Magnetic resonance mapping of transplanted endothelial progenitor cells
for therapeutic neovascularization in ischemic heart diseaseq
Alberto Webera, Ivan Pedrosab, Atsuhiko Kawamotoa, Nathan Himesb, Jeeva Munasingheb,
Takayuki Asaharaa, Neil M. Rofskyb, Douglas W. Losordoa,*
aDivisions of Cardiovascular Research and Cardiovascular Medicine, St. Elizabeth’s Medical Center, Tufts University School of Medicine,
Boston, MA 02135, USA
bDepartment of Radiology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA
Received 8 January 2004; received in revised form 22 February 2004; accepted 2 March 2004; Available online 18 May 2004
Objective: Intramyocardial transplantation of endothelial progenitor cells (EPCs) has been previously correlated with significant
augmentation of vascularity and improvement of left ventricular function following myocardial ischemia. However, precise intramyocardial
localization of the transplanted cells and the extent of in situ cell migration are unknown. We present a novel technique using magnetic
resonance imaging (MRI) to localize transplanted EPCs in ischemic hearts. Methods: CD34-positive cells were isolated from human
peripheral blood by magnetic bead selection: CD34-positive cells adhere to CD34-negative antibody coated magnetic beads, while CD34-
negative cells do not.All cells were labeled with fluorescentDiI-dye forhistological localization. CD34-positive cells or CD34-negative cells
(105, 1 £ 106 and 2 £ 106 cells) were transplanted into non-ischemic ðn ¼ 6Þ or ischemic myocardium ðn ¼ 2Þ of Sprague–Dawley rats.
Rats were sacrificed 24 h after cell transplantation. The resected hearts were imaged ex vivo using 3 and 8.5 T magnets. Morphological
correlation between the MRI findings and fluorescent microscopy for identification of retained CD34-positive cells was evaluated. Results:
CD34-positive cells were identified as areas oflow signalintensity on T2*-weighted images within the myocardium. These areas increased in
size with the gradual increase in the echo time due to susceptibility effect. The extent of the low signal intensity at a given echo time was
proportional to cell dosage. No areas of low signal were identified in the CD34-negative cell transplanted hearts. Histological localization of
DiI-labeled CD34-positive cells documented a direct anatomic correlation with the localization of transplanted cells on the MRI images.
Conclusions: Magnetically labeled EPCs transplanted for therapeutic neovascularization in myocardial ischemia can be visualized with
ex vivo MRI at high-field strengths.
q 2004 Elsevier B.V. All rights reserved.
Keywords: Angiogenesis; Ischemic heart disease; Stem cell tracking; Magnetic resonance imaging
Whereas most tissues in adult organs are composed of
differentiated cells, stem or progenitor cells are maintained
in a quiescent status locally or in the systemic circulation
and are activated during physiological and pathological
tissue regeneration. Haematopoietic stem cells (HSCs)
and endothelial progenitor cells (EPCs) are derived from
a common precursor, the hemangioblast (Fig. 1). HSCs and
EPCs share certain antigenic determinants, including Flk-1,
Tie 2, cKit, Sca-1, CD133 and CD34. These markers are
subsequently lost as HSCs differentiate . EPCs were first
isolated, by Asahara et al., as CD34-positive mononuclear
cells (MNCs) from adult peripheral blood by means of
magnetic beads coated with antibody to CD34, as EPCs-
enriched fraction .
Circulating EPCs have been shown to home to sites of
neovascularization where they differentiate into endothelial
cells (ECs) in situ, consistent with ‘vasculogenesis’ [3–5].
Intravenous transplantation of cultured human EPCs applied
1010-7940/$ - see front matter q 2004 Elsevier B.V. All rights reserved.
European Journal of Cardio-thoracic Surgery 26 (2004) 137–143
qPresented at the 16th Annual Meeting of the European Association for
Cardio-thoracic Surgery, Monte Carlo, Monaco, September 22–25, 2002.
* Corresponding author. Address: Division of Cardiovascular Research,
St. Elizabeth’s Medical Center, 736 Cambridge Street, Boston, MA 02135,
USA. Tel.: þ1-617-789-3474; fax: þ1-617-779-6362.
