Original Article
Evaluating The Expression of Self-Renewal Genes in Human
Endothelial Progenitor Cells
Hamid Bayat, M.Sc.1, Fardin Fathi, Ph.D.2, Habibollah Peyrovi, M.D.3, Seyed Javad Mowla, Ph.D.1*
1. Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran
2. Cellular and Molecular Research Center, Kurdistan University of Medical Sciences, Sanandaj, Iran
3. Nanomedicine and Tissue Engineering Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
* Corresponding Address: P.O.Box: 14115-145, Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat
Modares University, Tehran, Iran
Email: sjmowla@modares.ac.ir
Received: 4/Jan/2012, Accepted: 16/Jul/2012
Abstract
Objective: Endothelial progenitor cells (EPCs) have a potential application for cell therapy, however, their biological nature is not well-understood. EPCs also possess some
stemness features, such as their clonogenicity and differentiation capacity. The main aim
of this study was to evaluate the expression of certain transcription factors regulating selfrenewal property of stem cells.
Materials and Methods: In this experimental study, peripheral blood mononuclear cells
were isolated from fresh human blood of several volunteers and were cultured in ibronectin-coated plates. EPCs were identiied based on their morphology and growth characteristic. Then, the expression of some markers implicated in self-renewal capacity was
assessed in the isolated cells using reverse transcription-polymerase chain reaction (RTPCR) and immunocytochemistry.
Results: Expression of the cell surface markers, CD31 and CD34, was determined by
RT-PCR and immunocytochemistry. Furthermore, these cells had the ability for Di-ACLDL incorporation as well as attachment to lectin I. EPCs did not express the main stem
cell markers, like OCT4-A, Nanog, and Sox2; nevertheless, they expressed the weaker
pluripotent markers, including OCT4B and OCT4-B1 spliced variants, such as Nucleostemin and ZFX. Furthermore, the expression of Nucleostemin and ZFX genes revealed
a decreasing pattern from days 4th to 11th.
Conclusion: The main regulators of stem cell self-renewal genes, including OCT4-A, Nanog, and Sox2 are not expressed in EPCs. Forced expression of these genes can elevate
the stemness property and clinical application of EPCs.
Keywords: Endothelial, Progenitor Cell, Marker, Stem Cells, Peripheral Blood
Cell Journal (Yakhteh), Vol 14, No 4, Winter 2013, Pages: 300- 307
Citation: Bayat H, Fathi F, Peyrovi Ho, Mowla SJ. Evaluating the expression of self-renewal genes in human endothelial
progenitor cells . Cell J. 2013; 14(4): 300-307.
Introduction
Vasculogenesis was initially thought to be as the
generation of new blood vessels by stem or progenitor cells, taking place only during embryonic
development (1). The belief is later revised after
Asahara et al. identiied the endothelial progenitor
cells (EPCs) in adult human peripheral blood (2).
EPCs, a heterogeneous subpopulation of bone marrow mononuclear cells, are mobilized in response
to ischemia and are able to differentiate into endothelial cells in ischemic organs (3). Conventionally, EPCs are recognized by the expression of
both hematopoietic stem cell and endothelial cell
markers (4). However their molecular criteria are
not well-understood.
EPCs play an essential role in wound healing
process as well as pathogenic conditions, such as
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Expression of Self-Renewal Genes in Human EPCs
tumor growth (5) and metastases (6). Dysfunction
of EPCs occurs by various risk factors of coronary
artery disease, such as aging, diabetes, hypercholesterolemia, and hypertension (7). Researchers have
now begun to estimate the potential therapeutic
use of EPCs in treatment of ischemic cardiovascular disease. Results indicate that EPCs are a safe
and powerful tool in cell therapy of cardiovascular
disease (8).
EPCs seem to have some features of stem
cells, such as their clonogenicity and differentiation capacity (4). Similar to stem cells, they
have the capacity of differentiation into several types of cells, including: cardiomyocytes,
smooth muscle cells and endothelial cells (9).
