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 pathophysio....

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) suppl....

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