E-mail address: firstname.lastname@example.org (D.W. Losordo).
in a model of myocardial ischemia in the nude rat
demonstrated that transplanted human EPCs can incorporate
into rat myocardial neovascularization, preserving left
ventricular (LV) function, and inhibiting myocardial fibrosis
. Recently, Kocher et al.  attempted intravenous
infusion of freshly isolated human CD34-positive MNCs
(EPC-enriched fraction) into nude rats with myocardial
ischemia. These experimental findings using immunodefi-
cient animals suggest that both cultured and freshly isolated
human EPCs have therapeutic potential in peripheral and
EPCs were always isolated by means of magnetic beads
coated with antibody to the CD34 antigen . The magnetic
beads were not detached from the selected cells before
transplantation. Previous data from our lab documents
preserved viability and functionality of transplanted CD34-
positive cells isolated by means of magnetic beads.
The development of progenitor and stem cell therapy in
humans would be greatly enhanced by a technique to
monitor their fate non-invasively thereby permitting serial
assessment of the cellular biodistribution and migratory
capacity . To monitor the fate of transplanted cells,
including their migration in vivo, cells are currently labeled
ex vivo using a vital dye (e.g. a fluorochrome), a thymidine
analog (e.g. BrdU), or a transfected gene (e.g. LacZ or green
fluorescent protein, GFP), for later visualization using
(immuno)histochemical procedures following tissue
removal at a single time point. Considering the different
delivery options for implementing myocardial cell trans-
plantation (epicardial via intraoperative intramyocardial
injection; endocardial, via catheter-based intramyocardial
injection; intracoronary; retroperfusion, via cannulation of
the coronary sinus; or intrapericardial, after transthoracic
access to the pericardium) , a technique that could
monitor the engraftment and localization of the transplanted
cells is crucial to assess the safety and success of these
Most MR scanners used in clinical practice have
magnetic fields equivalent to 1.5 T. Recent advances in
the development of MR systems have allowed for scanners
that operate at high magnetic fields. High-field MR
scanners, including whole body clinical 3 T magnets for
human use, provide excellent signal that allow for near
microscopic resolution. The magnetic tagging of cells that
facilitates separation also creates an opportunity to visualize
these cells with magnetic resonance imaging (MRI).
MR signal results from the behaviour of protons in a
magnetic environment. One behaviour is referred as the T2
of a tissue, an innate feature based upon its constituent
molecules. The T2 characteristics of tissue are related to
local interactions amongst protons. These so called ‘spin–
spin’ interactions cause dephasing of the protons, which
leads to the decay of MR signal over time. Disturbances in
the local magnetic field affect the spin–spin interactions so
that dephasing occurs more rapidly and therefore, the MR
signal decay is also faster. This faster decay, or T2* decay,
yields a lower signal intensity on the image. Thus, local
magnetic field distortions caused by the tagged cells can be
captured with MRI as a dark signal intensity particularly
when T2* weighted sequences are used. An increase in the
time between the excitation of the protons and the collection
of the MR signal, or echo time (TE), allows for more
dephasing of the protons. Hence, the longer the TE the
greater decay in MR signal. By selecting gradually
increasing echo times, the T2* effect resulting from the
magnetic beads can be appreciated as an increase in the area
of dark signal intensity or ‘blooming effect’ within the MR
images. Gradient echo sequences (GRE) are a class of MRI
strategies that are particularly sensitive to T2* effects.
Finally, higher field strengths are more sensitive to T2*
effects as compared with lower field strengths.
Conventional magnetic cell labeling techniques rely on
surface attachment of magnetic beads ranging in size from
several hundred nanometers to micrometers . There are
several prior reports describing magnetic resonance tracking
of progenitor cells, in neural tissue, tumor or inflammatory
It has been established that bone marrow-derived EPCs
present in the systemic circulation home to and incorporate
into sites of neovascularization, and may be useful in
therapeutic strategies of ‘supply-side angiogenesis’, for
example, after myocardial ischaemia (MI) [2,10,15–20]. A
technique that could monitor the engraftment and migration
of intramyocardial injected EPCs, serially and non-invasive,
could guide further advances for clinical application. We
hypothesized that magnetic cell labeling would allow for
intramyocardial visualization and localization of EPCs on
The data presented in this study represent just a
preliminary proof of principle where we try to define
whether or not intramyocardial EPCs visualization and
localization is possible with MRI.
