Repair of Old Myocardial Infarction by Intracoronary Transplantation of Autologous Bone Marrow Mesenchymal Stem Cells: A Pilot Clinical Trial

INTRODUCTION
Myocardial infarction is the leading cause of congestive
heart failure and death in developed countries. World
Health Organization (WHO) predicts that the disease
will be the main cause of death world wide in the near
future (Lee and Makkar, 2004; Lee et al., 2004).
Several studies have documented the ability of the
human heart to regenerate, albeit to a degree that is
without clinical benefit, in patients with end stage
heart failure, and after myocardial infarction.
Therefore stem cell transplantation offers a simple and
cost-effective way of repairing the infarcted heart (Lee
and Makkar, 2004; Lee et al., 2004; Mathur and
Martin, 2004).
Both animal and human studies suggest that stem
cells capable of improving cardiac function do exist in
adults. Stem cells such as mesenchymal stem cells
have the potential to differentiate into cardiomyocytes
(Mohyeddin Bonab et al., 2006 and 2005). Preliminary
data from animal models and human studies also indicate that infarcted myocardium can be regenerated by
implanting embryonic or/adult stem cells (Lee and
Makkar, 2004; Lee et al., 2004; Liu et al., 2004;
Hodgson et al., 2004; Mangi et al., 2003; Mathur and
Martin, 2004; Soukiasian et al., 2004).
Different cell types have been used in cell transplantation. Meanwhile, autologous bone marrow stem
cells and skeletal myoblasts have generated the most
data (Liu et al., 2004; Mathur and Martin, 2004;
Soukiasian et al., 2004; Chierchia and Deferrari, 2004;
Stamm et al., 2003b). In this study, separated mesenchymal stem cells (MSCs) from the bone marrow of
patients who had suffered from old myocardial infarction were grown (expanded) in vitro and then injected

into the infarcted region via the infarct related artery
by the balloon angioplasty method (mean ± SD, 5.2 ±
3.11 months, time from MI to cell therapy).
MATERIALS AND METHODS
From May 2004 to March 2005, implantation of autologous bone marrow mesenchymal stem cells (BMSC)
was performed in five patients (4 males and one female,
mean ± SD, age = 48.4 ± 11.28 years) via percutaneous
transluminal coronary angioplasty (PTCA). All patients
were suffering from old anterior myocardial infarction.
Time from acute myocardial infarction (AMI) to stem
cell transplantation was 5.2 ± 3.11 months. Majority of
patients had moderate to severe left ventricular dysfunction. Their LVEF were 34% ± 10.83% (mean ±
SD). Their functional class were Ш-IV (NYHA class).
Inclusion criteria: 1- Patients with a history of old
myocardial infarction, 2- Age <70 years old, 3-
Presence of regional wall motion abnormality (WMA),
severe akinesia or dyskinesia, documented by left ventriculography and echocardiography, 4- Absence of
viable myocardium in ≥ 2 segments documented by
radionuclide scintigraphy.
Exclusion criteria: 1- Age >70 years old, 2- Coronary
anatomy unsuitable for percutaneous coronary intervention (PCI), 3- Severe co-morbidity, 4- Presence of
viable myocardium at the territory of the infarcted
zone, 5- Presence of cardiogenic shock or life threatening cardiac dysrhythmias, 6- Unwilling to do stem cell
transplantation.
Written informed consent has been signed by each
participant in the study. The study gained the approval
of the ethical committee of the Tehran University of
Medical Sciences under the supervision of the diagnostic digestive disease research center (FWA00001331).
Before making a decision for BMSC transplantation, diagnostic coronary angiography was performed
in all patients. After evaluation of their coronary
angiography and making sure that the infarct related
artery (IRA) is suitable for PCI, they were referred for
bone marrow (BM) aspiration and BMSC expansion.
Sample collection and MSC expansion: Under local
anesthesia, 30 ml of BM was obtained from the iliac
crest of each patient in a sterile and standard condition.
The BM mononuclear cells (MNCs) were separated by the ficoll density gradient method. Eight average
vented flasks (75 cm2) with 21 ml of MSC medium,
consisting of Dulbecco’s Modified Eagle’s Medium
(DMEM) with 10% (v/v) Fetal Bovine Serum (FBS)∗
and 1% penicillin and streptomycin (all from Gibco,
Sigma, Germany), were seeded with 1×106 MNC/ml
for the purpose of primary culture. Flasks were incubated at 37ºC in a humidified atmosphere containing
5% CO
2 and were fed by complete medium replacement every 4 days, until the fibroblast-like cells at the
base of the flask reached confluence.
