European Journal of Cell Biology 95 (2016) 153163

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European Journal of Cell Biology

journal homepage: www.elsevier.com/locate/ejcb

Research paper

Effects of mesenchymal stem cell-derived cytokines on the functional

properties of endothelial progenitor cells
Witchayaporn Kamprom a,b , Pakpoom Kheolamai b,c,d , Yaowalak U-Pratya b,e ,
Aungkura Supokawej f , Methichit Wattanapanitch g , Chuti Laowtammathron b ,
Surapol Issaragrisil b,e,
a
Department of Immunology, Faculty of Medicine Siriraj hospital, Mahidol University, Bangkok, Thailand
b
Siriraj Center of Excellence for Stem Cell Research, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
c
Division of Cell Biology, Faculty of Medicine, Thammasat University, Pathumthani, Thailand
d
Center of Excellence in Stem Cell Research, Faculty of Medicine, Thammasat University, Pathumthani, Thailand
e
Division of Hematology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
f
Faculty of Medical Technology, Mahidol University, Bangkok, Thailand
g
Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Human mesenchymal stem cell (hMSC) is a potential source for cell therapy due to its property to promote
Received 27 August 2015 tissue repair. Although, it has been known that hMSCs promote tissue repair via angiogenic cytokines, the
Received in revised form interaction between hMSC-derived cytokines and the endothelial progenitor cells (EPCs), which play an
28 December 2015
important role in tissue neovascularization, is poorly characterized. We investigate the effect of cytokine
Accepted 3 February 2016
released from different sources of hMSCs including bone marrow and gestational tissues on the EPC
functions in vitro. The migration, extracellular matrix invasion and vessel formation of EPCs were studied
Keywords:
in the presence or absence of cytokines released from various sources of hMSCs using transwell culture
Mesenchymal stem cell
Neovascularization
system. The migration of EPCs was highest when co-culture with secretory factors from placenta-derived
Gestational tissues hMSCs (PL-hMSCs) compared to those co-culture with other sources of hMSCs. For invasion and vessel
formation, secretory factors from bone marrow-derived hMSCs (BM-hMSCs) could produce the maximal
enhancement compared to other sources. We further identied the secreted cytokines and found that
the migratory-enhancing cytokine from PL-hMSCs was PDGF-BB while the enhancing cytokine from BM-
hMSCs on invasion was IGF-1. For vessel formation, the cytokines released from BM-hMSCs were IGF1
and SDF-1. In conclusion, hMSCs can release angiogenic cytokines which increase the migration, invasion
and vessel forming capacity of EPCs. We can then use hMSCs as a source of angiogenic cytokines to induce
neovascularization in injured/ischemic tissues.
2016 Elsevier GmbH. All rights reserved.

Abbreviations: AM-hMSCs, amnion-derived human mesenchymal stem cells; 1. Introduction

ANGPT1, angiopoietin 1; ANGPT2, angiopoietin 2; BM-hMSCs, bone marrow-derived
human mesenchymal stem cells; CFSE, 5-(6)-carboxyuorescein diacetate succin-
imidyl ester; EPCs, endothelial progenitor cells; FGF2, broblast growth factor 2;
GAPDH, glyceraldehydes-3-phosphate dehydrogenase; HUVECs, human umbilical
vein endothelial cells; IGF1, insulin-like growth factor 1; IGF2, insulin-like growth
factor 2; IL6, interleukin 6; IL8, interleukin 8; PDGF, platelet-derived growth factor
beta; PL-hMSCs, placenta-derived human mesenchymal stem cells; PlGF, placenta
growth factor; SDF1, stromal cell-derived factor 1; TGF, transforming growth factor
beta; UC-hMSCs, umbilical cord-derived human mesenchymal stem cells; VEGFA,
vascular endothelial growth factor A; VEGFR2, vascular endothelial growth factor
receptor 2; vWF, Von willebrand factor; WJ-hMSCs, Whartons jelly-derived human
mesenchymal stem cells.
Corresponding author at: Division of Hematology, Department of Medicine,
Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Tel.: +66 2 419 4448 50; fax: +66 2 411 2012.
E-mail address: surapolsi@gmail.com (S. Issaragrisil).
Human mesenchymal stem cells (hMSCs) are multipotent
stem/progenitor cells which are present in bone marrow, adipose
tissue, and postnatal sources including umbilical cord, placenta, the
Whartons jelly, and amnion (Bunnell et al., 2008; Int Anker et al.,
2004; Lee et al., 2004; Wang et al., 2004). hMSCs are considered to
be a potential source for cellular therapy in several disorders such
as acute graft-versus-host disease (acute GvHD), myocardial infarc-
tion, and neurological diseases (Uccelli et al., 2008; Williams et al.,
2013) due to their immunomodulatory property and multilineage
differentiation capacity (Jiang et al., 2002; Nauta and Fibbe, 2007;
Pittenger et al., 1999). The therapeutic effects of hMSCs are believed
due to soluble factors or cytokines released from hMSCs rather

