Cardiac Regeneration after mesenchymal stem cell therapy

Multimodality Noninvasive Imaging Demonstrates In Vivo Cardiac Regeneration After Mesenchymal Stem Cell Therapy

Cardiac Regeneration after mesenchymal stem cell therapy OBJECTIVES:

The purpose of this study was to test the hypothesis, with noninvasive multimodality imaging,
that allogeneic mesenchymal stem cells (MSCs) produce and/or stimulate active cardiac
regeneration in vivo after myocardial infarction (MI).

Cardiac Regeneration after mesenchymal stem cell therapy BACKGROUND:

Although intramyocardial injection of allogeneic MSCs improves global cardiac function
after MI, the mechanism(s) underlying this phenomenon are incompletely understood.

Cardiac Regeneration after mesenchymal stem cell therapy METHODS:

We employed magnetic resonance imaging (MRI) and multi-detector computed tomography
(MDCT) imaging in MSC-treated pigs (n 10) and control subjects (n 12) serially for
a 2-month period after anterior MI. A sub-endocardial rim of tissue, demonstrated with
MDCT, was assessed for regional contraction with MRI tagging. Rim thickness was also
measured on gross pathological specimens, to confirm the findings of the MDCT imaging,
and the size of cardiomyocytes was measured in the sub-endocardial rim and the non-infarct
zone.

Cardiac Regeneration after mesenchymal stem cell therapy RESULTS:

Multi-detector computed tomography demonstrated increasing thickness of sub-endocardial
viable myocardium in the infarct zone in MSC-treated animals (1.0 0.2 mm to 2.0 0.3 mm,
1 and 8 weeks after MI, respectively, p 0.028, n 4) and a corresponding reduction in
infarct scar (5.1 0.5 mm to 3.6 0.2 mm, p 0.044). No changes occurred in control
subjects (n 4). Tagging MRI demonstrated time-dependent recovery of active contractility
paralleling new tissue appearance. This rim was composed of morphologically normal
cardiomyocytes, which were smaller in MSC-treated versus control subjects (11.6 0.2 m
vs. 12.6 0.2 m, p 0.05).

Cardiac Regeneration after mesenchymal stem cell therapy CONCLUSIONS:

With serially obtained MRI and MDCT, we demonstrate in vivo reappearance of myocardial
tissue in the MI zone accompanied by time-dependent restoration of contractile function.
These data are consistent with a regenerative process, highlight the value of noninvasive
multimodality imaging to assess the structural and functional basis for myocardial regenerative strategies, and have potential clinical applications. (J Am Coll Cardiol 2006;48:
2116–24) © 2006 by the American College of Cardiology Foundation

