E X P E R I M E N TA L R E S E A R C H
CORONARY INTERVENTIONS
EuroIntervention 2014;10:709-716
Coronary stents seeded with human trophoblastic
endovascular progenitor cells show accelerated strut
coverage without excessive neointimal proliferation in
a porcine model
Tushar Raina1, MD, MRCP; Javaid Iqbal1, MRCP, PhD; Nadine Arnold1, VN; Harry Moore2, BSc, PhD;
Behrouz Aflatoonian2, BSc, PhD; Jim Walsh3, BSc, PhD; Sam Whitehouse4, BSc, PhD;
Kadem Al-Lamee4, MSc, PhD; Sheila Francis1, BSc, PhD; Julian Gunn1*, MD, MRCP
1. Department of Cardiovascular Science, University of Sheffield, Sheffield, United Kingdom; 2. Centre for Stem Cell Biology,
University of Sheffield, Sheffield, United Kingdom; 3. Intercytex Ltd, Sheffield, United Kingdom; 4. Lombard Medical
Technologies PLC, Sheffield, United Kingdom
This paper also includes accompanying supplementary data published online at: http://www.pcronline.com/eurointervention/77th_issue/123
KEYWORDS
• endothelialisation
• endothelial
progenitor cells
• neointimal
hyperplasia
• percutaneous
coronary
intervention
• restenosis
Abstract
Aims: The success of percutaneous coronary intervention (PCI) has been limited by restenosis and stent
thrombosis. Delayed or incomplete endothelial regeneration is believed to be a key factor responsible for
these events. Developing a stent with an accelerated healing profile may be of benefit. We aimed to evaluate
the feasibility and safety of seeding a bare metal stent (BMS) with human trophoblastic endovascular progenitor cells (hTEC) derived from human embryonic stem cells.
Methods and results: A porcine coronary artery model was used to compare the rate and extent of endothelial regeneration and the degree of neointimal proliferation. Characterisation of hTEC confirmed a mixed progenitor and endothelial cell phenotype. The biodistribution and fate of hTEC were studied using radiolabelled
111
Indium oxine and fluorescent in situ hybridisation. Scanning electron microscopy showed earlier endothelial
coverage in hTEC-seeded stents as compared to similar BMS. hTEC-seeded BMS achieved complete stent coverage in three days. Quantitative coronary angiography, intravascular ultrasound assessment and histomorphometry showed no difference in neointimal hyperplasia between hTEC-seeded and control BMS.
eration without increasing neointimal proliferation.
*Corresponding author: Department of Cardiovascular Science, University of Sheffield, Sheffield, S10 2RX, United Kingdom.
E-mail: J.Gunn@sheffield.ac.uk
© Europa Digital & Publishing 2014. All rights reserved.
DOI: 10.4244 / EIJV10I6A123
Conclusions: hTEC seeding of coronary stents is a novel and safe approach to accelerate endothelial regen-
SUBMITTED ON 10/10/2013 - REVISION RECEIVED ON 1st 13/11/2013 / 2nd 23/11/2013 - ACCEPTED ON 25/11/2013
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EuroIntervention 2014;10:709-716
Abbreviations
-SMA
BMS
CD
Cpm
DAPI
DES
DMEM
EDTA
EPC
FACS
FCS
FISH
FITC
HLA-G
hTEC
IVUS
LAD
LLL
MLA
MLD
MSA
PBMC
PAoEC
PCI
QCA
RCA
SM-MHC
VEGF
vWF
alpha smooth muscle actin
bare metal stents
cluster of differentiation
counts per minute
4’,6 diamidino-2-phenylindole
drug-eluting stents
Dulbecco’s modified eagle medium
ethylenediaminetetraacetic acid
endothelial progenitor cells
fluorescence activated cell sorting
foetal calf serum
fluorescent in situ hybridisation
fluorescein isothiocyanate
human leucocyte antigen-G
human trophoblastic endovascular progenitor cell
intravascular ultrasound
left anterior descending artery
late lumen loss
minimal lumen area
minimal lumen diameter
minimal stent area
peripheral blood mononuclear cells
porcine aortic endothelial cell
percutaneous coronary intervention
quantitative coronary angiography
right coronary artery
smooth muscle myosin heavy chain
vascular endothelial growth factor
von Willebrand factor
Introduction
Percutaneous coronary intervention (PCI) with angioplasty and
stenting is the mainstay in the treatment of significant and symptomatic coronary artery disease1,2. Neointimal hyperplasia within bare
metal stents (BMS), leading to in-stent restenosis, has been a longstanding limitation of PCI3. Drug-eluting stents (DES) have significantly reduced in-stent restenosis and the need for repeat PCI4,5.