http://dx.doi.org/10.1016/j.ejcb.2016.02.001
0171-9335/ 2016 Elsevier GmbH. All rights reserved.
154 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163
than their direct effect (Meirelles Lda et al., 2009). hMSC-derived
soluble factors can exert immunomodulatory effect, reduce tissue
inammation and prevent apoptosis of several cell types in injured
tissues which might be the underlying mechanism in the improve-
ment of tissue repair and inammatory disease (Gnecchi et al.,
2008; Wang et al., 2014). hMSCs can stimulate neovascularization
in the injured ischemic tissue via pro-angiogenic cytokines which
enhance endothelial cell proliferation and migration leading to neo-
vascularization and tissue restoration (Hung et al., 2007; Kamihata
et al., 2001; Li et al., 2010). It was the belief that mature endothelial
cells in the local vasculature are responsible for postnatal neovascu-
larization. However, recent studies have shown the important role
of endothelial progenitor cells (EPCs) in vascular homeostasis and
postnatal neovascularization in both physiological and pathological
conditions (Asahara et al., 1999; Melero-Martin et al., 2007; Tepper
et al., 2005). There are no reported information on the effects of
cytokines released from hMSCs on the endothelial progenitor cells.
In order to better understand the therapeutic effect of hMSCs on
neovascularization, we investigated the effects of soluble factors
released from hMSCs on the EPC functions including proliferation,
migration, invasion and vessel formation capacity in vitro, and also
identied those factors that play roles on various EPC functions. Sol-
uble factors released from gestational tissues-derived hMSCs were
chosen to study due to easily and non-invasive collection compared
to bone marrow-derived hMSCs (BM-hMSCs).
2. Materials and methods
2.1. Subjects
This study was approved by the Siriraj Institutional Review
Board, Faculty of Medicine Siriraj Hospital, Mahidol University
which was in accordance with the Declaration of Helsinki, the
Belmont Report, CIOMS Guidelines, and ICH-GCP. Human bone
marrow samples were obtained from healthy volunteers after giv-
ing written informed consent. The gestational tissues (umbilical
cord, Whartons jelly, placenta, and amnion) were obtained from
healthy newborns after receiving written informed consent from
their mothers.
2.2. Isolation and culture of hMSCs
Ten milliliter of heparinized bone marrow was collected for
hMSC isolation. Bone marrow-derived mononuclear cells were
then isolated using IsoPrep (Robbins Scientic Corporation,
USA) density gradient centrifugation, washed twice with PBS
(GIBCOTM , Invitrogen Corporation, USA), and re-suspended in com-
plete medium which is Dulbeccos Modied Eagle Medium (DMEM)
(GIBCOTM , Invitrogen Corporation, USA) supplemented with 10%
(v/v) Fetal Bovine Serum (FBS) (Lonza, USA), 100 U/ml penicillin
(General Drug House CO., Ltd, Thailand), and 100 g/ml strepto-
mycin (General Drug House CO., Ltd, Thailand). Cell suspensions
were then plated in 25 cm2 culture ask (Corning, USA) at a den-
sity of 2 105 cells/cm2 . Cultures were maintained at 37 C in a
humidied atmosphere containing 5% CO2 and the medium was
replaced every 3 days throughout the entire culture period.
For hMSC isolation from gestational tissues, umbilical cord,
Whartons jelly, placenta, and amnion were cut into small
pieces and digested by incubation with 0.25% (w/v) trypsin-EDTA
(GIBCOTM , Invitrogen Corporation, USA) for 30 min at 37 C. Cell
suspensions were washed twice with PBS, re-suspended in com-
plete medium and plated in 25 cm2 culture ask (Corning, USA).
Cultures were maintained at 37 C in a humidied atmosphere
containing 5% CO2 and the medium was replaced every 3 days
throughout the entire culture period.
2.3. Characterization of cultured hMSCs by ow cytometry
The 3rd5th passage of hMSCs were characterized for hMSC sur-
face markers by incubation with the following mouse anti-human
antibodies: anti-CD45-PerCP (BD Pharmingen, USA), anti-CD34-PE
(Biolegend, USA), anti-CD90-FITC (AbD Serotec, USA), anti-CD73-
PE (BD Pharmingen, USA), and anti-CD105-PE (Miltenyi Biotec,
Germany) for 30 min at 4 C in the dark. After incubation with the
antibodies, cell pellets were washed twice with PBS and xed with
1% (w/v) paraformaldehyde in PBS. Flow cytometry was performed
by FACS caliburTM Flow cytometer using CellQuestTM software
(Becton Dickinson, USA).
2.4. Osteogenic and adipogenic differentiation of cultured hMSCs
The 3rd5th passage hMSCs were used to assess their adi-
pogenic and osteogenic differentiation potentials. For adipogenic
differentiation, 5 104 cells were cultured in NH AdipoDiff
Medium (Miltenyi Biotec, Germany). Medium was replaced every
3 days according to the manufacturers instruction. After culture
for 3 weeks, cells were stained with 0.5% (w/v) Oil Red O (Sigma
Aldrich, USA), in isopropanol for 20 min at room temperature, to
determine the number of hMSC-derived adipocytes in culture.
For osteogenic differentiation, 5 104 cells were cultured in
NH OsteoDiff Medium (Miltenyi Biotec, Germany). Medium was
replaced every 3 days according to the manufacturers instruction.
After culture for 3 weeks, cells were stained with 40 mM Alizarin
Red S (Sigma Aldrich, USA) for 20 min at room temperature to deter-
mine the number of hMSC-derived osteocytes in culture.
2.5. Cultures and characterization of EPCs from umbilical cord
blood
Twenty milliliter of heparinized umbilical cord blood was col-
lected for EPC isolation. Umbilical cord blood-derived mononuclear
cells were then isolated using IsoPrep (Robbins Scientic Corpora-
tion, USA) density gradient centrifugation, washed twice with PBS
(GIBCOTM , Invitrogen Corporation, USA), re-suspended in endothe-
lial cell growth medium [endothelial basal medium-2 (LONZA,
Germany), supplemented with EGM-2 single aliquots (LONZA,
Germany) containing 2% (v/v) fetal bovine serum (FBS), 5 g/ml
epidermal growth factor, 200 g/ml hydrocortisone, 0.5 g/ml vas-
cular endothelial growth factor, 10 g/ml basic broblast growth
factor, 20 g/ml long R3 Insulin-like growth factor 1 and 1 mg/ml
ascorbic acid], and plated in an individual well of 6-well plate
coated with 10 g/ml human bronectin (Amersham Biosciences,
USA) at a density of 1 106 cells/well. After culture for 3 days, the
non-adherent cells were removed and fresh medium was added.
Cultures were maintained at 37 C in a humidied atmosphere con-
taining 5% CO2 and medium was replaced every 3 days throughout
the entire culture period.
The cultured cells were characterized for EPC surface markers
by incubation with the following mouse anti-human antibodies:
anti-CD34-PE (R&D Systems, USA), anti-VEGFR2-PE (R&D Systems,
