Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

Summary

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.

Abstract

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 marrow^2,3. 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 blood^4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs^7,8. 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.

Introduction

Several investigators have studied the characteristics and potential of human EPCs^{5,10,11,12,13}. 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 structures^4,14. 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 applications^4,15,16. The number of EPCs in an individual has been shown to be correlated with cardiovascular pathology^{9,15,16,17,18,19,20}. Other studies have also differentiated EPCs into a valve fibroblast-like phenotype and proposed that these cells could be used for tissue-engineering heart valves^7,21.

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 groups^1,17,22,23, 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 markers^13,24,25. 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 (α-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 vivo^4,7. 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 α-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's Modified Eagle Medium (DMEM).

Protocol

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

1. Preparation of reagents.
   1. 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 µg/mL ascorbic acid, 5 ng/mL recombinant human epidermal growth factor (rhEGF), 22.5 µg/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 µg/mL hydrocortisone, and 2% penicillin-streptomycin-glutamine. Sterilize the culture medium using a 0.2 µm polyethersulfone (PES) membrane vacuum filter.
   2. Prepare rat tail I collagen solution at a concentration of 50 µg/mL using 0.02 N acetic acid. Sterilize this solution using a 0.2 µm syringe filter.
   3. 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 °C for at least 1 h before seeding.
   4. Dilute approximately 25 mL of cord blood with Hanks Balanced Salt Solution (HBSS) at a 1:1 concentration.
   5. Prepare radio-immuno precipitation assay (RIPA) Buffer using 150 mM sodium chloride, 1% Triton X-100, 0.5% sodium deoxycholate, 1 mM β-glycerophosphate, 0.1% SDS, and 50 mM Tris (pH 8.0).
2. Isolation of endothelial progenitor cells.
   1. Warm all prepared reagents in a water bath maintained at 37 °C prior to isolation.
   2. Add 20 mL of density gradient medium reagent to a 50 mL centrifuge tube.
   3. 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.
   4. 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.
   5. 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.
   6. 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.
   7. Add an equal volume of EBM to the collected MNCs. Centrifuge these tubes at 500 x g for 10 min at 4 °C. Discard the supernatant.
      NOTE: The cell pellet will appear red because of the presence of red blood cells/erythrocytes.
   8. 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.
   9. Centrifuge the tubes at 500 x g for 10 min at 4 °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.
   10. Resuspend the pellet in prepared EGM and use the hemocytometer to count the MNCs.
   11. 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).
   12. 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 °C, 5% CO₂ incubator.
   13. 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 °C, 5% CO₂ incubator.
   14. Replenish the plate with fresh EGM medium every day for 7 days. After 7 days, change the EGM medium every other day.
   15. 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.
3. Expansion of endothelial progenitor cells.
   1. Coat the T-25 culture flask with rat tail I collagen (50 µg/mL) and place it in the 37 °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.
   2. Trypsinize the endothelial cell colonies when they reach about 3 mm in size using 150 µL of 0.05% Trypsin-EDTA solution in each well. Place the 6-well plate back into the 37 °C, 5% CO₂ incubator for 2 - 3 min.
   3. 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.
   4. Count the cells using a hemocytometer and calculate the initial seeding density.
   5. 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 °C, 5% CO₂ incubator.
      NOTE: Cells can be plated in EBM and DMEM to compare the growth with EGM.
   6. 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).

2. Characterization of Isolated Cells Using Western Blotting

1. Add 50 - 100 µL 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.
2. Pipette up and down vigorously to rupture the cells and release the proteins. Collect and transfer the buffer to a microcentrifuge tube.
3. 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 °C for long-term storage.
4. Quantify the amount of proteins in each tube using either Bradford's or BCA assay^27,28,29.
5. Carry out Western blotting using standard procedures^27,28,29. Analyze 5 µg of total protein for the expression of CD31, CD34, VEGFR2, and α-SMA. Use α-tubulin for normalizing the data.

3. Indirect Immunofluorescence

1. 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.
2. Coat the coverslips with 50 µg/mL of rat tail I collagen and transfer the plate to a 37 °C, 5% CO₂ incubator for at least 60 min.
3. 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.
4. Seed 250,000 cultured EPCs in each well and place them back in the 37 °C, 5% CO₂ incubator for at least 3 days before carrying out immunostaining.
5. 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.
6. Carry out immunostaining using a standard protocol^27,28,29. Use the antibodies at their appropriate dilutions (e.g., CD31 (1:20), CD34 (1:50), α-SMA (1:100), and VEGFR2 (1:50)).
7. Take images of the immunostained coverslips using an epifluorescence microscope.

Results

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...

Discussion

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 cells^5...

Materials

List of materials used in this article

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