There are, however, concerns regarding the safety of DES, notably
related to late stent thrombosis6-8, which is associated with adverse
clinical outcomes and a mortality rate of 17-45%9,10.
Delayed or incomplete endothelial regeneration is one of the key
factors responsible for late stent thrombosis. The therapeutic agents
incorporated in DES are antiproliferative, and therefore inhibit healing and prevent or delay endothelial regeneration11. Autopsy studies
of DES-treated arteries have revealed incomplete endothelialisation
of stent struts with adherent platelets and inflammatory cells12,13.
Post mortem examination of coronary segments from patients who
underwent DES and BMS implantation has revealed delayed arterial healing and incomplete endothelialisation after DES implantation as compared to BMS implantation of similar duration12,13.
The newer generation of DES with biocompatible or biodegradable polymers, bioresorbable scaffolds and potent antiplatelet agents
710
have reduced but not eliminated the risk of stent thrombosis. The
development of stents bearing pro-healing agents is, therefore, of
clinical interest, but has gained limited success to date. An endothelial progenitor cell (EPC) capture stent was safe and effective in
initial studies14, but larger observational studies and randomised
clinical trials revealed an excess of restenosis15-18. An alternative
approach is to use stents already coated with progenitor cells. We
have previously derived an enriched endovascular cell phenotype from cytotrophoblastic stem cell lines generated from human
embryonic stem cells19. These human trophoblastic endovascular
cells (hTEC) express human leucocyte antigen-G (HLA-G), potentially endowing them with immune-tolerance properties. The hTEC
also exhibit distinct morphological and immunological similarities
to cells of an endothelial progenitor phenotype20. We surmised that
the pseudo-endothelial vasculogenic and invasive characteristics
might make these cells applicable for seeding a stent.
We hypothesised that a cell-based regenerative strategy using
hTEC seeding would accelerate endothelial regeneration without
increasing neointimal hyperplasia after stent implantation in a porcine model of coronary stenting.
Methods
All animal work was carried out under UK Home Office licence
in accordance with the Animals (Scientific Procedures) Act, 1986,
and conforms with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health.
CULTURE, PREPARATION AND CHARACTERISATION OF
hTEC STEM CELLS
hTEC were derived by the terminal differentiation of trophoblast
stem cells while the latter were derived as distinct trophoblast cell
lines from human embryonic stem cells (Shef 3 and six cell lines)21
and maintained in culture, using previously described protocols19,20
(Online Appendix). Immunological characterisation of hTEC was
carried out by staining for endothelial progenitor markers: CD133,
CD34 and vascular endothelial growth factor receptor-2 (VEGFR2) and mature endothelial markers; CD31, E-selectin and von
Willebrand factor (vWF). hTEC were also stained for alpha smooth
muscle actin (ɑ-SMA) and smooth muscle myosin heavy chain
(SM-MHC).
We then aimed to demonstrate that hTEC could survive in coculture with porcine endothelial and smooth muscle cells, isolated
from porcine thoracic aortae (Online Appendix).
CELL SEEDING OF STENTS
hTEC were recovered from the culture flask by incubation in a solution of 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid
(EDTA). After five minutes digestion was stopped with Dulbecco’s
Modified Eagle Medium (DMEM) supplemented with 10% foetal calf serum (FCS) and the cells washed by centrifugation (×3)
and counted (Neubauer counting chamber). Under a laminar flow
hood and strict asepsis, BMS (Invastent Volo®; Invatec S.p.A.,
Roncadelle, Italy) pre-mounted on a balloon catheter were inserted
Stem cell-seeded stents accelerate healing
IMMUNOLOGICAL ISSUES
Our experiments involved inserting a human stem cell-coated
stent into a pig, so it was important to demonstrate any immunological ill-effects. The effect of any pre-existing circulating antihuman porcine antibodies upon hTEC was determined by exposing
hTEC to porcine serum followed by flow cytometry analysis with
fluorescein isothiocyanate (FITC) labelled anti-porcine IgG and
IgM antibodies (Online Appendix).