USA), anti-CD146-FITC (R&D Systems, USA), and anti-vWF-FITC
(R&D Systems, USA) for 15 min at 4 C in the dark. Cell pellets were
then washed twice with PBS and xed with 1% (v/v) paraformalde-
hyde in PBS. Flow cytometry was performed by FACScaliburTM ow
cytometer (Becton Dickinson, USA) using CellQuest software.
2.6. Study of hMSC-derived cytokines on the functional properties
of EPCs
2.6.1. Effect on proliferation
2 105 hMSCs derived from ve distinct tissues were cultured
in 2 ml DMEM supplemented with 2% (v/v) FBS for 48 h to generate
 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163
Table 1
The sequences of primers for qRT-PCR.
Gene name Sequence of forward primer
VEGFA 5 -TCCTCACACCATTGAAACCA-3
ANGPT1 5 -AGGAACCAGCCTCCTCTCTC-3
ANGPT2 5 -AACCAGACGGCTGTGATGAT-3
FGF2 5 -AGCGGCTGTACTGCAAAAAC-3
SDF1 5 -GTGGTCGTGCTGGTCCTC-3
IGF1 5 -CCGGAGCTGTGATCTAAGGA-3
PlGF 5 -GTTCAGCCCATCCTGTGTCT-3
IGF2 5 -TGCTGGTGCTTCTCACCTTC-3
IL6 5 -CAGGAGCCCAGCTATGAACT-3
IL8 5 -AAGAAACCACCGGAAGGAAC-3
TGF 5 -GAGCCTGAGGCCGACTACTA-3
PDGF 5 -CCGGAGTCGGCATGAATC-3
GAPDH 5 -GTCAACGGATTTGGTCGTATTG-3
hMSC conditioned media for proliferation assay. Briey, 2.5 104
EPCs were cultured in an individual well of culture E-Plate (Roche
Applied Science, USA) containing 100 l hMSC conditioned media
for 7 days. The number of EPCs in each well was continuously
monitored throughout the entire culture period by xCELLigence
Real-Time Cell Analyzer (Roche Applied Science, USA) and reported
as cell index (CI). EPCs cultured in DMEM containing 2% (v/v) FBS
served as controls.
2.6.2. Effect on migration and invasion
Five distinct sources of hMSCs were co-cultured with EPCs
through 8 m transwell inserts (Corning, USA) to assess the effect of
hMSC-derived cytokines on the migratory and invasive capacity of
EPCs. For migration assay, hMSCs were seeded in the lower cham-
ber containing 600 l DMEM supplemented with 2% (v/v) FBS at
the density of 2.5 104 cells/cm2 , while 4 104 EPCs were seeded
in the transwell inserts. After 6 h of co-culture, numbers of EPCs
that migrate to the other side of the transwells membrane were
determined by hematoxylin staining.
The procedure of invasion assay was similar to that of migra-
tion assay, except that the membrane of the transwell inserts were
pre-coated with 0.4 g/l Matrigel (BD Biosciences, USA) to serve
as an extracellular matrix barrier. The numbers of invasive EPCs
which are able to degrade coated Matrigel and migrate to the other
side of the transwells membrane were determined by hematoxylin
staining.
2.6.3. Effect on vessel-forming capacity
Five distinct sources of hMSCs were co-cultured with EPCs
through 0.4 m transwell inserts (Corning, USA) to assess the
effect of hMSC-derived cytokines on the vessel-forming capac-
ity of EPCs using an in vitro vessel formation assay as previously
described (Jiraritthamrong et al., 2012). Briey, 3.5 104 EPCs
labeled with 5-(6)-carboxyuorescein diacetate succinimidyl ester
(CFSE, CellTraceTM ; Invitrogen, USA) were mixed with an equal
number of non-labeled human umbilical vein endothelial cells
(HUVECs) and plated into an individual well of 6-well plate pre-
coated with MatrigelTM (BD Bioscience, USA) while hMSCs were
seeded in the transwell inserts at the density of 1 104 cells/cm2 .
After 12 h of co-culture, the extent of incorporated EPCs in the
generated capillary-like structure was determined by uorescent
microscopy (Nikon, Japan) using NIS-Elements D software (Nikon,
Japan).
2.7. Expression levels of angiogenic-related genes in hMSCs by
quantitative real-time PCR (qRT-PCR)
Total RNA were isolated from hMSCs using TRIzol reagent
(Invitrogen Corporation, USA). cDNA was synthesized from
2 g of RNA using the SuperScriptTM III Reverse Trancriptase
155
Sequence of reverse primer
5 -GATCCTGCCCTGTCTCTCTG-3
5 -TTCTCCAGCAGCTGTATCTCAA-3
5 -TTGTCGAGAGGGAGTGTTCC-3
5 -AGCCAGGTAACGGTTAGCAC-3
5 -ATCTGAAGGGCACAGTTTGG-3
5 -CCTGCACTCCCTCTACTTGC-3
5 -AACGTGCTGAGAGAACGTCA-3
5 -AGACGAACTGGAGGGTGTCC-3
5 -GTGAGTGGCTGTCTGTGTGG-3
5 -AAATTTGGGGTGGAAAGGTT-3
5 -CACGTGCTGCTCCACTTTTA-3
5 -CGTTGGAGATCATCAAAGGAG-3
5 -CATGGGTGGAATCATATTGGAA-3
(Invitrogen Corporation, USA). MicroAmp fast optical 96-well
reaction plate (Applied Biosystem, USA) was used for qRT-PCR. Each
well contained 3 l cDNA, 1 l 10 M forward and reverse primer
mix, and 10 l SYBR Green PCR Mastermix (Applied Biosystem,
USA). The plate was then sealed with MicroAmp clear adhesive
lm (Applied Biosystem, USA) to prevent evaporation of the reac-
tant. PCR was performed using 7500 Fast Real-time PCR system
(Applied Biosystem, USA) using the following protocol: 95 C ini-
tial denaturation for 10 min, followed by 40 cycles of denaturation
(95 C, 10 s), annealing (60 C, 10 s), and extension (72 C, 40 s). The
relative quantity of a target gene was calculated by normaliza-
tion with glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
using the 7500 software version 2.0.5 (Applied Biosystem, USA).
The primer sequences were described in Table 1.
2.8. Study of PDGF-BB on EPC migration
4 104 EPCs were seeded into 8 m transwell inserts that were
placed into an individual well of 24-well plates containing 600 l
DMEM supplemented with 2% (v/v) FBS and various concentra-
tions (0, 20, 50, and 100 ng/ml) of platelet-derived growth factor-BB
(PDGF-BB; R&D system, USA). After 6 h of culture, the effect of
PDGF-BB on EPC migration was determined by migration assay as
previously described.
To further determine the role of PDGF- on EPC migration,
4 104 EPCs were treated with 10 g/ml anti-PDGFR- neutraliz-
ing antibody (R&D system, USA) for 30 min to inhibit the action
of PDGF-BB before co-cultured with hMSCs through 8 m tran-
swell. After 6 h of culture, the migration assay was performed as
previously described.
2.9. Study of SDF1, IGF1 and PlGF on EPC invasion
4 104 EPCs were seeded into 8 m Matrigel-coated transwell
inserts that were placed into an individual well of 24-well plates
containing 600 l DMEM with 2% (v/v) FBS which were supple-
mented with either 100 ng/ml stromal derived factor1 (SDF1) (R&D
system, USA), 100 ng/ml insulin-like growth factor1 (IGF1; R&D
system, USA), 100 ng/ml placenta growth factor (PlGF; R&D sys-
tem, USA), or combination of those three factors (100 ng/ml each).
After culture for 18 h, the effect of those factors on capacity of EPC
invasion was determined by invasion assay as previously described.
To further determine the roles of SDF1, IGF1 and PlGF on
EPC invasion, 4 104 EPCs were pre-treated with either 10 M
AMD3100 octahydrochloride (inhibitor of SDF1; Tocris Bioscience,
UK), 10 g/ml anti-hIGF1R (inhibitor of IGF1; R&D system, USA),
5 g/ml anti-hVEGFR1 (inhibitor of PlGF; R&D Systems, USA), or
the combination of all those three inhibitors for one hour before co-
