Figure 5: Immunostaining. Representative images of EPCs cultured in EGM for 7 days and immunostained at various passages for CD31, CD34, VEGFR2, and &#945;-SMA. Scale bar - 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 1" src="/files/ftp_upload/56021/56021supfig1.jpg" /> 
Supplemental Figure 1: The graph depicts the number of cells per unit area over a period of 7 days when cultured in EGM, EBM, and DMEM. EGM showed higher cell growth compared with the media (EBM and DMEM). The data showed statistical significance, with p &#60;0.01, using one-way ANOVA (n = 2). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021supfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 2" src="/files/ftp_upload/56021/56021supfig2.jpg" /> 
Supplemental Figure 2: Representative images of EPCs cultured in EBM for 7 days and immunostained for CD31, CD34, VEGFR2, and &#945;-SMA. Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021supfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplemental Figure 3" src="/files/ftp_upload/56021/56021supfig3.jpg" /> 
Supplemental Figure 3: Representative images of EPCs cultured in DMEM for 7 days and immunostained for CD31, CD34, VEGFR2, and &#945;-SMA. Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56021/56021supfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

isolation-endothelial-progenitor-cells-from-human-umbilical-cord

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

11.1K Views.  University of Arkansas. Het doel van dit protocol is te isoleren van endotheliale voorlopercellen van bloed van de navelstreng. Enkele van de toepassingen zijn het gebruik van deze cellen als een biomarker voor het identificeren van patiënten met cardiovasculaire risico, behandeling van ischemische ziekten, en creëren weefsel-engineered vasculaire en hart ventiel construeert.

Video: Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

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 primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood 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 Human Umbilical Vein Endothelial Cells (HUVEC)

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, lumen formation, anastomosis, and formation of a new basement membrane. Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels often do not resemble vessels in vivo. Here we demonstrate an optimized in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts. This model recapitulates all of the key early stages of angiogenesis, and importantly the vessels display patent intercellular lumens surrounded by polarized EC. Vessels can be easily observed by phase-contrast and time-lapse microscopy, and recovered in pure form for downstream applications.

Isolation of Human Umbilical Vein Endothelial Cells HUVEC

This video protocol illustrates the isolation and culture of human umbilical vein endothelial cells (HUVEC) from human umbilical cord. Once isolated these cells can be used for in vitro angiogenesis assays like the Optimized Fibrin Gel Bead Assay also demonstrated by the Hughes lab.

Angiogenesis is a complex multi-step process, where in response to angiogenic stimuli, new vessels are created from the existing vasculature. These steps ...

Biology

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation of Human Umbilical Vein Endothelial Cells and Their Use in the Study of Neutrophil Transmigration Under Flow Conditions

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, neutrophils are the first inflammatory cells to migrate to the site of injury. Recruitment of neutrophils to an injury site is a stepwise process that includes first, dilation of blood vessels to increase blood flow; second, microvascular structural changes and escape of plasma proteins from the bloodstream; third, rolling, adhesion and transmigration of the neutrophil across the endothelium; and fourth accumulation of neutrophils at the site of injury2,3. A wide array of in vivo and in vitro methods has evolved to enable the study of these processes4. This method focuses on neutrophil transmigration across human endothelial cells.
One popular method for examining the molecular processes involved in neutrophil transmigration utilizes human neutrophils interacting with primary human umbilical vein endothelial cells (HUVEC)5. Neutrophil isolation has been described visually elsewhere6; thus this article will show the method for isolation of HUVEC. Once isolated and grown to confluence, endothelial cells are activated resulting in the upregulation of adhesion and activation molecules. For example, activation of endothelial cells with cytokines like TNF-&alpha; results in increased E-selectin and IL-8 expression7. E-selectin mediates capture and rolling of neutrophils and IL-8 mediates activation and firm adhesion of neutrophils. After adhesion neutrophils transmigrate. Transmigration can occur paracellularly (through endothelial cell junctions) or transcellularly (through the endothelial cell itself). In most cases, these interactions occur under flow conditions found in the vasculature7,8.
The parallel plate flow chamber is a widely used system that mimics the hydrodynamic shear stresses found in vivo and enables the study of neutrophil recruitment under flow condition in vitro9,10. Several companies produce parallel plate flow chambers and each have advantages and disadvantages. If fluorescent imaging is needed, glass or an optically similar polymer needs to be used. Endothelial cells do not grow well on glass.
Here we present an easy and rapid method for phase-contrast, DIC and fluorescent imaging of neutrophil transmigration using a low volume ibidi channel slide made of a polymer that supports the rapid adhesion and growth of human endothelial cells and has optical qualities that are comparable to glass. In this method, endothelial cells were grown and stimulated in an ibidi &mu;slide. Neutrophils were introduced under flow conditions and transmigration was assessed. Fluorescent imaging of the junctions enabled real-time determination of the extent of paracellular versus transcellular transmigration.

This article first describes a procedure for isolating human endothelial cells from umbilical veins and then shows how to use these cells to examine neutrophil transmigration under flow conditions. By using a low-volume flow chamber made from a polymer with the optical characteristics of glass, live-cell fluorescent imaging of rare cell populations is also possible.

Neutrophils are the most abundant type of white blood cell. They form an essential part of the innate immune system1. During acute inflammation, ...

Immunology and Infection

Isolation of Precursor B-cell Subsets from Umbilical Cord Blood

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation.	
This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Here we describe a protocol for isolating subsets of precursor B-cells from umbilical cord blood. A sufficient quantity and quality of nucleic acids may be extracted from the cells and used in subsequent assays utilizing DNA or RNA.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ cells.

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

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.

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 ...

Medicine

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.

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 ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

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