Fig. 1. Differentiation profile of endothelial lineage cells. Embryonic
endothelial progenitor cells (EPCs) and hematopoietic stem cells (HSCs)
share among others CD34, lost by the second as they differentiate.
A. Weber et al. / European Journal of Cardio-thoracic Surgery 26 (2004) 137–143138
2. Materials and methods
2.1. Animal models
All procedures were performed in accordance with
St. Elizabeth’s Institutional Animal Care Committee. We
utilized male Sprague–Dawley rats (200–250 g weight) as
our animal model.
2.2. Fresh isolation and intramyocardial transplantation
of human EPCs
Human total peripheral blood MNCs were isolated from
healthy volunteers by density-gradient centrifugation.
CD34-positive mononuclear blood cells were isolated
from total MNCs by means of colloidal super-paramagnetic
beads (MACS-Microbeads) conjugated to monoclonal
mouse anti-human CD34 antibody (isotype: mouse IgG1,
clone: QBEND/10) (Miltenyi Biotec) as EPC-enriched
fraction. Mononuclear cell isolation by density-gradient
centrifugation is necessary for the initial step, because
mononuclear cells in the systemic circulation contain a
fraction capable of differentiation to endothelial lineage
cells. After mononuclear cell isolation, total mononuclear
cells are incubated with anti-CD34 antibodies coated with
magnetic microbeads for fresh isolation. A magnetic
column is used to collect only the cells binding to the
antibodies with microbeads. After the isolation, CD34-
negative MNCs were also collected. Both populations of
CD34-positive and CD34-negative MNCs were labeled
with fluorescent DiI-dye. Cells were counted using a
haemocytometer and resuspended in 100 ml PBS. The
magnetic beads were never detached from the isolated
CD34-positive cells before transplantation. Non-ischemic
(A) and ischemic (B) rat hearts were treated. (A) Non-
ischemic: Sprague–Dawley rats ðn ¼ 4Þ were anaesthetized
with ketamine i.p. (0.6 ml/100 g) and intubated. Left
parasternal longitudinal thoracotomy was performed. After
pericardectomy, DiI-labeled CD34-positive MNCs in
100 ml of PBS were injected intramyocardially in the
anterior and/or lateral wall of the LV using a 27G needle.
These four rats were treated with intramyocardial injection
of 105, 2 £ 105, 1 £ 106or 2 £ 106CD34-positive cells,
respectively. Two additional rats received 1 £ 106or
2 £ 106DiI-labeled CD34-negative MNCs in 100 ml of
PBS as negative controls. (B) Ischemic: Two rats underwent
ligation of the left anterior descending coronary artery
(LAD). Ten minutes after the operation these rats were
injected with 105DiI-labeled CD34-positive MNCs in
100 ml of PBS. Cells were injected in two sites within the
ischemic vascular territory of the LAD using a 27G needle.
The ischemic zone was macroscopically identified by the
pale color of the anterior and lateral walls after LAD
All rats were sacrificed 24 h after intramyocardial
transplantation of CD34-positive EPCs or CD34-negative
MNCs. From our experience, it takes about 24 h for the rat
heart to absorb the injected saline and that is why 24 h was
chosen as time point for sacrifice. The hearts were resected
and fixed with 4% paraformaldehyde.
2.3. MR imaging and histopathological correlation
To obtain a completely dark background we embedded
the specimens in a perfluoropolyether (Fomblin, Fluortek
AB, Sweden) devoid of proton signals. This polymer was
found to be inert, effectively sealing the specimens from
dehydration, with no observed effects on tissue morphology.