culture with hMSCs through 8 m Matrigel-coated transwell. After
156 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163
culture for 18 h, the invasion assay was performed as previously
described.
2.10. Study of SDF1, IGF1 and PlGF on vessel-forming capacity of
EPCs
3.5 104 CFSE-labeled EPCs were co-cultured with an equal
number of non-labeled HUVECs in endothelial basal medium sup-
plemented with either 100 ng/ml SDF1, 100 ng/ml IGF1, 100 ng/ml
PlGF, or the combination of all those three factors (100 ng/ml each).
The mixtures of cells were then seeded into an individual well
of Matrigel-coated 24-well plate. After 12 h of culture, an in vitro
vessel formation assay was performed as previously described.
To further verify the roles of SDF1 and IGF1 on vessel-forming
capacity of EPCs, 105 EPCs were treated with either 10 M
AMD3100 octahydrochloride (SDF1 inhibitor; Tocris Bioscience,
UK) or 10 g/ml anti-hIGF1R (IGF1 inhibitor; R&D system, USA) for
one hour before co-culture with BM-hMSCs through transwell cul-
ture system. An in vitro vessel formation assay was performed as
previously described.
2.11. Statistical analysis
Data were presented as mean standard error of the mean
(SEM). The paired Students t-test and one-way ANOVA were used
to assess the signicance of differences between observed data.
P < 0.05 was considered to be statistically signicant.
3. Results
3.1. Characteristics of hMSCs derived from gestational tissues and
bone marrow
hMSCs derived from gestational tissues, including placenta
(PL-hMSCs), umbilical cord (UC-hMSCs), Whartons jelly (WJ-
hMSCs) and amnion (AM-hMSCs) exhibited similar characteristics
to those of bone marrow-derived hMSCs (BM-hMSCs). The gesta-
tional tissue-derived hMSCs displayed broblast-like morphology
(Fig. 1AE), expressed typical hMSC surface markers (positive for
CD73, CD90, CD105 and negative for hematopoietic markers CD34
and CD45; Fig. 1P). Moreover, the gestational tissue-derived hMSCs
could differentiate toward adipocyte- and osteocyte-lineages as
demonstrated by Oil Red O (Fig. 1FJ) and Alizarin Red S staining
(Fig. 1KO), respectively.
3.2. Expression of pro-angiogenic genes in gestational tissue and
bone marrow-derived hMSCs
The expression levels of pro-angiogenic genes among vari-
ous sources of hMSCs were determined by quantitative real-time
PCR. The expression levels of stromal cell-derived factor 1 (SDF1),
insulin-like growth factor 1 (IGF1), and placental growth factor
(PlGF) genes in the BM-hMSCs were signicantly higher than those
of other hMSC sources (Fig. 2). In contrast, the expression level
of platelet-derived growth factor beta (PDGF-) was signicantly
higher in PL-hMSCs than the rest of the hMSC sources. Apart from
those 4 genes, the expression levels of several pro-angiogenic
genes, including vascular endothelial growth factor A (VEGF-
A), broblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1),
angiopoietin 2 (ANGPT2), insulin-like growth factor 2 (IGF2), inter-
leukin 6 (IL6), interleukin 8 (IL8), and transforming growth factor
beta (TGF-) were not signicantly different among the various
hMSC sources.
3.3. Characteristics of umbilical cord blood-derived endothelial
progenitor cells (EPCs)
EPCs were cultured from mononuclear cell populations that
had been separated from umbilical cord blood by density gradi-
ent centrifugation. After culture in endothelial growth medium for
710 days, several EPC-like colonies of cobblestone morphology
were observed (Fig. 3A). These cells expressed typical EPC surface
markers, including CD34, CD146, Von Willebrand factor (vWF) and
vascular endothelial growth factor receptor 2 (VEGFR2) (Fig. 3B),
and were able to form capillary-like structures on MatrigelTM
(Fig. 3C).
3.4. Effects of hMSC-derived soluble factors on the functional
properties of EPCs
3.4.1. Effect of hMSC secreted factors on EPC proliferation
The conditioned medium derived from hMSCs from various
sources were collected and used to culture EPCs. Similar to con-
trol group (cultured in DMEM supplemented with 2% [v/v] FBS), the
numbers of EPCs cultured in various hMSC-conditioned media were
increased initially during the rst 48 h of culture before rapidly
decreasing toward the baseline level at the end of culture (Fig. 4),
indicating that the soluble factors secreted from bone marrow and
gestational tissue-derived hMSCs did not promote EPC prolifera-
tion.
3.4.2. Effect of hMSC secreted factors on EPC migration
EPCs were co-cultured with hMSC from various sources using
the transwell culture system (Fig. 5A). The numbers of EPCs that
migrate toward various hMSC sources were determined after 6 h
of co-culture. Interestingly, only the soluble factors derived from
PL-hMSCs signicantly enhanced EPC migration in comparison to
controls (306.2 60% vs. 100% of control, P < 0.001) (Fig. 5BC). In
contrast, the number of EPCs, that migrated after co-culture with
BM-hMSCs (98.8 11.9% vs. 100% of control), UC-hMSCs (80 5.2%
vs. 100% of control), WJ-hMSCs (107.1 22.8% vs. 100% of control),
and AM-hMSCs (80.3 10% vs. 100% of control), were not statis-
tically different from their controls, suggesting that the soluble
factors which were uniquely released from PL-hMSCs enhanced
EPC migration.
According to the gene expression results (Fig. 2), PDGF- is a
possible candidate for the EPC migration-inducing factor. To deter-
mine the effect of PDGF- on EPC migration, various concentrations
of PDGF-BB were added to the lower chamber of the transwell
system (Fig. 5A) to induce EPC migration (Fig. 5C). As shown in
Fig. 5D, 20-, 50-, and 100 ng/ml PDGF-BB signicantly increased
the numbers of EPC migrants compared to control (120.01 4.76%,
137.26 7.31%, and 125.39 2.04% vs. 100% of control). All the
various concentrations of PDGF-BB used in this study enhanced
EPC migration at the equivalent levels (Fig. 5D). EPCs were also
treated with PDGFR- neutralizing antibody for 30 min before co-
culture with PL-hMSCs and the anti-PDGFR- signicantly reduced
the ability of PL-hMSCs to induce EPC migration compared to con-
trols (71.62 7.37% vs. 100% of control) (Fig. 5E). Taken together,
the results demonstrate that PL-hMSCs enhanced the EPC migra-
tion and this enhancing effect can be mediated, at least in part, by
PDGF-BB.
3.4.3. Effect of hMSC secreted factors on EPC invasion
EPCs were co-cultured with various sources of hMSCs using
the transwell culture system (Fig. 6A). The ability of EPCs to
degrade MatrigelTM and migrate toward various hMSC sources
was determined after 18 h of co-culture. In contrast to the
migration assay, only the soluble factors released from BM-
hMSCs signicantly enhanced EPC invasion in comparison to
 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163 157