An important characteristic of stem cells is
their self- renewal property, controlled by certain transcription factors which are exclusively
expressed in stem cells (10). The main stem
cell self-renewal regulatory genes are OCT4,
Nanog, and Sox2, as well as Nucleostemin and
ZFX. Evaluating the expression of these genes
is one of the first steps in assigning the stemness
property to stem cells.
While EPCs seem to be an important cell
source for cell therapy, many aspects of their
identity and characteristic still remain unclear.
For example, a potential expression of selfrenewal genes in EPCs has not been comprehensively examined. In this study, we isolated
endothelial progenitor cells from human peripheral blood and characterized the cellular
and molecular identity of isolated cells as putative stem/progenitor cells.
Materials and Methods
Cell isolation and culture
In this experimental study, peripheral blood
(100 ml) was obtained from some healthy volunteers. Mononuclear cells were separated from
other components of the peripheral blood by
density-gradient centrifugation on Histopaque
1077 (Sigma Aldrich, Belgium). Isolated cells
were resuspended in EGM-2-MV Bullet Kit
(Clonetics, USA) medium consisting of endothelial basal medium, 5% fetal bovine serum
human epidermal growth factor (hEGF), vascular epidermal growth factor (VEGF), human
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growth factor-basic recombinant (hFGF-B),
insulin-like growth factor 1 (IGF-1), ascorbic
acid, and heparin. The peripheral blood mononuclear cells (PBMNCs) (1 × 107 per well)
were then cultured in the coated (2.5µg per cm2
fibronectin; Sigma/Aldrich, Japan) six-well
plates, and incubated in a 5% CO2 incubator
at 37˚C. After 4 days of culture, the medium
containing non-adherent cells were collected,
centrifuged, washed with phosphate-buffered
saline (PBS), resuspended in fresh medium, and
returned to the cell culture until 7th day. The attached cells were cultured for 3 weeks. Culture
medium was changed every 3 day. The experimental procedure was approved by the Ethical
Board of Tarbiat Modares University.
RNA extraction and RT-PCR
Total RNA was extracted on days 4th, 7th and 11th
after seeding of mononuclear cells (MNCs) using
the RNX Plus kit according to the manufacturer’s
instruction (CinnaGen, Iran). The quality of RNA
was estimated by gel electrophoresis as well as
by optical density at 260 nm and 280 nm, respectively.
Therefore, 1 µg of the extracted RNA was
treated with RNase-free DNase (Fermentas,
Germany) and used for cDNA synthesis using
0.2 µg random hexamer and MMuLV Reverse
Transcriptase (Fermentas, Germany) in a 20
µg reaction according to the manufacturer’s
instructions. For each sample, a negative control, without addition of MMuLV, was also
employed. PCR was performed using 2µg of
synthesized cDNA with 1U of Taq polymerase
(CinnaGen, Iran), 2.5 μl of 10x PCR buffer, 0.5
μl of 20 mM deoxy nucleotide tri-phosphates
(dNTPs), 0.5 μl of each primer and deionized
distilled water in a 25μl PCR reaction. The
PCR amplification was carried out for 35 cycles for all genes, except for the internal control gene GAPDH, where the number of cycles
was 30. The cycling conditions were as follows:
94˚C for 30 seconds, 55- 64˚C (depending on
primers used, Table 1) for 30 seconds, 72˚C for
45 seconds, and a final extension of 72˚C for 5
minutes. Each experiment was repeated at least
three times.
Bayat et al.