BIODISTRIBUTION AND LATE FATE OF hTEC
Initial porcine experiments were focused on assessing whether the
hTEC remained on the stent. BMS were seeded with hTEC, which
had been radiolabelled with 111Indium oxine and maintained in cell
culture conditions for three days. Stents were implanted in the left
anterior descending (LAD) coronary artery of three Yorkshire White
pigs, which were sacrificed at one hour, one day and seven days.
The radioactivity over the stented segment of the explanted LAD
and other organs was measured using a gamma counter. In addition, BMS seeded with hTEC derived from a male cell line (XY)
were implanted in female pigs (XX). hTEC-seeded stents were
implanted in the LAD or right coronary artery (RCA) and a BMS
without seeded cells deployed in the contralateral artery for comparison. The animals were sacrificed at 28 days. Fluorescent human
X, Y and 18 chromosome probes were used to detect the human or
pig origin of cells within the neointima. The stented vessels were
dissected, fixed and cut longitudinally using tungsten scissors and
opened en face. Tissue lining the inner surface of the stent halves
(neointima) was removed, embedded and cut into sections. Paraffin
sections from the neointimal tissue within the implanted BMS in
the contralateral coronary artery were used as negative control and
actively proliferating early passage hTEC used as positive controls.
Fluorescent in situ hybridisation (FISH) analysis was performed
using centromere specific chromosome X, Y and 18. The samples
were counterstained with 4’,6 diamidino-2-phenylindole (DAPI)
and the images captured with a fluorescence microscope.
STENT IMPLANTATION
Normolipaemic, genetically normal, juvenile Yorkshire White pigs
(20-30 kg) of either sex were anaesthetised and vascular access
obtained through the right carotid artery. The stents used in this
study (both for coating and for control groups) were 3.5×11 mm
Invastent Volo® pre-mounted on a rapid-exchange balloon stent
delivery system. Invastent Volo® is a slotted-tube, closed cell design
stent made up of stainless steel (316L) with a metallic surface area
of 13.3% and strut thickness of 95 μm. The coronary arteries were
intubated using standard guide catheters. Stent implantation sites
were chosen to achieve a stent:artery ratio of 1.25:1 in suitable segments of LAD and RCA. The animals received 150 mg aspirin and
75 mg clopidogrel throughout the experiment.
ASSESSMENT OF ENDOTHELIAL REGENERATION
For endothelial regeneration experiments, eight animals received
two stents each: hTEC-seeded 3.5×11 mm BMS and uncoated identical BMS in the LAD or the RCA in alternating sequence. A coronary angiogram was performed at the end of each procedure to
document arterial patency. The animals were sacrificed at one
hour, one day, three days and seven days; the hearts were explanted
and the stented arterial segments examined for stent strut coverage using a scanning electron microscope. Each stented vessel segment was cut longitudinally using tungsten scissors with the stent
in situ and opened en face. After fixation and dehydration, samples were examined under a scanning electron microscope (Philips
XL-20). Stent strut coverage was quantified in each stented vessel
half. Images acquired under low magnification (×25) were divided
into four quadrants. Stent strut coverage was quantified under high
magnification (×100) in all four quadrants for the four samples for
each time point. Each quadrant was assigned a strut coverage score
from 1 to 4 according to the percentage covered struts: 1=(0-25%),
2=(25-50%), 3= (50-75%) and 4=(75-100%). The mean of the strut
coverage scores for the four individual quadrants was taken as the
strut coverage score for each sample.
EuroIntervention 2014;10:709-716
into sterile Nunc® CryoTubes (Sigma-Aldrich, Dorset, UK), seeded
with 1×106 hTEC and maintained in standard cell culture conditions for three days with gentle agitation using a rocker tray (IKA®
MTS-4; IKA®-Werke GmbH & Co, Staufen, Germany). For each
batch of stents cultured with hTEC, two were fixed and processed
for scanning electron microscopy for estimation of cell adherence
and coverage.