On reaching confluence, the adherent cells were
resuspended using 0.025% trypsin and reseeded at
1×104 cells/ml (1 st passage). These were incubated
again until confluence, and were once again trypsinized
and reseeded at 1×104 cells /ml. The number of passage
of cells depending on the required amount of cells can
be repeated, (normally 1-3 passages).
At the end of the last passage, when the cells
reached confluence, they were washed with tyrode salt
and incubated with MI99 medium for an hour. Cells
were detached by trypsinization and washed with normal saline supplemented with 1% human serum albumin and heparin and then resuspended at 1-1.5×
106/ml density. This washing process eliminates trace
amounts of FBS as well.
Immunophenotyping: At the end of the last passage,
expression of CD166, CD105, CD44 and CD13-which
are MSC surface markers, and CD34 and CD45-which
are Hematopoietic Stem Cell (HSC) surface markers
were determined in culture-expanded MSCs. The monoclonal antibodies used were anti-CD44, CD45, CD34
fluorescein isothiocyanate (FITC) and anti-CD13 phycoerythrin (PE) (all purchased from Dako, Denmark),
anti-CD166 FITC and anti CD105 RPE (from Serotec,
Germany). Relevant isotope control antibodies were
also used. Flowcytometry was performed on a FACS
calibur system (Becton Dickinson) and data were analyzed with Cellquest software.
Safety assessment: To make sure that the cells are not
contaminated, bacteriological tests were performed on
the samples for every passage and at the time of injection. Viability of the cells was assessed by the
Methylene Blue dye exclusion test just before injection.
We used high grade FBS from Gibco Co. which has
been checked for virus, micoplasma, sterility, bacteriophage and endotoxin contamination.
Intracoronary injection of BMSC: Patients were
treated with antiplatelets (325 mg of Aspirin on the day
before transplantation followed by 300 mg of a loading
dose of clopidogrel, at least 6 hours before the procedure). In addition 75-100 U/kg of heparin was given
during the procedure to maintain activated clotting time
(ACT) between 250-300 seconds.
The procedure was initiated with diagnostic coronary and left ventricular (LV) angiography to re-evaluate coronary anatomy and LV function. Then, a diagnostic catheter was replaced with a guiding catheter.
After crossing the lesion with a long (300 cm),
0.014 inch diameter guide wire, an over-the-wire
(OTW) balloon was introduced into the infarct related
artery and positioned at the occlusion site. Then, the
guide wire was drawn out from the central lumen of
the OTW balloon. The procedure of BMSC injection
into the infarcted area began with sequential inflation
and deflation of OTW balloon every 2-3 minutes.
During inflation of the balloon, injection of 1-2 ml of
the prepared suspension of bone marrow MSC in to the
infarcted region was performed through the central
lumen of the balloon. The balloon remained inflated
for at least 2-3 minutes in order to prevent backflow of
BMSCs suspension, and thus facilitate distribution of
the suspension into the infarcted zone. After each
inflation, the balloon was deflated and remained so for
2-3 minutes in order to permit perfusion of the area.
This cycle was repeated for 5-6 times. The mean number of 8 × 106 (ranging 5-12 × 106) bone marrow mesenchymal cells was injected into the infarcted area
(Table 2). After the cell injection process, the procedure was completed with stenting the lesion with an
appropriate sized bare metal stent (BMS). Procedures
were tolerated well by all patients without any hemodynamic or rhythm disturbances.
Follow up: Patients have been visited every month for
three months, and then every 3 months during follow
up. In each visit, clinical and paraclinical evaluation in
terms of physical examination and 12 leads of electrocardiography were carried out.
Selective coronary angiography and left ventriculography were performed between 6 to 9 months after
BMSC transplantation in order to evaluate regional
and global left ventricular ejection fraction (LVEF)
and binary instent restenosis (binary restenosis defined
as > 50% luminal loss).
Statistical analysis: Statistical analysis was performed with SPSS program (version 11.5). A comparison of pre and post-procedure data were carried out by
using the paired t-test and nonparametric test of
wilcoxon. Discrete variables were compared as rates.
