Research Article

Originally Published 5 July 2005

Free Access

Allogeneic Mesenchymal Stem Cell Transplantation in Postinfarcted Rat

Myocardium: Short- and Long-Term Effects

Wangde Dai, MD, Sharon L. Hale, BS, Bradley J. Martin, PhD, Jin-Qiang Kuang, MS, Joan
S. Dow, BS, Loren E. Wold, PhD, and Robert A. Kloner, MD, PhDAuthor Info & Affiliations

Circulation

Volume 112, Number 2

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Abstract
Background— Mesenchymal stem cells (MSCs) have the potential to replace infarct scar, but
the long-term effects are unknown. We studied short- and long-term effects of MSC
transplantation on left ventricular (LV) function in a rat myocardial infarction model.

Methods and Results— Saline (n=46) or MSCs labeled with 1,1′-dioctadecyl-3,3,3′3′-

testramethylindocarbocyanine perchlorate (DiI; n=49, 2×106 cells each) were injected into
the scar of a 1-week-old myocardial infarction in Fischer rats. The presence and
differentiation of engrafted cells and their effect on LV ejection fraction was assessed. At 4
weeks, LV stroke volume was significantly greater in the MSC-treated group (145±9 μL) than
in the saline group (122±3 μL, P=0.032), and LV ejection fraction was significantly greater in
MSC-treated animals (43.8±1.0%) than in the saline group (38.8±1.1%, P=0.0027). However,
at 6 months, these benefits of MSC treatment were lost. DiI-positive cells were observed in
the MSC group at 2 weeks and at 3 and 6 months. Expression of the muscle-specific markers
α-actinin, myosin heavy chain, phospholamban, and tropomyosin was not observed at 2
weeks in DiI-positive cells. At 3 and 6 months, the DiI-positive cells were observed to express
the above muscle-specific markers, but they did not fully evolve into an adult cardiac
phenotype. Some of the DiI-positive cells expressed von Willebrand factor.

Conclusions— Allogeneic MSCs survive in infarcted myocardium as long as 6 months and

express markers that suggest muscle and endothelium phenotypes. MSCs improved global
LV function at 4 weeks; however, this benefit was transient, which suggests a possible early
paracrine effect.

Cellular cardiomyoplasty may be a promising approach to improve cardiac regeneration or

vascularization after myocardial infarction.1 Our group has reported that transplantation of
fetal or neonatal cardiomyocytes can increase the thickness of the infarct wall and left
ventricular (LV) stroke volume, decrease LV end-systolic volume, and improve LV ejection
fraction in a rat model of myocardial infarction2,3 and induce neoangiogenesis.4 However,
transplanted allogenic cardiomyocytes can cause immunorejection in recipient hearts, and
sources of these immature cardiomyocytes are likely to be limited.5 Therefore, the beneficial
effects of fetal or neonatal cardiomyocyte transplantation in clinical use are limited.

See p 151

In clinical practice, bone marrow cells are easily obtained from bone marrow aspirate drawn
through the skin, and marrow contains multipotential progenitor cells, which can
differentiate into various kinds of cells, including myogenic cells.1 Furthermore,
mesenchymal stem cells (MSCs) from bone marrow cells are thought to be immune-
privileged and have been successfully transplanted into hearts without immunosuppression
(for review, see Pittenger and Martin6). Autologous or allogeneic MSCs are considered to be
one of the potential cell sources for cellular cardiomyoplasty; however, few studies have
investigated the long-term effects of this type of therapy. In the present study, we
investigated the engraftment, survival, and differentiation of allogeneic MSCs obtained from
a commercial source and their short- and long-term effects on LV function in a rat myocardial
infarction model.

Methods

The Heart Institute at Good Samaritan Hospital is accredited by the American Association for
Accreditation of Laboratory Animal Care. All procedures were approved by the Institutional
Animal Care and Use Committee and performed in accordance with the “Guide for the Care
and Use of Laboratory Animals” (NIH publication No. 85-23, National Academy Press,
Washington, DC, revised 1996).