ASSESSMENT OF NEOINTIMAL GROWTH
For neointimal proliferation experiments, 10 animals received
two stents each, using the protocol described above. They underwent repeat coronary angiography and an intravascular ultrasound
(IVUS) study at 28 days. QCA measurements were performed using
the automated edge-detection algorithms of CAAS-5 software (Pie
Medical, Maastricht, The Netherlands) by two independent experienced observers blinded to the treatment groups. The artery and
stent diameters were measured at baseline and at 28 days. Minimal
lumen diameter (MLD) was measured after stent implantation and
at 28 days. Late lumen loss (LLL) was calculated as the difference
between the MLD at 28 days and the MLD after stent implantation.
The IVUS imaging runs were acquired from 5 mm distal from
the stented segment to 5 mm proximal to the stented segment at
a constant pullback speed of 1 mm/sec. Still image frames were
analysed using the contour detection software supplied with the
imaging system. The minimal stent area (MSA) and minimal lumen
area (MLA) were calculated. The minimal lumen diameter (MLD)
was derived assuming a circular model and using the formula:
MLD=2×√MLA/π. The hearts were explanted and the stented arterial segments fixed, embedded and serially sectioned into 20-30 µm
thick sections, as previously described22. Stented sections were
examined microscopically and images captured using a high-resolution digital camera with output to a computerised image analysis system (NIS-Elements software; Nikon, Kingston upon Thames,
UK). For each section, the vessel area, lumen area, neointimal
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EuroIntervention 2014;10:709-716
area and the mean injury score were measured. The arterial injury
caused by the stent overstretch was quantified using Schwartz23.
The mean of the individual stent strut injury scores was taken as the
injury score for each section.
Statistical analysis
Data are expressed as mean±SEM. Comparisons between groups
were made using two-tailed Student’s t-tests. A probability of
p<0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism® software version 5.02
(GraphPad Software, San Diego, CA, USA).
Results
PREPARATION OF STEM CELLS (hTEC)
hTEC demonstrated a mixed progenitor and mature endothelial
phenotype, expressing both progenitor (CD133 and VEGFR-2) and
mature endothelial (CD31, E-selectin and vWF) markers. These
cells survived in co-culture with porcine endothelial (PAoEC)
and vascular smooth muscle cells (VSMC). Only 2.0% of hTEC
showed any binding to porcine anti-porcine IgG or IgM antibodies
as compared to 12-15% of porcine peripheral blood mononuclear
cells (PBMC) (Figure 1).
BIODISTRIBUTION AND FATE OF hTEC AFTER STENT
IMPLANTATION
For the stents seeded with 111Indium-labelled hTEC, the radioactivity measured over the explanted stented LAD was 77,642 cpm
at one hour, 20,048 cpm at one day and 2,323 cpm at seven days,
with no significant radioactivity detected at any of the distal sites,
including blood vessels, heart, lung, spleen, liver and kidneys.
Neointimal tissue obtained from within the hTEC (XY) seeded
stents implanted in the female pigs (XX) did not show any fluorescent signal when human probes specific for X, Y and 18 chromosomes were applied (Figure 2). No fluorescence was seen in the
paraffin sections of the neointimal tissue obtained from within the
BMS implanted in the contralateral coronary artery. By comparison, FISH of early passage hTEC showed a fluorescent signal for
all three probes (Figure 2).
hTEC ACCELERATES STRUT COVERAGE
Scanning electron microscopy of the hTEC-seeded stent group
showed an increased coverage of cells as compared to the BMS
controls, at all early time points, the difference being most marked
at one hour (Figure 3). At day three, the hTEC-seeded stents were
completely covered by a layer of tissue whereas the BMS struts
could still be seen. At day seven, both groups were well covered with tissue (Figure 3). Quantification of stent strut coverage
revealed a significantly accelerated tissue coverage in hTEC-seeded
stents (Figure 4).
hTEC DO NOT INCREASE NEOINTIMAL PROLIFERATION
Angiographic assessment with QCA did not show any significant difference in minimal lumen diameter at baseline, post stent
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Figure 1. Flow cytometry analysis of hTEC. Flow cytometry analysis
of hTEC with FITC labelled anti-porcine IgG and IgM antibodies
showing hTEC+anti-IgG:FITC (A) and hTEC+anti-IgM:FITC (B).