Fig. 1. Characteristics of hMSCs derived from bone marrow and gestational tissues. (A)(E) Fibroblast-like morphology of the various hMSC sources (Scale bar: 500 m). (F)(J)
Adipogenic differentiation of various hMSC sources as determined by Oil Red O staining (Scale bar: 100 m). (K)(O) Osteogenic differentiation of various hMSC sources as
determined by Alizarin Red S staining (Scale bar: 500 m). (P) Immunophenotypes of various hMSC sources as determined by ow cytometry.

controls (223.3 44.3% vs. 100% of control) (Fig. 6BC). The inva- AM-hMSCs (84.15 2.29% vs. 100% of control) was not statisti-
sive capacity of EPCs which were co-cultured with PL-hMSCs cally different in comparison to controls. Soluble factors, uniquely
(100.9 6.4% vs. 100% of control), UC-hMSCs (89.7 10% vs. 100% released from BM-hMSCs appear to signicantly enhance EPC
of control), WJ-hMSCs (59.46 11.08% vs. 100% of control), and invasion.
158 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163

Fig. 2. Expression of angiogenic genes in various hMSC sources. Total RNA were isolated from hMSCs using TRIzol reagent and expression levels of various angiogenic genes
were determined by qRT-PCR. The relative quantity of a target gene was calculated by normalization with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), using the
7500 software version 2.0.5. Data are presented as mean SEM of three independent experiments. One-way ANOVA was used to assess the signicance of differences between
observed data. * P < 0.05 vs. other hMSC sources.

Fig. 3. Characteristics of umbilical cord blood-derived EPCs. (A) EPC colonies with a cobblestone-like appearance (Scale bar: 500 m). (B) Immunophenotype of EPCs as
determined by ow cytometry. (C) The capillary-like structures generated from EPCs which were cultured on MatrigelTM (Scale bar: 200 m).
 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163 159

Fig. 4. Effect of hMSC-derived cytokines on EPC proliferation. The number of EPCs, which were cultured in conditioned medium derived from various hMSC sources, as
determined by xCELLigence Real-Time Cell Analyzer and reported as cell index (CI) were plotted against culture time. Data are presented as mean SEM of three independent
experiments.

According to the gene expression results (Fig. 2), SDF1, IGF1
and PlGF are the possible candidates for the EPC invasion-
inducing factor. To determine the effect of those three factors
on EPC invasion, 100 ng/ml SDF1, 100 ng/ml IGF1, and 100 ng/ml
PlGF were added to the lower chamber of the transwell sys-
tem, either separately or in combination, to determine their
capacity to induce EPC invasion. Indeed, 100 ng/ml IGF1 sig-
nicantly increase the number of invaded EPC in comparison
to controls (164.8 10.72% vs. 100% of control). In contrast,
the number of invaded EPCs that are treated with 100 ng/ml
SDF1 (116.4 14.4% vs. 100% of control) and 100 ng/ml PlGF
(120.5 21.9% vs. 100% of control) were not signicantly different
when compared to controls (Fig. 6D). Moreover, the combination
of all three factors did not show any additional benecial effect
when compare with the effect of IGF1 alone (157.9 20.4% of
control vs. 164.8 10.7% of control) (Fig. 6D). Additionally, EPCs
were treated with AMD3100, antibody to hIGF1R and antibody to
hVEGFR1, either separately or in combination, before co-culture
with BM-hMSCs to perform invasion assay. AMD3100, anti-
hIGF1R, anti-hVEGFR1 or the combination of all three inhibitors
failed to inhibit the enhancing effects of BM-hMSCs on EPC
invasion (157.2 12.6% vs. 100% of control for AMD3100 treat-
ment, 119.8 8.5% vs. 100% of control for anti-hIGF1R treatment,
165.3 29.5% vs. 100% of control for anti-hVEGFR1 treatment, and
174.4 10.4% vs. 100% of control for the combination of three
inhibitors) (Fig. 6E), indicating that BM-hMSCs enhanced EPC inva-
sion and that enhancing effect was mediated, at least in part, by
IGF1.

Fig. 5. Effect of hMSC-derived cytokines on EPC migration. (A) The transwell culture system used for migration assay. (B) EPCs which migrated to the other side of transwell
membrane after co-culture with various hMSC sources as determined by hematoxylin staining (Scale bar: 100 m.). (C) Levels of EPCs migration after co-culture with various
hMSC sources. One-way ANOVA was used to assess the signicance of differences between observed data. * P < 0.05 vs. other hMSC sources. (D) Effect of various concentrations
of PDGF-BB on the levels of EPC migration in the absence of hMSCs. Paired Students t-test was used to assess the signicance of differences between observed data. (E)
Effect of 10 g/ml anti-PDGFR- (PDGF- inhibitor) on the migration of EPCs after co-culture with placenta-derived hMSCs. Paired Students t-test was used to assess the
signicance of differences between observed data. (Scale bar: 100 m) Data are presented as mean SEM of three independent experiments.
160 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163

Fig. 6. Effect of hMSC-derived cytokines on EPC invasion. (A) The transwell culture system used for invasion assay. (B) EPCs which invade the coated Matrigel and migrate to the
other side of transwell membrane after co-culture with various hMSC sources as determined by hematoxylin staining. (Scale bar: 200 m). (C) Levels of EPC invasion after
co-culture with various hMSC sources. One-way ANOVA was used to assess the signicance of differences between observed data. * P < 0.05 vs. other MSC sources. (D) Effect
of SDF1, IGF1, and PlGF on the levels of EPC invasion in the absence of hMSCs. Paired Students t-test was used to assess the signicance of differences between observed data.
(E) Effect of SDF1 inhibitor (AMD3100), IGF1 inhibitor (anti-hIGFIR), and PlGF inhibitor (anti-hVEGFR1) on the invasion of EPCs after co-culture with bone marrow-derived
hMSCs. One-way ANOVA was used to assess the signicance of differences between observed data. Data are presented as mean SEM of three independent experiments.