Three-dimensional spin echo MR images were obtained by
using a 9 cm bore 8.5 T Bruker magnet (Bruker Biospin,
parameters were: TR/TE ¼ 1700/25 ms, 256 £ 128 £ 128
matrixwithanFOV ¼ 30 £ 15 £ 15 mm,affordinga117 m3
resolution. Additionally, the hearts were scanned on a
clinical 3 T magnet (General Electric Medical Systems,
Milwaukee) using a 3-inch surface coil. Two-dimensional
gradient echo MR images were obtained with the following
parameters:TE ¼ 10
angle ¼ 308, slice thickness of 1 mm, matrix 512 £ 256,
FOV ¼ 6 mm,NEX ¼ 1.TheGREsequencewasperformed
with two echoes to facilitate an evaluation of the suscepti-
bility effect of the magnetic beads, the longer echo time
having greater sensitivity to this effect.
Images were obtained in the long and short axes of the
resected hearts. The area of T2/T2* effect (marked
hypointensity) on each short axis slice-image was
measured for each heart, dividing each heart in 1 mm
thick slice-images; this analysis was performed on a
Macintosh computer using the public domain NIH Image
program (developed at the US National Institutes of Health
and available on the Internet at http://rsb.info.nih.gov/
nih-image/). The marked hypointensity is presented as
percentage of the left ventricular volume, excluding the
intra-ventricular cavity, after measuring all short axis
slices. Imaging analyses were always done by a blind
observer (MR technician). Correlation analysis by Pearson
was performed. After MR imaging, the (fixed) heart
specimens were embedded in OCT compound (Miles
Scientific) and snap-frozen in liquid nitrogen and stored at
280 8C until it was analyzed in cryosections under
and20 ms, TR ¼ 325,flip
Super-paramagnetic particles induce a T2/T2* effect that
is detected by MR as regions with low signal intensity and
appearing black relative to adjacent myocardium. Regions
with CD34-positive cells were identified as intramyocardial
areas of low signal intensity on T2*-weighted images at 8.5
A. Weber et al. / European Journal of Cardio-thoracic Surgery 26 (2004) 137–143139
and 3 T. Areas of susceptibility effect were localized within
the anterior and lateral walls of the LV.
Susceptibility effect related to the presence of
magnetic beads was confirmed by imaging the hearts
with different echo times on the 3 T magnet. Intramyo-
cardial areas of low signal intensity demonstrated
increased magnetic susceptibility (blooming effect) with
longer echo time. An increase in the echo time from
10 to 20 ms, resulted in a doubling of the area of low
signal intensity, independently of the cell dosage
injected. Computational planimetric analysis (NIH ima-
ging software) demonstrated a proportional increase of
the intramyocardial areas of low signal intensity on the
MR images with the increase in the number of EPCs
transplanted (cell dosage). Areas of low signal intensity
related to transplanted EPCs increased proportionally to
the cell dosage. MR images of each heart acquired with
similar echo time demonstrated a linear relation between
the number of cells injected and the area of low signal
intensity ðP , 0:0001Þ:
For each heart, transverse cryosections corresponding to
the injection sites were analysed under a fluorescence
microscope. MR images were visually correlated with the
results from histopathological analysis by fluorescence
microscopy. There was an excellent correlation between
the location of the areas of low signal intensity on MR
images and the identification of labeled cells in the injection
sites by fluorescent microscopy (Fig. 2).
There was no difference in depiction of the labeled cells
between ischemic and non-ischemic myocardium, since it
was not possible to enhance the MR signal of ischemic
myocardium within 24 h postoperative ex vivo (data not
Areas engrafted with fluorescent DiI CD34-positive cells
matched closely with the areas of low signal intensity seen
on the MR images. The areas of low signal intensity on the
MR images were slightly larger than the areas with CD34-
positive cells on fluorescence microscopy. This was likely
caused by the blooming effect secondary to an extended-
range susceptibility effect on the magnetic particles (Fig. 3).
Two hearts transplanted with CD34-negative cells
demonstrated no areas of low signal intensity within the
myocardium. Fluorescence microscopy confirmed the pre-
sence of the fluorescence DiI CD34-negative cells in areas
of myocardium that showed no susceptibility effect on MR
(Fig. 4). No areas of susceptibility effect were noted at the
sites of the intramyocardial injection. The lack of suscep-
tibility effect in these two hearts confirmed that the areas of
low signal intensity were related to the magnetic beads
attached to CD34-positive cells.
Several clinical trials are being conducted that utilize
exogenous stem or progenitor cells transplanted into
damaged myocardium to augment myocardial performance
and/or neovascularization after infarction or heart failure.