Table 1: The sequences, annealing temperatures, and product sizes of the primers used to amplify genes of interest
Genes
Product
size (bp)
Annealing
temperature
(°C)
Forward primer sequence
Reverse primer sequence
Oct4-A
496
63.5
CTTCTCGCCCCCTCCAGGT
AAATAGAACCCCCAGGGTGAGC
Oct4-B
267
64
AGACTATTCCTTGGGGCCACAC
CTCAAAGCGGCAGATGGTCG
Oct4-B1
492
64
AGACTATTCCTTGGGGCCACAC
CTCAAAGCGGCAGATGGTCG
Sox2
426
60
ATGGGTTCGGTGGTCAAGTC
GTGGATGGGATTGGTGTTCTC
Nanog
470
59
ACCTATGCCTGTGATTTGTGG
AAGAGTAGAGGCTGGGGTAGG
Nucleostemin
747
60
CAGAGATCCTCTTGGTTGCAG
AATGAGGCACCTGTCCACTC
ZFX
465
60
TGATTCCAGGCAGTACCAAAC
TGACGAAAACCCTTACCACAC
CD31
578
59.5
AGGACATCCATGTTCCGAGA
TGAACCGTGTCTTCAGGTTG
CD34
219
56
TGAGCCTCTCACCTGTACTC
AGGAGCTGATCTGGGCTATG
KDR
180
61
CCCTGCCGTGTTGAAGAGTT
GGACAGGGGGAAGAACAAAA
VCAM1
375
58
AGAAATGCCCATCTATGTCC
CGGCATCTTTACAAAACCTG
Tie1
173
57
GCAAACTCTGCTGTCTAACC
GGATGCCCAGGATAGCTATG
GAPDH (1)
139
64
GCCCCAGCAAGAGCACAAGA
TAGGCCCCTCCCCTCTTCAA
GAPDH (2)
224
60
AGGGTCTCTCTCTTCCTCTTGTGCTC
CCAGGTGGTCTCCTCTGACTTCAACA
Immunocytochemistry
Immunocytochemistry was performed on EPCs at
7th day of seeding. Briely, the cells were ixed with
4% paraformaldehyde for 20 minutes at room temperature. Cells were then washed with PBS containing 1% bovine serum albumin (BSA), 0.02% Triton
X-100 and 10% rabbit serum while incubated with
blocking solution for 30 minutes at room temperature. The cells were then incubated with primary polyclonal antibodies (anti-CD31, 1:40; anti-CD34, 1:50
from Dako, USA and anti-OCT4, 1:50 from Santa
Cruz Biotechnology, USA) for 2 hours at 37˚C. After washing with PBS, the cells were incubated with
the secondary antibodies (dilution 1:100) as follows:
anti-mouse IgG conjugated with cy3 (Cmicon, CA,
USA), anti-goat IgG conjugated with FITC (Jackson
Immuno Research Labs, USA), and anti-rabbit IgG
conjugated with luorescein isothiocyanate (FITC)
(Sigma-Aldrich, USA), respectively, for attachment
to anti-CD31, anti-CD34 and anti-OCT4 for 45 minutes at 37˚C followed by washing further three times
with PBS at room temperature.
Finally, the immunoreactivity was analyzed with
a luorescent microscope (Nikon TE300, Japan).In
a similar approach, the negative control were prepared, except for the omission of primary antibodies.
Results
Isolation of endothelial progenitor cells
On the 7th day of MNCs culture, ex vivo expanded EPCs showed two kinds of morphology, including
spindle-shape cells and round cells, yet some EPC
colonies were observable. With continued culture,
the number of round cells decreased and spindleshape cells become dominant cell type (Fig 1).
By the 7th day, the identity of EPCs was conirmed
through double staining for Di-AC-LDL and lectin
binding (Fig 2). The obtained data was further validated by immunocytochemistry results revealing that the
isolated cells express CD31 and CD34, cell surface
markers (Fig 3). Moreover, further characterization
of EPCs was carried out by studying the expression
of CD34, KDR, VCAM-1, CD31 and Tie-1 by RTPCR, where all of these markers were expressed in
isolated cells (Fig 4). All together, our data conirmed
the genuine nature of the isolated cells as EPCs.
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Expression of Self-Renewal Genes in Human EPCs
A
B
C
D
Fig 1: Morphological features of isolated endothelial progenitor cells before and after differentiation. A. Mononuclear cells
isolated from human peripheral blood appeared small and round. B. Most cells were attached to the plates coated with human
ibronectin (day 4). The attached cells gradually exhibited a spindle-shaped, endothelial cell-like morphology on day 7th C. and
day14th D. after seeding (This igure has also been printed in full-color at the end of this issue).
A
B
C
Fig 2: Fluorescence images of isolated endothelial progenitor cells on day 7th after seeding showing positive staining for: A.
acetylated LDL (DiI-acLDL) and B. lectin. C. The phase contrast counterpart of A and B (This igure has also been printed in
full-color at the end of this issue).