Bone Marrow Harvest, MSC Isolation, Expansion, and Labeling

Allogeneic MSCs were isolated, characterized, cultured, and labeled according to established
methods at Osiris Therapeutics, Inc (Baltimore, Md).6–8 Briefly, 25 male ACI rats (weight 250
to 300 g) were used in this study as MSC donors. The MSCs were isolated from the femoral
and tibial bones of donor ACI rats as described previously.7,8 MSCs were expanded to
passage 3 before infusion to recipient animals. At this time, cultures were 95% homogenous
for rat MSCs. The multipotentiality of the resulting cells was verified with the use of in vitro
assays to differentiate MSCs into osteogenic (alkaline phosphatase activity), adipogenic (oil
red O staining), and chondrogenic (type II collagen staining) lineages.8

To label cells with 1,1′-dioctadecyl-3,3,3′3′-testramethylindo- carbocyanine perchlorate (DiI),

0.25 μg of DiI per microliter of dimethylsulfoxide (DMSO; Sigma) stock solution was added to
MSC suspensions at 1×106 cells/mL in Plasmalyte A (Baxter) to yield a final concentration of
1 μg of DiI solution/mL. This suspension was incubated at 37°C for 5 minutes, then at 4°C for
15 minutes with occasional mixing. MSCs labeled with DiI were washed 3 times with DPBS
before preparation for frozen storage in freezing medium containing 90% fetal bovine serum
(Valley Biomedical)/10% DMSO. All cells were kept frozen until use. MSC viability on thawing
was routinely measured to range from 90% to 95%.

Model of Myocardial Infarction and Injection of MSCs

Myocardial infarction was performed in female Fischer CDF rats as described

previously.2 Briefly, rats were anesthetized with ketamine (75 mg/kg IP) and xylazine (5
mg/kg IP). After endotracheal intubation and initiation of ventilation (room air, rate 60
cycles/min, tidal volume 1 mL per 100 g of body weight, Harvard Apparatus Rodent
Ventilator, model 683), the heart was exposed through a left thoracotomy, and the proximal
left coronary artery was ligated. The rats were allowed to recover under care. One week
later, rats were reanesthetized and hearts reexposed (as described above) for the injection
of MSCs or saline. Saline (≈70 μL, n=46) or MSCs (2×106 cells in ≈70 μL of saline, n=49; Osiris
Therapeutics Inc) were injected directly into the infarct area with a 28-gauge needle
attached to an insulin syringe. Successful injection was typified by the formation of a bleb
covering the infarct zone.
Assessment of Survival and Differentiation of MSCs in Myocardial Infarction

Some rats were euthanized (KCl 2 mEq IV to arrest the heart in diastole) under deep
anesthesia at 2 weeks, 3 months, and 6 months after saline or stem cell injection. Hearts
were harvested, frozen, and sent to Osiris to blindly examine the survival and differentiation
of the cells in the infarction. The survival of the transplanted MSCs was demonstrated by the
presence of DiI-labeled cells. Immunohistochemical staining with antibodies against α-
actinin (Sigma, 1:200), MF-20 (myosin heavy chain; Developmental Studies Hybridoma Bank
at University of Iowa, 1:2), phospholamban (Affinity BioReagents, 1:100), and tropomyosin
(Sigma, 1:100) was performed to show muscle-specific marker expression. Endothelial cells
were identified by the expression of von Willebrand factor (Dako, 1:100). Colocalization of
the DiI label and muscle-specific marker expression were examined with a confocal
microscope (Nikon Eclipse TE 300, Simple PCI image software).

The number of blood vessels positive for von Willebrand factor was assessed in the scar area
at 6 months after MSC or saline treatment. Five fields on the slide representing infarcted
area from each heart were randomly chosen for counting stained blood vessels. The slides
were first examined at ×100 magnification to identify the infarcted area, and then the
number of blood vessels was counted at ×400 magnification. All stained vessels oriented
with the lumen cut transversely were counted. The blood vessel density was expressed as
vessel number/field ×400.