Recently, Stamm et al. (EACTS 2002) reported results from
ongoing clinical studies for intramyocardial transplantation
of bone marrow progenitor cells isolated using antibodies
coated with magnetic beads. These studies point to a need
Fig. 2. T2-weighted images on 8.5 T Bruker magnet; representative
transversal heart sections only. CD34-positive cells were identified as
anterolateral intramyocardial areas of low signal intensity. (A) (Arrows)
105 CD34-positive cells. (B) (Arrows) 1 £ 106 CD34-positive cells.
(C) (Arrows) 2 £ 106 CD34-positive cells. (D) The area of low signal
intensity is proportional to the cell dosage. (LV) Left ventricle cavity. (RV)
Right ventricle cavity. (Stars) Some areas of low signal intensity, far away
from the injection sites, are usually seen in ex vivo preparations and are
well known as haemosiderine precipitations after blood coagulation.
Fig. 3. T2-weighted image on 8.5 T Bruker magnet and fluorescent
microscopy analysis. Excellent agreement between the areas of MR low
signal intensity (black) (A) and histopathological fluorescence DiI staining
for CD34-positive transplanted cells (red) (B) and (negative; black) (C).
The cell location area in the MR image shows a blooming effect compared
to the corresponding histological section caused by an extended-range
susceptibility effect of the magnetic particles. (LV) Left ventricular cavity.
(For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
A. Weber et al. / European Journal of Cardio-thoracic Surgery 26 (2004) 137–143140
for techniques that permit an evaluation of the efficiency of
transplantation and an evaluation of the potential post-
operative migration of the transplanted cells. Such a
technique would provide an important tool for assessing
patients following treatment. The recent occurrence of
postoperative arrhythmic complications in patients post-
cell-transplant underscores the requirement for careful
evaluation of cell location and fate.
The technique we investigate in this study, could be one
such method for tracking the location of these cells in
treated myocardium. It has the advantage of using magnetic
beads both to isolate EPCs from the mononuclear cell
fraction of peripheral blood (a standard technique) and to
serve as the magnetic label for MRI.
Further studies will be needed to determine whether cell
surface magnetic labeling, previously used only for cell
selection, is stable enough to allow mid- and long-term in
cultured with attached beads and the cells grow and adhere
EPCs were also always isolated by means of magnetic beads
coated with antibody to the CD34 antigen .
In these studies the magnetic beads were never
de-attached from the selected cells before transplantation.
Therefore, based on our previous data we have established
that there is a preserved cell viability and functionality of
CD34-positive cells isolated by means of magnetic beads.
However, some authors [21,22] claim that after approxi-
mately three passages in culture the beads are diluted out.
Others suggest that after in vivo administration there is a
more rapid reticuloendothelial recognition and clearance of
cells thus surface-labeled . Conversely, there is some
evidence that the magnetic coated antibodies might be
internalised to the cytoplasm and remain intracellular.
There are several works reporting the possibility of
intracellular labeling with superparamagnetic ironoxide
nanoparticles or magnetodendrimers [9,11,23] using fluid-
phase or receptor-mediated endocytosis. These methods
allow in vivo cell tracking and the magnetic beads are stably
retained intracellularly over time (up to 6 months has been
reported). Unfortunately, labeling efficiency is generally
low and cells need to be exposed to culture media for long
incubation periods. This methodology would not be
compatible with the transplantation of autologous freshly
isolated cells, a consideration that avoids any contact with
potential immunogenic agents.
In the present study we limited our resources and goals to
realize whether it is possible or not to identify the magnetic
labeled cells by means of MRI just to establish a proof of
principle. The selected cell dosages were established aiming
previous functional studies in a range from low to relatively
high dosage. We first used a small bore 8.5 T magnet, which
is currently used for experimental purposes only, still far
from clinical use, but allowed us to draw on the full
potential of MRI. In a next step we were able to corroborate
these initial results with the 3 T magnets currently in clinical
use. This is especially encouraging taking into account that
we were measuring small rat hearts with a machine that is
used for human adults.