Porcine PBMC+anti-IgG:FITC (C) and porcine PBMC+antiIgM:FITC (D). hTEC+anti-IgG:FITC+50% porcine serum (E) and
+anti-IgM:FITC+50% porcine serum (F).
implantation or at 28 days (Table 1). IVUS assessment at day 28
also showed no difference in minimal stent area or minimal lumen
area between the two groups (Table 1). Histological examination
of these sections revealed a variable amount of neointimal tissue
Figure 2. Fate of hTEC after stent implantation. Fluorescent
micrographs of neointimal tissue at 28 days from hTEC (XY) seeded
stents in female (XX) pigs. A) Early passage hTEC for comparison.
B) The X chromosome was detected by green, the Y chromosome by
red and the 18 chromosome by aquamarine fluorescence.
Stem cell-seeded stents accelerate healing
EuroIntervention 2014;10:709-716
Figure 3. Scanning electron microscopy for assessment of strut coverage. Representative images from scanning electron microscopy of BMS
and hTEC-seeded stents under low (×25) and high (×100) magnification at one hr (hTEC-seeded [A,B]; BMS [C,D]), one day (hTEC-seeded
[E,F]; BMS [G,H]), three days (hTEC-seeded [I,J]; BMS: [K,L]) and seven days (hTEC-seeded [M,N]; BMS [O,P]) after stent implantation.
growth, in keeping with vascular injury that ranged from mild to
severe (Figure 5). Histomorphometric examination of 120 analysable sections from 10 pairs of BMS and hTEC-seeded stents showed
Table 1. Vessel measurements using quantitative coronary
angiography (QCA) and intravascular ultrasound (IVUS).
BMS
hTEC stent
n=10
n=10
Reference vessel diameter (mm)
2.78±0.01
2.79±0.01
0.83
Minimal lumen diameter post
implantation (mm)
3.39±0.05
3.33±0.05
0.47
QCA
A
Strut coverage at 1 hour
B
4
Strut coverage at 1 day
4
p<0.0001
2
1
Strut coverage score
Strut coverage score
p<0.0001
3
0
2.23±0.10
2.11±0.20
0.61
Diameter stenosis (%)
36.20±4.14
45.40±6.82
0.26
Late lumen loss (mm)
1.17±0.15
1.23±0.18
0.80
n=9*
n=9*
Minimal stent area (mm2)
7.68±0.23
7.48±0.13
0.45
Minimal lumen area (mm2)
5.09±0.28
5.04±0.37
0.92
Strut coverage at 7 days
Minimal stent diameter (mm)
3.12 ±0.05
3.09±0.03
0.47
p=0.70
Minimal lumen diameter (mm)
2.54±0.07
2.52±0.09
0.88
2
1
hTEC
IVUS at day 28
Strut coverage at 3 days
p<0.0001
BMS
D
4
Strut coverage score
4
Strut coverage score
Minimal lumen diameter at day 28 (mm)
0
BMS
C
3
3
2
1
0
hTEC
*IVUS measurements could not be obtained in one hTEC-seeded stent so stent pair excluded
from analysis. BMS: bare metal stents; hTEC: human trophoblastic endovascular progenitor cells
3
2
1
0
BMS
hTEC
p-value
BMS
hTEC
Figure 4. Stent strut coverage of bare metal and hTEC-seeded stents.
Stent strut coverage of BMS compared to hTEC-seeded stents at one
hr (A), one day (B), three days (C) and seven days (D) after stent
implantation. This demonstrates earlier strut coverage in hTECseeded stents.
that the vessel size and Schwartz injury score were similar between
the groups receiving BMS and the hTEC-seeded stents (Figure 6).
The lumen and neointimal area were also similar between the two
groups (Figure 6).
Discussion
Our study has demonstrated the feasibility and safety of a novel
approach to accelerate stent healing in the coronary artery, by
implanting metal stents pre-coated with stem cells of human
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EuroIntervention 2014;10:709-716
Figure 5. Tissue response to stent implantation at 28 days. Representative light microscopy images of sections from the BMS group: A) mild
injury, B) moderate injury, C) severe injury; and from the hTEC-seeded group: D) mild injury, E) moderate injury, F) severe injury.
embryonic trophoblastic endovascular progenitor cells. In our
study, the hTEC-seeded stents showed earlier stent strut coverage,
without excessive neointimal proliferation, compared to BMS.