LV Angiography

At 4 weeks and 6 months after treatment, rats were anesthetized and a catheter was
inserted into the left jugular vein. LV contrast angiography was performed after injection of 1
mL of nonionic contrast into the left jugular vein with a XiScan 1000 C-arm x-ray system
(XiTec, Inc; 3-inch field of view). Video images of anterior-posterior and lateral projections
were acquired on half-inch super-VHS videotape at 30 frames per second under constant
fluoroscopy. LV volumes in systole and diastole were calculated blindly from the video
images. All parameters were averaged over 3 consecutive cycles in both projections. Ejection
fraction (%) was calculated as [100×(volume in diastole−volume in systole)/volume in
diastole] and averaged over both projections.

Assessment of Regional Wall Motion by LV Angiography

Regional wall motion after myocardial infarction was also assessed by LV angiography.
Tracings of the LV circumference during end diastole and end systole of the same cardiac
cycle were superimposed on transparent film in both the anterior-posterior and lateral views
with the base of the heart as a reference. If the tracing from end systole was not confined
within that of end diastole, it was defined as bulging. Akinesis was evident when the tracing
from end systole was superimposable on the tracing for end diastole. The size of paradoxical
LV systolic bulging (dyskinesis) or akinetic motion was calculated by measuring the length of
total LV diastolic circumference and circumferential length of the bulging or akinetic segment
with computerized planimetry, expressed as % of total LV diastolic circumference.

Hemodynamics

To record arterial and LV hemodynamic parameters, a 2F high-fidelity, catheter-tipped

micromanometer (model SPR-869, Millar, Inc) was advanced into the ascending aorta and
into the LV through the right carotid artery.

Regional Myocardial Blood Flow

To measure regional myocardial blood flow (RMBF), 103Ru-labeled radioactive microspheres

(≈500 000) were injected directly into the LV, and a reference blood sample was withdrawn
simultaneously from an arterial catheter (0.361 mL/min) for 1 minute. Radioactivity in the
scar tissue, the noninfarcted myocardium, and the reference blood sample was measured in
a multichannel pulse-height analyzer (model ND62, Nuclear Data). After correction for
background, RMBF was calculated as the ratio of counts in the tissue and the reference
blood sample multiplied by pump flow (0.361 mL/min) and divided by the weight of the
tissue.

Postmortem LV Volume, Wall Thickness, and Infarct Size

After intravenous injection of 0.6 mL of 50% Unisperse blue, a suspension of blue particles
obtained from Ciba Geigy was performed for confirmation of a perfusion defect of the scar
area, the rats were euthanized (KCl 2 mEq IV to arrest the heart in diastole) under deep
anesthesia at 4 weeks (n=12 in each group) and 6 months (n=21 in each group). The hearts
were excised and pressure-fixed with formalin (pressure equal to 13 cm H2O column).
Postmortem LV volumes were measured by filling the cavity with water and weighing, which
was repeated 3 times.

The hearts were cut into 3 transverse slices after LV volume measurement. The middle slice
was embedded in paraffin and processed for histology, and the other 2 slices were cut for
RMBF measurement of the infarct and noninfarct areas. Sections (5-μm thickness) of the
paraffin-embedded tissue were stained with hematoxylin and eosin, as well as picrosirius
red. The density of arterioles and small arteries in the scar area (but excluding capillaries)
also was calculated on hematoxylin and eosin–stained slides (expressed as vessel
number/mm2). Computerized planimetry of the histological images of the stained sections
was used to measure and calculate (1) scar thickness (average of 5 equidistant
measurements) and septum thickness (average of 3 equidistant measurements); (2)
epicardial and endocardial circumference and circumference occupied by infarcted wall
(infarct size was expressed as percentage of total LV circumference); (3) expansion index, as
defined by Hochman and Choo, which is expressed as [LV cavity area/total LV area×septum
thickness/scar thickness].