3.4.4. Effect of hMSC secreted factors on vessel-forming capacity
of EPC
To investigate the effect of hMSC-derived soluble factors on
the vessel-forming capacity of EPCs, EPCs were co-cultured with
hMSCs from various sources using the transwell culture sys-
tem and then subjected to an in vitro vessel formation assay
(Fig. 7A). The vessel-forming capacity of EPCs co-cultured with
BM-hMSCs was signicantly higher than those co-cultured with
PL-hMSCs (121.3 9.7% of control vs. 96.9 4.7% of control) and
WJ-hMSCs (121.3 9.7% of control vs. 82.4 8.4% of control)
(Fig. 7BC), suggesting that there are soluble factors, uniquely
released from BM-hMSCs to enhance the vessel-forming capacity
of EPCs.
According to the gene expression results (Fig. 2), SDF1, IGF1 and
PlGF are possible candidate factors. To determine their effects on
the vessel-forming capacity of EPCs, 100 ng/ml SDF1, 100 ng/ml
IGF1, and 100 ng/ml PlGF were added to the lower chamber
of the transwell system, either separately or in combination,
before performing the vessel formation assay. Indeed, the vessel-
forming capacity of EPCs was signicantly increased by the addition
of 100 ng/ml SDF1 (150.7 13.3% vs. 100% of control, P < 0.05),
100 ng/ml IGF1 (144.2 12.2% vs. 100% of control, P < 0.05) and the
combination of all three factors (157.9 10.9% vs. 100% of con-
trol, P < 0.05) while the addition of PlGF alone did not increase the
vessel-forming capacity of EPCs (122.37 12.93% vs. 100% of con-
trol) (Fig. 7DE), indicating that BM-hMSCs enhance vessel-forming
capacity of EPCs and that enhancing effect was mediated, at least
in part, by IGF1 and SDF1.
The effect of IGF1 and SDF1 on vessel-forming capacity of
EPCs was further determined by treating EPCs with either anti-
hIGF1R (IGF1 inhibitor) or AMD3100 (SDF1 inhibitor) before
co-culture with BM-hMSCs (Fig. 7A). SDF1 inhibitor could signif-
icantly reduce the ability of BM-hMSCs to promote vessel-forming
capacity of EPCs (85.62 2.44% vs. 100% of control, P < 0.05). IGF1
inhibitor could diminish the enhancing effect of BM-hMSCs on
EPC vessel-forming capacity but the difference was not signicant
(90.31 0.61% vs. 100% of control, P > 0.05) (Fig. 7FG). These nd-
ings indicate that BM-hMSCs enhance the vessel-forming capacity
of EPCs, at least in part, through the secreted SDF1.
4. Discussion
Recently, hMSCs have been used to treat tissue injury caused
by ischemia and inammation. Despite evidence suggesting that
 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163 161

Fig. 7. Effect of hMSC-derived cytokines on the vessel-forming capacity of EPCs. (A) The competitive vessel formation assay used to determine the vessel-forming capacity of EPCs.
(B) Extent of CFSE-labeled EPCs in the capillary-like structures after co-culture with various hMSC sources as determined by uorescent microscopy. (Scale bar: 200 m). (C)
The vessel-forming capacity of EPCs after co-culture with various hMSC sources. Paired Students t-test was used to assess the signicance of differences between observed
data. (D) Effect of SDF1, IGF1, and PlGF on the extent of CFSE-labeled EPCs in capillary-like structures in the absence of hMSCs. (Scale bar: 200 m). (E) Effect of SDF1, IGF1,
and PlGF on the vessel-forming capacity of EPCs in the absence of hMSCs. Paired Students t-test was used to assess the signicance of differences between observed data. (F)
Effect of SDF1 inhibitor (AMD3100) and IGF1 inhibitor (anti-hIGFIR) on the extent of CFSE-labeled EPCs in capillary-like structures after co-culture with bone marrow-derived
hMSCs. (Scale bar: 200 m). (G) Effect of SDF1 inhibitor (AMD3100) and IGF1 inhibitor (anti-hIGFIR) on vessel-forming capacity of EPCs. One-way ANOVA was used to assess
the signicance of differences between observed data. Data are presented as mean SEM of three independent experiments.

