Standardization of a Colony Assay to Further Characterize Endothelial Precursor Cells in Blood
and Bone Marrow
SM Faulkes1, C Pereira1, CE Peters1, TE Thomas1, AC Eaves2 and E Clarke1
StemCell Technologies Inc, Vancouver, Canada, and 2Terry Fox Laboratory, BC Cancer Agency, Vancouver, Canada
1
Background
Results
Blood vessel development is a regulated process involving the proliferation,
migration, and remodelling of endothelial cells from adjacent pre-existing blood
vessels (angiogenesis) or the differentiation of endothelial progenitor cells (EPCs)
from mesodermal precursors (vasculogenesis). EPCs or angioblasts were originally
thought to be present only during embryonic development, however accumulating
evidence in the past several years suggests that they can persist in adult life. This
has generated interest in the use of EPCs for neovascularization of ischemic or
injured tissue and for the clinical assessment of risk factors for various diseases.
There is currently no standardized procedure for the isolation and in vitro culture of
EPCs. The most commonly used isolation method is culturing mononuclear cells on
a variety of substrates. There has been no systematic study regarding the
physiological variations in the number of EPCs in healthy individuals. Recently, Hill et
al. (NEJM 348:593, 2003) described an in vitro colony assay that showed an inverse
correlation between the number of circulating endothelial colony forming cells
(CFU-EC) and the risk of cardiovascular disease. We have used this assay in
conjunction with flow cytometry to characterize the CFU-EC in normal peripheral
blood and bone marrow and evaluated the cell populations removed following
adherence on fibronectin coated plates. Although there is a general consensus of the
phenotype of the mature endothelial cell, less is known about the phenotype of the
circulating CFU-EC. We further attempted to evaluate the cell surface markers
expressed on the circulating CFU-EC using a panel of antibodies.
Methods
Figure 1. 7 day EPC colony assay
unprocessed BM / PB
Table 1. Flow cytometric analysis on bone marrow samples
Ficolled BM
prior to culture
(% mean ± SD)
Ficolled BM
following adherence
(% mean ± SD)
CD14+
7.8 ± 1.6
0.97 ± 0.50
CD105+/CD45-
2.0 ± 0.49
0.28 ± 0.14
CD146+/CD45+/CD3+
0.54 ± 0.29
0.42 ± 0.18
cell phenotype
Incubation for 48 hours on fibronectin coated plates removed 33.1 ± 5.3 % of
mononuclear cells including monocytes (CD14+) and mature endothelial cells
(CD105+CD45-).
Table 2. Frequency of endothelial progenitors in bone marrow and peripheral
blood
sample
number
frequency of CFU-EC
bone marrow
5
1:10,000 ± 2500
peripheral blood
16
1:120,000 ± 170,000
Bone marrow samples were purchased from Cambrex, MD. All samples were
obtained from young volunteer donors and cultured as described in Figure 1.
Peripheral blood samples were obtained with consent from male donors (age range
20-40 years) and cultured as described in Figure 1. The data in Table 2 suggests
bone marrow samples have a higher frequency of CFU-EC, although the relative low
sample number and limited age range may explain the low variability in this data.
Peripheral blood results suggest a significant lower frequency of CFU-EC. The large
variability in the data maybe due to the broader age range and unknown clinical
situation.
Figure 2. Representative colonies after 7 days of culture
All photographs were taken at 125X magnification on day 7. Photograph 3 shows von
Willebrand Factor (vWF) staining using the APAAP procedure in which colonies were
fixed with 4% paraformaldehyde and stained at 1:50 dilution with vWF (clone 2F2-A9
from BD Pharmingen).
Ficoll
collect buffy coat
wash 2x with PBS + 2% FBS
EndoCult™
100mL
FACS
FOR RESEARCH USE ONLY
StemCell Technologies Inc
Vancouver, BC (604)877-0713
resuspend in EndoCultTM
plate 5x106 cells/well on
BD BioCoatTM fibronectin 6 well dish
day -2
2 days
collect non-adherent cells
1. Peripheral blood-derived CFU-EC
FACS
Plate 1x106 cells/well (PB)
5x105 cells/well (BM) on
BD BioCoatTM fibronectin 24 well dish
day 0
3 days
change media
day 3
2-4 days
count colonies
day 5-7
Flow cytometric analysis (FACS) was performed on ficolled bone marrow (BM) and
peripheral blood (PB) samples prior to culture and following 48 hours of adherence
on fibronectin coated plates.
2. Bone marrow-derived CFU-EC
3. Peripheral blood CFU-EC stained
with vWF
Which cell is the circulating CFU-EC?
Although there is a general consensus on the phenotype of the mature endothelial
cell, the phenotype of the circulating progenitor remains incompletely defined and
may depend on the assay used. We attempted to evaluate using a panel of
antibodies, specific cell populations in ficolled peripheral blood or bone marrow that
may contain candidate endothelial cell precursors. Since the frequency of CFU-EC
in bone marrow is approximately 10 times higher than that of peripheral blood, we
specifically looked for a population of cells from both sources showing similar
proportions (Figure 3).
Figure 3. FACS analysis of fresh ficolled peripheral blood and bone marrow
peripheral blood
R4=CD45-/loSSCloPIlo
R3=CD45+PIlo
IgG1 FITC
IgG1 FITC
0.41%
<0.01%
0.16%
0.07%
CD146 PE
0.58%
CD146 PE
0.02%
CD3 FITC
CD3 FITC
0.07%
0.28%
0.22%
0.46%
CD146 PE
CD146 PE
0.50%
2.04%
CD34 FITC
0.21%
CD146 PE
CD146 PE
0.08%
0.23%
18.53%
0.13%
10.54%
0.22%
4.06%
CD144 PE
CD144 PE
CD105 FITC
7.82%
1.50%
CD133 PE
CD34 FITC
0.69%
CD105 FITC
0.07%
0.10%
0.42%
1.24%
CD105 FITC
0.62%
`1.06%
7.51%
CD105 FITC
0.03%
2.27%
CD34 FITC
0.54%
CD133 PE
R4=CD45-/loSSCloPIlo
IgG1 PE
IgG1 PE
R3=CD45+PIlo
bone marrow
1.44%
1.58%
1.00%
1.13%
CD34 FITC
Cells were stained with antibodies in the dark, and washed with 1 µg/mL propidium iodide (PI). 100,000 events were collected per
tube on a FACSCalibur and files analyzed using CellQuest. % indicates % of total viable cells (defined as PIlo) that fall within the given
quadrant. Antibodies used: CD146 clone P1H12 (BD Pharmingen), CD105 clone SH2 (StemCell Technologies Inc), CD144 clone F8
(Santa Cruz Biotechnology), AC133 (Miltenyi).
Conclusions
• CFU-EC frequency is higher in bone marrow than peripheral blood
• Pre-plating on fibronectin coated dishes results in a decrease in CD14+ and CD105+CD45- populations
• Although CFU-EC morphology was variable, all colonies expressed von Willebrand Factor
• Phenotypic characterization remains difficult since antibodies associated with endothelial cells may be co-expressed on other cell populations
StemCell Technologies
Tel: 604.877.0713 • Fax: 604.877.0704
Toll Free Tel: 1.800.667.0322 • Toll Free Fax: 1.800.567.2899
E-mail: info@stemcell.com
www.stemcell.com