There was a series of major challenges to achieve this outcome in
our study. First, the animal experiments were, perhaps, even more
stringent than will be seen in clinical trials, because there was the
potential for immune-mediated cell rejection by testing human cells
in pigs. However, because of the “immune protection” afforded by
expression of HLA-G in the cells, there was hardly any binding of
A
Vessel area
B
15
Schwartz score
2.0
p=0.279
1.5
Injury score
Area in mm2
p=0.363
10
5
1.0
0.5
0
0.0
BMS
C
hTEC
Lumen
p=0.275
5
BMS
D
4
Neointima
p=0.748
3
Area in mm2
Area in mm2
4
hTEC
3
2
2
1
1
0
0
BMS
hTEC
BMS
hTEC
Figure 6. Histomorphometric outcome of bare metal and hTECseeded stents. Histomorphometric measurements of tissue sections at
28 days after stent implantation (mean±SEM). Total vessel area (A),
Schwartz injury score (B), lumen (C) and neointimal area (D). This
demonstrates no difference in injury score, vessel area, lumen area
and neointimal proliferation between the two groups.
714
hTEC to pre-existing porcine anti-human IgG and IgM antibodies.
Second, the cells were required to adhere to the stent surface. We
demonstrated that this was possible in cell culture. Third, it was
important to demonstrate cell survival after stent implantation. We
showed that a sizeable proportion of seeded cells remained at the
implantation site, with little or no detection elsewhere, using radioactive labelling. Finally, we showed accelerated coverage of stent
struts at early time points, together with neutrality of neointimal
growth at 28 days, compared with BMS.
The alternative technique, of attracting coverage of endogenous
circulating endothelial progenitor cells (EPC), has gained limited
success24. Stents loaded with anti-CD34 antibodies (Genous™;
OrbusNeich Medical Technologies, Fort Lauderdale, FL, USA)
have shown good healing response at the price of restenosis, which
is greater than that seen in first-generation DES16. The TRIAS HR
(TRI-stent adjudication study-high risk of restenosis) trial comparing the Genous™ EPC capture stent with DES had to be terminated early due to excess restenosis in the EPC capture stent-treated
patients17. This technology is dependent upon the capacity of the
patient to mobilise endothelial precursors. Yet patients with severe
coronary artery disease have fewer EPCs with reduced migratory
potential and lower functional capacity25,26. The number of circulating EPCs in peripheral blood in all subjects is extremely low.
A study estimated early CD133+ / CD34+ / VEGFR-2+ cells in
healthy human subjects to be 0.002% of the total PBMC with
a reported increase in the number to 0.02% of the total PBMC after
stimulation with granulocyte colony stimulating factor (G-CSF)27.
Moreover, a homing response of EPCs has not been confirmed after
PCI performed for acute coronary syndromes28. The advantage of
pre-seeding stents with stem cells is that no reservoir or mobilisation of circulating and functional EPCs capable of homing to the
site of vascular injury is required.
Our findings are positive compared with previously attempted
cell-seeding stents. Seeding with endothelial cells has been shown to
increase neointimal proliferation in a rabbit model29. Mesenchymal
Stem cell-seeded stents accelerate healing
Limitations
This study has several limitations. The porcine coronary stenting
model lacks atherosclerosis and relies upon the de novo growth of
neointima due to vascular injury caused by stent implantation in
a disease-free artery. Additionally, results from juvenile healthy
animals may not be directly extrapolated to ageing human patients
with comorbidities. Characterisation of the progenitor cell phenotype is challenging. The hTEC used in our study possessed a mixed
progenitor and mature endothelial phenotype, expressing both progenitor and mature endothelial markers, but surface marker patterns
remain controversial.
Conclusion
This study has demonstrated the safety and feasibility of implanting a novel intracoronary stent seeded with hTEC in a large animal
model. This approach can accelerate tissue regeneration over the
stent struts without provoking excessive neointimal growth.
Impact on daily practice
This is a preclinical study in a clinically relevant large animal
model demonstrating a novel approach to enhance stent strut
coverage by coating stents with human trophoblastic endovascular progenitor cells. Further studies are warranted before this
technology can be used in clinical practice.