All data were presented as mean ± SD and median,
with p ≤ 0.05 considered as significant.
RESULTS
From the beginning of May 2004 to the end of March
2005, intracoronary injection of BMSC was performed
in five patients, with old anterior myocardial infarction. Baseline and clinical characteristics of patients
are presented in Table 1.
At the end of the processes of immunophenotyping
and safety assessment, the mean number of 8 × 106
(ranging 5-12 ×106) cells representing the prepared suspension of BM mesenchymal cells was utilized for
transplantation (Table 2). The results of flowcytometry
analyses of CD13 (78%), CD44 (79%), CD166 (51%),
and CD 105 (56%) were positive and for the hematopoetic cell markers, CD34 and CD45 they were negative.
The results of bacteriological analyses were negative for all samples. The viability of injected cells was
over 95%.
Procedural success rate was 100% (there were no
failures and no major complications in terms of death,
nonfatal myocardial infarction or urgent CABG).Three
patients had single vessel disease, left anterior
descending (LAD) and two patients had multivessel
disease LAD, LCX (left circumflex) and LAD,OM
(left anterior descending,obtuse marginal).
Angiographic characteristics of LV and coronary arteries as well as procedural results are shown in Table 2.
Follow up LV and coronary angiography are carried out 6 to 9 months (mean ± SD, 7 ± 1.4 months)
after BMSC transplantation in four patients (80%).
One patient has been lost regarding follow up (couldn't be accessed during the follow up). Therefore, follow up coronary angiography was carried out in four

patients, during wich there was one target lesion revascularization (TLR) (patient number 2). Therefore, the
major adverse cardiac event (MACE) i.e. death, nonfatal MI and TLR) during follow up in four patients was
identified only as TLR. In four patients, follow up
angiography revealed improvement in their LV ejection fraction (EF) (mean ± SD from 34 ± 10.8% to
46.25 ± 9.46%, p= 0.051 and median from 35% to
42.5%). Baseline and follow up angiographic characteristics are presented in Table 3.
Figure 1 shows baseline and follow up LV, wall
motion abnormalities (WMA), and ejection fraction of
patient number 4.
Clinical follow up was carried out for 12 to 18
months. During clinical follow up, no cardiac arrhythmia, death or nonfatal myocardial infarction occurred.
There was only one TLR due to in-stant restenosis,
revascularization was carried out by repeat percutaneous coronary intervention (rePCI) and implanting a
drug eluting stent (DES).
The symptoms (dyspnea and chest discomfort) of
all patients improved and their functional class
changed from NYHA and /CCS class Ш-IV to class I-
П, during clinical follow up. Baseline and follow up
clinical characteristics are presented in Table 4.
DISCUSSION
The possibility of stem cell therapy for repairing
myocardial infarction has created a new situation,
quite unlike any previous therapeutic development
process. Open collaboration amongst basic scientists
and clinicians around the world are crucial for this
superior procedure to be carried out successfully.
The traditional concept implies that the heart muscle itself has no house keeping mechanism to repair
any minor damage, but recent investigations suggest
that large numbers of mitotic figures are present in the
adult heart. The source of dividing cells in the
myocardium is unclear. Two sources have been suggested, first, bone marrow stem cells, second, cardiac
stem cells (Lee and Makkar, 2004; Lee et al., 2004;
Mathur and Martin, 2004; Wu et al., 2003; Caplice and
Gersh, 2003; Forrester et al., 2003; 15-Barbash et al.,
2003). Studies in animals and patients with heart transplant, have revealed that, donor-derived cardiomyocytes were present in the recipient heart. Investigators
believe that these cells are donor MSCs which have
differentiated into cardiomyocytes (Bayes-Genis et al.,
2002; Laflamme et al., 2002; Muller et al., 2002; 20-
Sauer et al., 2002; Toma et al., 2002; ).
The most consistent improvement in myocardial
function combined with safety has come from studies
using autologous bone marrow stem cell transplantation in myocardial infarction. Compared to other current methods that have been used for cell transplantation in patients with myocardial infarction, the intracoronary approach is one of the safest, the most feasible and minimally invasive method for cell transplantation. In most studies of intracoronary injection of
autologous bone marrow stem cells (Siminiak et al.,
2004, Chen et al., 2004; Wollert et al., 2004; Assmus
et al., 2002; Strauer et al., 2002) no adverse effect was
reported. Only in one study (Bartunek et al., 2005)
increased incidences of coronary events following
intracoronary administration of enriched CD133 cells
were reported.