MSCs could differentiate to endothelial cells (ECs) and smooth
muscle cells, which are essential parts of the newly generated
vessels (Davani et al., 2003), most studies suggest that the major
mechanism underlying the effects of MSCs is the induction of
neovascularization in the injured/ischemic tissues by angiogenic
factors released from MSCs (Au et al., 2008; Kinnaird et al., 2004;
Yao et al., 2015). Neovascularization of the ischemic tissues could be
accomplished by either angiogenesis, which generates new blood
vessels from the pre-existing vessels, or vasculogenesis that gener-
ates new vessels de novo from endothelial progenitor cells (EPCs).
The multi-step neovascularization processes are tightly regulated
by many angiogenic factors, such as vascular endothelial growth
factor (VEGF), basic broblast growth factors (bFGF), angiopoietin
(ANGPT), and transforming growth factor (TGF-), among others
(Carmeliet and Jain, 2011).
Although several previous studies showed that BM-MSCs pro-
duced and secreted several angiogenic factors, including VEGF,
HGF, IGF1, MCP1, IL-6 and PlGF which play important roles during
the processes of neovascularization and wound healing (Kinnaird
et al., 2004; Wang et al., 2006; Wu et al., 2007), the effect of those
factors on the functional properties of EPCs during neovascular-
ization processes has yet to be characterized. In the present study,
we used in vitro culture model to study the effect of hMSCs from
various sources on the proliferation, migration, invasion and vessel-
forming capacity of EPCs, important steps during the process of
vasculogenesis. The expression levels of several angiogenic genes
in gestational tissues-derived hMSCs (PL-hMSCs, UC-hMSCs, WJ-
hMSCs and AM-hMSCs) are different from those of BM-hMSCs.
Moreover, the effect of gestational tissues-derived hMSCs on the
migration, invasion capacity and vessel-forming capacity of EPCs
were also different from those of BM-hMSCs.
The BM-hMSCs expressed signicantly higher levels of IGF1,
SDF1, and PlGF gene and induced higher level of EPC invasion
compared to gestational tissue-derived hMSCs. IGF1 released from
162 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163
BM-hMSCs were responsible, at least in part to the enhancing effect
of BM-hMSCs on EPC invasion. A recent previous study showed
that IGF1 increased the colony formation, migration and vessel-
formation of EPCs (Thum et al., 2007). To our knowledge, this is the
rst report on the effect of IGF1 on EPC invasion.
For EPC migration, the PL-hMSCs signicantly induced higher
level of EPC migration compare to other sources of hMSCs. The abil-
ity of PL-hMSCs to induce EPC migration was mediated, at least in
part, through the action of secreted PDGF-BB. The effect of PDGF-
BB observed in this study is in agreement with a previous study
which reported that the overexpression of PDGFR- gene in EPCs
enhanced their proliferative, migratory and vessel forming capacity
(Wang et al., 2012). Our ndings suggested that the higher level of
PDGF- in PL-hMSCs might make this particular source of hMSCs
more efcient in inducing EPC migration compared to other hMSC
sources.
The addition of PDGF-BB, SDF1, and IGF1 could signicantly
enhance the migration, invasion and vessel-forming capacity of
EPCs but the enhancing effects were only 5070% of those with PL-
hMSC- and BM-hMSC-secretome. Moreover, the inhibition of SDF-1
and IGF-1 failed to abolish the enhancing effects of hMSCs on EPC
functions. Taken together, these results suggest that, apart from
PDGF-BB, SDF-1 and IGF-1, PL-MSCs and BM-MSCs might secrete
other additional unidentied factors that enhance the migration,
invasion and vessel-forming capacity of EPCs.
Our in vitro vessel formation assay showed that SDF1 and IGF1
which were highly expressed in BM-hMSCs could signicantly
enhance vessel-forming capacity of EPCs either individually or in
combination. Previous studies showed that IGF1 could increase
proliferative, migratory and vessel-forming capacity of EPCs (Thum
et al., 2007) while SDF1 was shown to regulate the mobilization of
EPCs toward ischemic tissue (Ceradini et al., 2004) and enhance
angiogenic potential of EPCs (Zemani et al., 2008). Although a
previous study demonstrated that the combination of VEGF and
PlGF could efciently enhance vessel formation in vitro (Li et al.,
2006), our results showed that PlGF, which also highly expressed
in BM-hMSCs, did not increase the vessel-forming capacity of EPCs.
Therefore, our results suggested that PlGF might exhibit its angio-
genic potential only in the presence of other angiogenic factors,
such as VEGF. Similar to the previous studies which indicate that
BM-hMSC-derived cytokines had no effect on the proliferation of
endothelial cells (Burlacu et al., 2013; Gruber et al., 2005), all ges-
tational tissue-derived hMSCs generated in the present study did
not promote EPC proliferation.
In conclusion, our ndings provide a better understanding of
the angiogenic potential of soluble factors secreted from bone mar-
row and gestational tissue-derived hMSCs. We demonstrate for the
rst time that hMSCs derived from distinct sources produced and
secreted a distinct combination of angiogenic factors which exert
different biological effects on EPC function. The knowledge gained
from this study might help to improve the therapeutic applica-
tions of hMSCs as sources of angiogenic cytokines for inducing
neovascularization in the injured/ischemic tissues. Furthermore,
the administration of hMSC-derived angiogenic factors which play
important role during the specic stages of vasculogenesis might be
used to induce neovascularization in the injured/ischemic tissues
without the need for cell transplantation.
Acknowledgements
This research project was funded by grants from Thailand
Research Fund (grant no. RTA 488-0007) and the Commission on
Higher Education (grant no. CHE-RES-RG-49). Witchayaporn Kam-
prom was supported by the Royal Golden Jubilee Ph.D. program of
the Thailand Research Fund.
References
Asahara, T., Masuda, H., Takahashi, T., Kalka, C., Pastore, C., Silver, M., Kearne, M.,
Magner, M., Isner, J.M., 1999. Bone marrow origin of endothelial progenitor
cells responsible for postnatal vasculogenesis in physiological and pathological
neovascularization. Circ. Res. 85, 221228.
Au, P., Tam, J., Fukumura, D., Jain, R.K., 2008. Bone marrow-derived mesenchymal
stem cells facilitate engineering of long-lasting functional vasculature. Blood
111, 45514558.
Bunnell, B.A., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C., 2008. Adipose-derived stem
cells: isolation, expansion and differentiation. Methods 45, 115120.
Burlacu, A., Grigorescu, G., Rosca, A.M., Preda, M.B., Simionescu, M., 2013. Factors
secreted by mesenchymal stem cells and endothelial progenitor cells have
complementary effects on angiogenesis in vitro. Stem Cells Dev. 22, 643653.
Carmeliet, P., Jain, R.K., 2011. Molecular mechanisms and clinical applications of
angiogenesis. Nature 473, 298307.