Limitations of this study are the lack of experiments with
big animals and, of course, the lack of measurements in
living animals. These are surely the next steps to follow,
being in vivo MRI of the heart a challenging issue because
of the need for cardiac and respiratory gating in order to
trigger the physiologic heart beats and lung movements.
In vivo imaging of animals would allow the investigator
to follow the migration of transplanted cells in the heart and
would provide considerable data related to the safety of cell
transplantation procedures. Knowledge of the migration
pattern of haematopoietic progenitor cells in vivo after
homing to ischemic areas would be of considerable
importance to understand their physiological impact.
In the present study, we demonstrate that magnetically
labeled EPCs intramyocardially transplanted for therapeutic
MRI at high-field strengths. This can be achieved with an
experimental, small bore 8.5 T magnet, as well as with the
3 T magnets currently in clinical use. We observed an
excellent agreement between the areas demonstrating MR
susceptibility effect and histopathological fluorescence
DiI staining for CD34-positive transplanted cells. This
study introduces MR tracking as a technique to monitor,
Fig. 4. No low signal intensity areas in hearts transplanted with 2 £ 106
CD34-negative cells. (A) Image with 3 T magnet. (B and C) Histopatho-
logical fluorescence DiI staining for 2 £ 106 CD34-negative cells.
(D) Image of sagittal section, 3D T2-weighted, on 8.5 T magnet. (Arrow)
Tube with PBS solution including CD34-positive cells as positive control.
A. Weber et al. / European Journal of Cardio-thoracic Surgery 26 (2004) 137–143141
non-invasively, the localization of magnetically labeled
cells after intramyocardial transplantation.
Hence, this ex vivo experiment constitutes a proof of
principle of the utility of cellular magnetic tags for tracking
cellular transplants currently being evaluated for use in
This study was supported by a grant from the Swiss
National Research Foundation. This work was supported by
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Appendix A. Conference discussion
Dr Hoerstrup (Zurich, Switzerland): My question is concerning the
clinical applicability of this concept. You mentioned that you have
sacrificed the animals after 24 hours, and you said that for future clinical
application you might use intracellular labeling with magnetic particles.
How long will it be possible of tracing down these cells in an in vivo
Dr Weber: Well, there are several studies, mainly at Johns Hopkins,
already tracing these cells in with intracellular labeling; this group even
started clinical studies recently. The imaging of these cells appears to be
much better than the surface tracing, and the in vivo tracking seems to be
possible for long term.
Dr Hoerstrup: What would be a time frame seen from these other
Dr Weber: The last studies point out that the intracellular magnetic
labeling is still available 6 months after implantation. However, these are
neural stem cells which don’t divide as fast as EPCs, so we don’t know, but
in our case the tracking follow up might be a little shorter.
Dr C. Stamm (Rostock, Germany): Am I right to say that you do see the
iron particle, not the cell? And if I am right, can you think of a way to image
a cell to say something about the viability of the cell instead of simply
saying that the iron particle that you injected is there?
Dr Weber: In this case, this experiment constitutes proof of principle of
the utility of cellular magnetic tags for tracking cells in their localization
A. Weber et al. / European Journal of Cardio-thoracic Surgery 26 (2004) 137–143 142
after intramyocardial injection. The next step will be to evaluate the follow- Download full-text
up and see, in the case of migration, if the correlation between cells
and magnetic beads persists. However, in vitro experiments suggested that
the magnetic-coated antibodies might be internalized to the cytoplasm and
remain intracellular, mimicking the ex vivo intracellular labeling
techniques. These methods, as I said before, demonstrated that
the magnetic beads are stably retained intracellularly over time (up to
6 months, as reported by Johns Hopkins).
Dr Stamm: And once you have injected the cells, what you do see is the
iron particle in the myocardium, right, you can’t say this is a living cell
inside the myocardium that you can image?
Dr Weber: In all our previous functional studies (see papers by Drs
Isner and Asahara) the magnetic beads were never detached from the
selected cells before transplantation. Therefore, based on our previous data
we have established that there is preserved in vivo cell viability and
functionality of CD34-positive cells isolated by means of magnetic beads.
A. Weber et al. / European Journal of Cardio-thoracic Surgery 26 (2004) 137–143 143