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Bayat et al.
A
B
C
D
control. Also, a no-reverse transcription (noRT) control was accompanying all PCR experiments, as a negative control.
Fig 3: A. Immunocytochemistry of EPCs double-stained
with anti-CD31, and B. anti-CD34 antibodies. C. Cells'
nuclei were visualized with DAPI . D. The same phase
contrast view of the cells in A-C (This igure has also been
printed in full-color at the end of this issue).
Fig 4: ART-PCR analysis of the expression of endothelial
markers in endothelial progenitor cells (EPCs) at day 7th
after seeding. As it is evident, EPCs express CD34, CD31,
KDR, VECAM-1 and Tie-1 genes. GAPDH was used as an
internal control.
Evaluating the expression of stem cell marker
genes in EPCs
RT-PCR analysis of EPCs on 7th day of culture revealed that the main regulators of self-renewal property in embryonic stem cells, namely
OCT4A; Nanog; and Sox2 are not expressed
in EPCs (Fig 5). However, the adult stem cell
markers, Nucleostemin and ZFX, and to a lesser
extent the other spliced variants of OCT4, like
OCT4B and OCT4B1 were expressed in the
cells (Fig 5). The expression of genes was compared to that of NT2, a human carcinoma cell
line used as a positive control. Moreover, the
expression of GAPDH, a housekeeping gene,
was monitored in all experiments as an internal
Fig 5: RT-PCR analysis of the expression of some selfrenewal genes in EPCs on day 7th after seeding. Note that
EPCs do not express OCT4A variant, Nanog, and Sox2, but
express Nucleostemin (NS), ZFX, OCT4B, and OCT4B1
variants. The embryonic carcinoma cell line NT2 was used
as a positive control for pluripotency. GAPDH was used as
an internal control.
Immunocytochemistry analysis of the subcellular
distribution of OCT4 demonstrated a cytoplasmic
signal in EPCs, with no immunoreactivity presence
within the nuclei of the cells. The observations further
conirmed the expression of OCT4B and/or OCT4B1
variants which are localized within the cytoplasm
of the cells. On contrary, the OCT4 immunoreactivity was primarily visualized within the nuclei of the
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Expression of Self-Renewal Genes in Human EPCs
pluripotent cell line NT2, which is consistent with
the expression of OCT4A variant within these
cells. As expected, no staining signal was detected
in the negative control, in which all of the conditions were kept the same, except for the omission
of the primary antibody (Fig 6).
The expression levels of the Nucleostemin
and ZFX genes seem to be down-regulated from
days 4th to 11th. For Nucleostemin and ZFX
genes, an apparent decrease in the expression
level was observed on days 4th and 7th of the
seeding (Fig 7).
A
B
C
a
b
c
Fig 6: Sub-cellular localization of OCT4 in EPCs was stained with a polyclonal antibody against C-terminal domain of OCT4.
A. Immunocytochemistry revealed a cytoplasmic immunoreactivity for EPCs. B. NT2 cells were used as a positive control and
demonstrated a primarily nuclear immunoreactivity. C. Negative control slide, all the conditions were the same as A and B, except for the elimination of the primary antibodies. a, b, and c depict the phase-contrast counterparts of A, B and C, respectively
(This igure has also been printed in full-color at the end of this issue).
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Bayat et al.
(10). Our results revealed that none of these genes
is expressed in EPCs; therefore, we suggest that
EPCs do not have self-renewal properties similar to ES cells. This inding is in accordance with
some recent reports that suggest OCT4A is exclusively expressed in pluripotent ES/EC cells (13).
The absence of OCT4A expression in already reported in some adult stem cells, such as peripheral
blood mononuclear cells (14) and rat bone marrow
stromal stem cells (15).
Fig 7: Changes in NS and ZFX genes expression during differentiation of EPCs. As it is evident, there is a decline in the
expression of NS and ZFX in the course of cell differentiation from the day 4th to day 11th of cell seeding.