The genetic and cellular mechanisms that initiate
transdifferentiation of stem cells are very complex and
poorly understood, nevertheless it has been shown that
transplanted stem cells undergo a “homing” process in
which they are attached to the site of injury (Lee et
al.,2004; Mangi et al., 2003; Wu et al., 2003) .
Strauer and his colleagues for the first time in 2002
reported cases of intracoronary injection of autologous
bone marrow derived progenitor cells for repairing
myocardial infarction. According to their study an
improvement in the left ventricular function including
a significant reduction in the infarct size has occurred
(from 30 ± 13% to 12 ± 7%, P = 0.005) (Strauer et al.,
2002).
To our knowledge phase-1 clinical studies of stem
cell transplantation (before 2004) included 13 studies.
These attempts have been undertaken in humans for
repairing injured myocardium in patients with acute
myocardial infarction. In these studies, different protocols such as PTCA, thoracotomy (during CABG) and
transcatheter intramyocardial injection have been
implicated, which in four of them PTCA was the prefered method (Chen et al., 2004; Lee and Makkar,
2004; Lee et al., 2004; Siminiak et al., 2004; S t a m m
et al., 2004a; Wollert et al., 2004; Menasche et al.,
2003; Assmus et al., 2002; Strauer et al., 2002).
In those studies that BMSC were used via the
PTCA technique. Although their methods were different but they reached the same results. Strauer and his
colleagues (2002) reported a significant decrease in the
infarct region within the cell therapy group compared
to the control group (p= 0.04) after 3 months.
In BOOST (intracoronary autologous BOne
marrOw cell tranSfer afTer and myocardial infarction)
and TOPCARE-AMI (Transplantation Of Progenitor
Cells And REgeneration in Acute Myocardial
Infarction) randomized trials, improvements in global
LVEF after 4-6 months follow up were significant
(from 50 ± 10% to 56.7 ± 12.5%, p= 0.0026 and from

51.6 ± 9.6% to 60.1 ± 8.6%, p= 0.003 respectively),
(Wollert et al., 2004; Assmus et al., 2002).
Chen and his colleagues (2004) also reported significant improvement in the LVEF of their cases 3
months after cell therapy (from 49 ± 9% to 67 ± 11%,
p = 0.01).
There are few investigations regarding stem cell
therapy in patients with old myocardial infarction.
Similar to attempts carried out in AMI; results of these
studies were surprising as well.
Improvement of coronary endothelial function and
enhanced reserve flow due to intracoronary application of BMSC after successful PCI (post-recanalization) in patients with chronic coronary total occlusion
has been reported by lenk and his colleagues (2004).
Manginas and his colleagues (2004) for the first
time in human study documented that intracoronary
administered radio-labeled BMSC were clearly seen
adhered to the infracted zone in patients with old
myocardial infarction, In another study they observed
reduction in the left ventricular dimensions and
improvement in perfusion of the previously infracted
region (Manginas et al., 2004a; Manginas et al.,
2004b).
The results of this study were comparable with
those that have been performed in patients with AMI
as well as with those studies in patients with old
myocardial infarction.
In our patients, as in other studies, improvement in
the left ventricular function and functional capacity
were consistent during the follow up period (12-18
months). In this period, no cardiac arrhythmia, death
or nonfatal MI has occurred. Also safety and feasibility of the application of BMSC for myocardial regeneration in patients with old myocardial infarction and
moderate to severe LV dysfunction have been elucidated in our, albeit small, study.
Limitation: The absence of a matched control group
and follow up positron emission tomography with
foulorodeoxyglucose (FDG-PET) study to quantitate
the magnitude of regional viability after cell therapy
and the small number of patients were the limitations of
our pilot study. For reaching a stronger conclusion, a
large randomized controlled clinical trial is necessary.
CONCLUSION
Application of autologous BMSC transplantation for
repairing myocardium in patients with old myocardial
infarction is safe, feasible and effective. Catheter
based percutaneous transluminal coronary approach is
one of the safest and minimally invasive techniques for
this novel method of myocardial regeneration in such
patients.