Acknowledgements
We are grateful to the Department of Trade and Industry, UK,
for funding this study and to D. Baker at the Sheffield Children’s
Hospital for performing cytogenetic analysis of the porcine neointimal tissue samples.
Conflict of interest statement
The authors have no conflicts of interest to declare.
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EuroIntervention 2014;10:709-716
stem cell seeding has been shown to promote endothelialisation and
inhibits the neointimal hyperplasia compared with BMS in a rabbit model, but no subsequent studies have been conducted in large
animals or in man30. It is encouraging that we achieved accelerated healing without excessive neointimal growth at a late time
point. Stents coated with hTEC may be clinically useful in patients
with high bleeding risk, planned surgery or anticipated problems
with prolonged dual antiplatelet therapy. One possibility might
be to coat stents with hTEC on the luminal aspect and an antiproliferative drug on the abluminal, to achieve accelerated coverage
with reduced neointimal proliferation. Further studies of this novel
device are warranted.
715
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716
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Online data supplement
Appendix. Methods.
Stem cell-seeded stents accelerate healing
EuroIntervention 2014;10
Online data supplement
Appendix. Methods
PREPARATION OF hTEC STEM CELLS
hTEC were derived by the terminal differentiation of trophoblast
stem cells while the latter were derived as distinct trophoblast cell
lines (TS) from human embryonic stem cells (Shef 3 and six cell
lines). A vial of TS cells (2×106 cells/ml) was recovered from longterm storage in liquid nitrogen and thawed into Dulbecco’s Modified
Eagle Medium (DMEM) supplemented with 10% foetal calf serum
(FCS), washed by centrifugation (×2) and cultured in 2×T25 culture flasks with fresh medium at 37ºC in 5% CO2 in air for 10 days.
During this time, the cells terminally differentiated to a proven endovascular phenotype, and further cell proliferation was arrested.
ISOLATION OF PORCINE ENDOTHELIAL AND SMOOTH
MUSCLE CELLS
Endothelial and smooth muscle cells were isolated from porcine
thoracic aortae. Porcine aortic endothelial cells (PAoEC) were
cultured in DMEM supplemented with 2.5 µg/mL amphotericin
B, 100 U/mL of penicillin and streptomycin, 2 mM L-glutamine,
20 µg/mL of endothelial cell growth supplement, 90 µg/mL of
heparin and 20% porcine serum, and incubated at 37ºC in 5% CO2
in air. Porcine vascular smooth muscle cells (PVSMC) were cultured in DMEM supplemented with 2.5 µg/mL amphotericin B,
100 U/mL penicillin and streptomycin, 2 mM L-glutamine, 1×nonessential amino acids and 20% porcine serum, and incubated at
37ºC in 5% CO2 in air. Both PAoECs and PVSMCs adhered to the
bottom of gelatine-coated tissue culture flasks and once confluent
were passaged using 0.1% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA). Fixed PAoEC were stained for endothelial cell markers; E-selectin and von Willebrand Factor (vWF).
For E-selectin staining, PAoEC were stimulated with recombinant human tumour necrosis factor-alpha (hTNF-α; AbD Serotec,
Kidlington, Oxford, UK) prior to methanol fixation. Staining of
fixed PVSMC was carried out for α-smooth muscle actin (α-SMA)
and smooth muscle myosin heavy chain (SM-MHC).
PORCINE ANTIBODIES AGAINST hTEC
The effect of any pre-existing circulating anti-human porcine antibodies upon hTEC was determined by exposing hTEC to porcine
serum followed by flow cytometry analysis with FITC labelled
anti-porcine IgG and IgM antibodies. hTEC (1×105) were incubated with increasing dilutions of porcine serum (50%, 20%, 10%,
5% and 2%) and treated with anti-pig IgG or IgM: FITC antibody
(AHP865F and AAI39F; AbD Serotec) and 2% goat serum at 4ºC.
The samples were centrifuged at 500 g for five minutes, supernatant
aspirated and a fluorescence activated cell sorting (FACS) analysis of the resulting cell suspension performed using a FAC Scan
(Becton Dickinson, Oxford, UK) analyser. Viability of samples
was confirmed by propidium iodide staining. Untreated hTEC and
hTEC incubated with only anti-pig IgG and IgM antibodies were
used as negative controls. Mononuclear cells isolated from freshly
obtained whole blood were used as positive controls.
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