Discussion
EPCs in the human peripheral blood are a potential source of stem cells to promote angiogenesis in
ischemic tissues (3). However, the cells have not
yet been well characterized and many questions
remain to be answered about their nature and molecular characteristics.
In the present study, the isolation of EPCs was
carried out by an ex vivo expansion of a heterogeneous PBMC fraction. In this method of cell isolation, the number of isolated cells is higher than
other methods (11). In this regard, using magnetic
beads for EPCs evaluation may inluence the viability of the cells. It can also preferentially exclude some cells with endothelial potential, thereby decrees the number of cells within an isolation
population (12). Moreover, it seems that cultivated
EPCs in this method are more heterogeneous. After 7 days of culture, the attached cells have the
ability of Dil-AC-LDL incorporation, lectin banding, as well as the expression of CD34, CD31,
VCAM-1, Tie-1 and KDR genes. All of the mentioned properties indicate an endothelial identity
for the expanded cells.
After isolation and expansion of EPCs, the expression of self-renewal genes, like OCT4, Nanog,
and Sox2 were evaluated by RT-PCR .These genes
form the core of main regulatory network that
induce the expression of genes maintaining the
pluripotency of embryonic stem cells (ESCs), and
also suppress some genes inducing differentiation
However, our data is in contrast with Romagnani et al. who have previously reported the expression of OCT4 and Nanog in a subpopulation
of EPCs, CD14 + CD34 LOW (16). Interestingly,
all the previous publications on OCT4A expression
in different cell types is doubted with the presence of several expressed pseudogenes of OCT4,
as well as with the presence of two other splicing
variants of the gene (OCT4B and OCT4B1). The
very high sequence conservation between OCT4A,
its pseudogenes, and splicing variants could be
likely a source of misinterpretation of RT-PCR and
immunostaining experiments, as genuine OCT4A
expression. Therefore, here we designed speciic
primers to avoid pseudogenes ampliication, and
also to discriminate OCT4 alternatively spliced
variants (13). Our data revealed no expression of
OCT4A, but expression of OCT4B and OCT4B1
spliced variants in endothelial progenitor cells.
While OCT4A has a well-known critical role in
maintaining the pluripotency of embryonic stem
cells, little is known about the functional role of
OCT4B and OCT4B1 variants in pluripotent and
non-pluripotent cells. However, it has been reported recently that OCT4B and OCT4B1 are also
expressed in non-pluripotent cells, where their
proteins are localized within the cytoplasm of the
cells (13, 17, 18).
In addition, we also detected for the irst time the
expression of ZFX, a newly identiied self-renewal regulatory gene, in EPCs. ZFX has been shown
to be expressed in both embryonic and adult (hematopoietic) stem cells, likely an anti-apoptotic role in these cells (19, 20). Hematopoietic stem cells are one of the major sources
of EPCs in peripheral blood. Therefore, it is
concluded that the expression of ZFX in this
pathway continues until completion of the
differentiation of EPCs which it likely seems
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Expression of Self-Renewal Genes in Human EPCs
to have the same role in self-renewal of EPCs. Furthermore, our results demonstrated that EPCs express Nucleostemin (NS) gene and that its expression was down-regulated from days 4th to 11th. NS
is highly expressed in bone marrow stromal cells,
and the expression is suddenly turned off with the
induction of neural differentiation in the cells (15).
Based on recent reports, NS was hypothesized to
be a proliferating factor in stem cells, where it regulates cell cycle proceeding (21).
7.
8.
9.
Conclusion
Our data revealed that EPCs express endothelial markers, an indication that they are in a late
stage of cell differentiation. The data also revealed
that none of the main ES-speciic regulatory genes
OCT4A, Sox2 and Nanog is expressed in EPCs;
a inding that indicates these cells have a limited
self-renewal capacity compared to embryonic
stem cells. The latter inding suggests that the cells
could be considered as a safer source compared to
ES cells for cell therapy, in terms of the likelihood
of teratoma generation.
Acknowledgments
10.
11.
12.
13.
14.
This study was supported by a joint research
grant from the Tarbiat Modares University and
the Kurdistan University of Medical Sciences.
The authors have no conflict of interest in this
paper.
15.
16.
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