Ceradini, D.J., Kulkarni, A.R., Callaghan, M.J., Tepper, O.M., Bastidas, N., Kleinman,
M.E., Capla, J.M., Galiano, R.D., Levine, J.P., Gurtner, G.C., 2004. Progenitor cell
trafcking is regulated by hypoxic gradients through HIF-1 induction of SDF-1.
Nat. Med. 10, 858864.
Davani, S., Marandin, A., Mersin, N., Royer, B., Kantelip, B., Herve, P., Etievent, J.P.,
Kantelip, J.P., 2003. Mesenchymal progenitor cells differentiate into an
endothelial phenotype, enhance vascular density, and improve heart function
in a rat cellular cardiomyoplasty model. Circulation 108 (Suppl. 1), Ii253Ii258.
Gnecchi, M., Zhang, Z., Ni, A., Dzau, V.J., 2008. Paracrine mechanisms in adult stem
cell signaling and therapy. Circ. Res. 103, 12041219.
Gruber, R., Kandler, B., Holzmann, P., Vogele-Kadletz, M., Losert, U., Fischer, M.B.,
Watzek, G., 2005. Bone marrow stromal cells can provide a local environment
that favors migration and formation of tubular structures of endothelial cells.
Tissue Eng. 11, 896903.
Hung, S.C., Pochampally, R.R., Chen, S.C., Hsu, S.C., Prockop, D.J., 2007. Angiogenic
effects of human multipotent stromal cell conditioned medium activate the
PI3K-Akt pathway in hypoxic endothelial cells to inhibit apoptosis, increase
survival, and stimulate angiogenesis. Stem Cells 25, 23632370.
Int Anker, P.S., Scherjon, S.A., Kleijburg-van der Keur, C., de Groot-Swings, G.M.,
Claas, F.H., Fibbe, W.E., Kanhai, H.H., 2004. Isolation of mesenchymal stem cells
of fetal or maternal origin from human placenta. Stem Cells 22, 13381345.
Jiang, Y., Jahagirdar, B.N., Reinhardt, R.L., Schwartz, R.E., Keene, C.D.,
Ortiz-Gonzalez, X.R., Reyes, M., Lenvik, T., Lund, T., Blackstad, M., Du, J., Aldrich,
S., Lisberg, A., Low, W.C., Largaespada, D.A., Verfaillie, C.M., 2002. Pluripotency
of mesenchymal stem cells derived from adult marrow. Nature 418, 4149.
Jiraritthamrong, C., Kheolamai, P., U-Pratya, Y., Chayosumrit, M., Supokawej, A.,
Manochantr, S., Tantrawatpan, C., Sritanaudomchai, H., Issaragrisil, S., 2012.
In vitro vessel-forming capacity of endothelial progenitor cells in high glucose
conditions. Ann. Hematol. 91, 311320.
Kamihata, H., Matsubara, H., Nishiue, T., Fujiyama, S., Tsutsumi, Y., Ozono, R.,
Masaki, H., Mori, Y., Iba, O., Tateishi, E., Kosaki, A., Shintani, S., Murohara, T.,
Imaizumi, T., Iwasaka, T., 2001. Implantation of bone marrow mononuclear
cells into ischemic myocardium enhances collateral perfusion and regional
function via side supply of angioblasts, angiogenic ligands, and cytokines.
Circulation 104, 10461052.
Kinnaird, T., Stabile, E., Burnett, M.S., Lee, C.W., Barr, S., Fuchs, S., Epstein, S.E., 2004.
Marrow-derived stromal cells express genes encoding a broad spectrum of
arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through
paracrine mechanisms. Circ. Res. 94, 678685.
Lee, O.K., Kuo, T.K., Chen, W.M., Lee, K.D., Hsieh, S.L., Chen, T.H., 2004. Isolation of
multipotent mesenchymal stem cells from umbilical cord blood. Blood 103,
16691675.
Li, B., Sharpe, E.E., Maupin, A.B., Teleron, A.A., Pyle, A.L., Carmeliet, P., Young, P.P.,
2006. VEGF and PlGF promote adult vasculogenesis by enhancing EPC
recruitment and vessel formation at the site of tumor neovascularization.
FASEB J. 20, 14951497.
Li, H., Zuo, S., He, Z., Yang, Y., Pasha, Z., Wang, Y., Xu, M., 2010. Paracrine factors
released by GATA-4 overexpressed mesenchymal stem cells increase
angiogenesis and cell survival. Am. J. Physiol. Heart Circ. Physiol. 299,
H1772H1781.
Meirelles Lda, S., Fontes, A.M., Covas, D.T., Caplan, A.I., 2009. Mechanisms involved
in the therapeutic properties of mesenchymal stem cells. Cytokine Growth
Factor Rev. 20, 419427.
Melero-Martin, J.M., Khan, Z.A., Picard, A., Wu, X., Paruchuri, S., Bischoff, J., 2007.
In vivo vasculogenic potential of human blood-derived endothelial progenitor
cells. Blood 109, 47614768.
Nauta, A.J., Fibbe, W.E., 2007. Immunomodulatory properties of mesenchymal
stromal cells. Blood 110, 34993506.
Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D.,
Moorman, M.A., Simonetti, D.W., Craig, S., Marshak, D.R., 1999. Multilineage
potential of adult human mesenchymal stem cells. Science 284, 143147.
Tepper, O.M., Capla, J.M., Galiano, R.D., Ceradini, D.J., Callaghan, M.J., Kleinman,
M.E., Gurtner, G.C., 2005. Adult vasculogenesis occurs through in situ
recruitment, proliferation, and tubulization of circulating bone
marrow-derived cells. Blood 105, 10681077.
Thum, T., Hoeber, S., Froese, S., Klink, I., Stichtenoth, D.O., Galuppo, P., Jakob, M.,
Tsikas, D., Anker, S.D., Poole-Wilson, P.A., Borlak, J., Ertl, G., Bauersachs, J., 2007.
Age-dependent impairment of endothelial progenitor cells is corrected by
growth-hormone-mediated increase of insulin-like growth-factor-1. Circ. Res.
100, 434443.
 W. Kamprom et al. / European Journal of Cell Biology 95 (2016) 153163
Uccelli, A., Moretta, L., Pistoia, V., 2008. Mesenchymal stem cells in health and
disease. Nat. Rev. Immunol. 8, 726736.
Wang, H., Yin, Y., Li, W., Zhao, X., Yu, Y., Zhu, J., Qin, Z., Wang, Q., Wang, K., Lu, W.,
Liu, J., Huang, L., 2012. Over-expression of PDGFR-beta promotes
PDGF-induced proliferation, migration, and angiogenesis of EPCs through
PI3K/Akt signaling pathway. PLoS ONE 7, e30503.
Wang, H.S., Hung, S.C., Peng, S.T., Huang, C.C., Wei, H.M., Guo, Y.J., Fu, Y.S., Lai, M.C.,
Chen, C.C., 2004. Mesenchymal stem cells in the Whartons jelly of the human
umbilical cord. Stem Cells 22, 13301337.
Wang, M., Crisostomo, P.R., Herring, C., Meldrum, K.K., Meldrum, D.R., 2006.
Human progenitor cells from bone marrow or adipose tissue produce VEGF
HGF, and IGF-I in response to TNF by a p38 MAPK-dependent mechanism. Am.
J. Physiol. Regul. Integr. Comp. Physiol. 291, R880R884.
Wang, Y., Chen, X., Cao, W., Shi, Y., 2014. Plasticity of mesenchymal stem cells in
immunomodulation: pathological and therapeutic implications. Nat. Immunol.
15, 10091016.
163
Williams, A.R., Hatzistergos, K.E., Addicott, B., McCall, F., Carvalho, D., Suncion, V.,
Morales, A.R., Da Silva, J., Sussman, M.A., Heldman, A.W., Hare, J.M., 2013.
Enhanced effect of combining human cardiac stem cells and bone marrow
mesenchymal stem cells to reduce infarct size and to restore cardiac function
after myocardial infarction. Circulation 127, 213223.
Wu, Y., Chen, L., Scott, P.G., Tredget, E.E., 2007. Mesenchymal stem cells enhance
wound healing through differentiation and angiogenesis. Stem Cells 25,
26482659.
Yao, Y., Huang, J., Geng, Y., Qian, H., Wang, F., Liu, X., Shang, M., Nie, S., Liu, N.,
Du, X., Dong, J., Ma, C., 2015. Paracrine action of mesenchymal stem cells
revealed by single cell gene proling in infarcted murine hearts. PLoS ONE 10,
e0129164.
Zemani, F., Silvestre, J.S., Fauvel-Lafeve, F., Bruel, A., Vilar, J., Bieche, I., Laurendeau,
I., Galy-Fauroux, I., Fischer, A.M., Boisson-Vidal, C., 2008. Ex vivo priming of
endothelial progenitor cells with SDF-1 before transplantation could increase
their proangiogenic potential. Arterioscler. Thromb. Vasc. Biol. 28, 644650.