Statistical Analysis
All data are presented as mean±SEM. Comparisons between groups were made by
Student t test or Fisher’s exact test, where appropriate. Results were considered statistically
significant if P<0.05.

Results

Of the 95 successful rats (46 in the saline group and 49 in the MSC group), 29 hearts were
sent to Osiris for assessment of survival and differentiation of MSCs (8 at 2 weeks, 9 at 3
months, and 12 at 6 months). Twenty-four hearts at 4 weeks (12 in each group) and 42
hearts at 6 months (21 in each group) were processed for postmortem analysis, and 2 rats in
the saline group and 1 rat in the MSC group were excluded because of small infarct sizes
(<20% of LV circumference) at 6 months. Successful LV angiographic images were obtained in
24 rats at 4 weeks and 49 rats at 6 months.

Survival and Differentiation of MSCs in Myocardial Infarction

At 2 weeks postimplantation, 3 of 4 hearts in the cell-treated group but none of the 4 hearts
in the saline group showed the presence of grafted MSCs with DiI labeling in the infarct
zone. In the 3 DiI-labeled–positive hearts in the cell-treated group, the muscle-specific
markers α-actinin, myosin heavy chain, phospholamban, and tropomyosin staining were
negative in the DiI-positive cells observed by confocal microscopy. At 3 months, 4 of 5 hearts
in the cell group but none of the 4 hearts in the saline group showed the presence of grafted
DiI-labeled MSCs in the infarct zone. In 3 of 4 hearts with DiI-labeled MSCs, colocalization of
the DiI label and the marker α-actinin was observed. In only 1 heart, DiI-positive cells also
expressed myosin heavy chain, phospholamban, and tropomyosin. At 6 months, 7 of 7
hearts in the cell group but none of the 5 hearts in the saline group contained DiI-labeled
MSCs. DiI-labeled cells in all 7 hearts in the cell group also expressed the muscle-specific
markers α-actinin, myosin heavy chain, phospholamban, and tropomyosin (Figures 1, 2, 3,
and 4). Although DiI-positive cells could be observed in both the center of the scar area and
the border zone, it was easier to identify DiI-positive cells in the center of the scar area. The
percentage of DiI-positive cells that expressed muscle markers was 46±5% for α-actinin,
40±2% for myosin heavy chain, 42±5% for phospholamban, and 38±5% for tropomyosin,
respectively. Although MSCs expressed the cardiac cell markers, differentiation was
incomplete, and myofibril organization was immature (Figure 5). In addition, all of these
hearts at 6 months were found to have MSCs that expressed von Willebrand factor (Figure
6), which indicates a role for MSCs in angiogenesis.
microscopy of fluorescent immunohistochemical staining of DiI-labeled cells in infarcted
myocardium for α-actinin at 6 months. A, DiI-labeled MSCs appear red (white arrows). B,
Sections stained with antibody to muscle marker α-actinin appear green. White arrows point
to MSC transplant. C, Merged image of A and B shows DiI-labeled MSCs express α-actinin
(yellow cells; white arrows). Original magnification ×[Link] in viewer
microscopy of fluorescent immunohistochemical staining of DiI-labeled cells in infarcted
myocardium for MF-20 at 6 months. A, DiI-labeled MSCs appear red (white arrows). B,
Sections stained with antibody to muscle marker MF-20 appear green. White arrows point to
MSC transplant. C, Merged image of A and B shows DiI-labeled MSCs express MF-20 (yellow
cells; white arrows). Original magnification ×[Link] in viewer
microscopy of fluorescent immunohistochemical staining of DiI-labeled cells in infarcted
myocardium for phospholamban at 6 months. A, DiI-labeled MSCs appear red (white
arrows). B, Sections stained with antibody to muscle marker phospholamban appear green.
White arrows point to MSC transplant. C, Merged image of A and B shows DiI-labeled MSCs
express phospholamban (yellow cells; white arrows). Original magnification ×400.