Cardiac Regeneration after mesenchymal stem cell therapy – Full Study

Multimodality Noninvasive
Imaging Demonstrates In Vivo Cardiac
Regeneration After Mesenchymal Stem Cell Therapy
Luciano C. Amado, MD,† Karl H. Schuleri, MD,† Anastasios P. Saliaris, MD,† Andrew J. Boyle, MBBS, PHD,† Robert Helm, MD,* Behzad Oskouei, MD,† Marco Centola, MD,†
Virginia Eneboe, VT,† Randell Young, MVD,‡ Joao A. C. Lima, MD, Albert C. Lardo, PHD,*
Alan W. Heldman, MD,† Joshua M. Hare, MD†
Baltimore, Maryland
OBJECTIVES The purpose of this study was to test the hypothesis, with noninvasive multimodality imaging,
that allogeneic mesenchymal stem cells (MSCs) produce and/or stimulate active cardiac
regeneration in vivo after myocardial infarction (MI).
BACKGROUND Although intramyocardial injection of allogeneic MSCs improves global cardiac function
after MI, the mechanism(s) underlying this phenomenon are incompletely understood.
METHODS We employed magnetic resonance imaging (MRI) and multi-detector computed tomography
(MDCT) imaging in MSC-treated pigs (n 10) and control subjects (n 12) serially for
a 2-month period after anterior MI. A sub-endocardial rim of tissue, demonstrated with
MDCT, was assessed for regional contraction with MRI tagging. Rim thickness was also
measured on gross pathological specimens, to confirm the findings of the MDCT imaging,
and the size of cardiomyocytes was measured in the sub-endocardial rim and the non-infarct
zone.
RESULTS Multi-detector computed tomography demonstrated increasing thickness of sub-endocardial
viable myocardium in the infarct zone in MSC-treated animals (1.0 0.2 mm to 2.0 0.3 mm,
1 and 8 weeks after MI, respectively, p 0.028, n 4) and a corresponding reduction in
infarct scar (5.1 0.5 mm to 3.6 0.2 mm, p 0.044). No changes occurred in control
subjects (n 4). Tagging MRI demonstrated time-dependent recovery of active contractility
paralleling new tissue appearance. This rim was composed of morphologically normal
cardiomyocytes, which were smaller in MSC-treated versus control subjects (11.6 0.2 m
vs. 12.6 0.2 m, p 0.05).
CONCLUSIONS With serially obtained MRI and MDCT, we demonstrate in vivo reappearance of myocardial
tissue in the MI zone accompanied by time-dependent restoration of contractile function.
These data are consistent with a regenerative process, highlight the value of noninvasive
multimodality imaging to assess the structural and functional basis for myocardial regenerative
strategies, and have potential clinical applications. (J Am Coll Cardiol 2006;48:
2116–24) © 2006 by the American College of Cardiology Foundation
Ischemic cardiomyopathy due to myocardial infarction (MI)
is the major cause of congestive heart failure and death in
the Western world (1). There is accumulating experimental
support for the application of cellular transplantation as a
strategy to improve myocardial function (2–8) and reduce
scar burden due to MI (2–4,8); however, controversy exists
regarding the underlying mechanism(s) for this effect. In
particular, the question of whether transplanted cells regenerate
new myocardium (either through differentiation or
cell– cell signaling to endogenous precursor cells) to replace
those lost to MI (9,10) remains unanswered. Additionally,
the use of different cell populations and varying clinical
protocols and their application in diverse patient groups has
hampered any effort to compare outcomes using the various
cell types with one another (11).
Critical in assessing the efficacy of any novel regenerative
therapy is the ability to noninvasively image tissues within
the heart at various time points. Moreover, it is essential to
determine whether regenerated tissue is functional (i.e.,
does the new tissue contract with the rest of the heart or is
it non-contractile and mechanically uncoupled from the rest
of the heart), in which case it might actually be detrimental
to cardiac function.
Implantation of cells such as bone marrow-derived mesenchymal
stem cells (MSC) restores the function of the
damaged heart and decrease necrotic tissue (8,12). Whether
they do so by generating new, electromechanically coupled
From the Department of Medicine, Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland; †Institute for Cell Engineering (ICE), Johns Hopkins University School of Medicine, Baltimore, Maryland; and ‡OsirisTherapeutics, Baltimore, Maryland. This work was supported by the Johns Hopkins University School of Medicine Institute for Cell Engineering (ICE); National Institutes of Health grants U54 HL081028 (Specialized Center for Cell-Based Therapy), R21 HL-72185 and RO1 NIA AG025017; and the Donald W. Reynolds foundation. Dr. Hare has received research grant funding from Osiris Therapeutics. Manuscript received February 27, 2006; revised manuscript received June 6, 2006, accepted June 29, 2006. Journal of the American College of Cardiology Vol. 48, No. 10, 2006 © 2006 by the American College of Cardiology Foundation ISSN 0735-1097/06/$32.00 Published by Elsevier Inc. doi:10.1016/j.jacc.2006.06.073 myocardium (13,14) or by altering the composition of the evolving scar, thereby preventing remodeling (15,16), remains controversial. Some groups advocate that implanted cells are not contractile but rather that new viable tissue serves to decrease myocardial wall tension at the infarcted region by strengthening the infarct scar and preventing ventricular dilatation and thus improving diastolic function and enhancing global cardiac function (16,17). Others suggest there is actual regeneration of functional myocardium that replaces the area occupied by scar (3). Whether this occurs by (trans)differentiation of the implanted cells into new cardiomyocytes, whether there is fusion of the donor cells with the surviving cells inducing cellular turnover, or whether the injected cells stimulate endogenous myocytes or myocyte precursors to divide via a paracrine or cell– cell signaling action is incompletely understood. To address this important issue we used multimodality imaging, with both magnetic resonance imaging (MRI) and contrast-enhanced multi-detector computerized tomography (MDCT) to serially monitor the regional and global structure and function of the porcine heart after MI in response to allogeneic MSC therapy. We have previously demonstrated that MSC therapy leads to the development of a sub-endocardial rim of muscle and improvement in overall cardiac function (12); however, whether this rim of myocardium is functionally coupled to the rest of the heart remains unknown. We therefore tested the hypothesis that MSC cellular cardiomyoplasty stimulates the regeneration of viable, contractile myocardium adjacent to evolving MI scar thereby enhancing regional systolic function. METHODS Experimental protocol. All animal studies were approved by the Johns Hopkins University School of Medicine Institutional Animal Care and Use Committee and comply with the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health [NIH] Publication no. 80-23, revised 1985). Twenty-three female Yorkshire pigs (25 to 30 kg) underwent left anterior descending coronary artery occlusion followed by reperfusion and then were randomized to receive MSCs (n 10) or not receive MSCs (n 12, non-treated group) 3 days later. Mortality was not different between groups. In the control group, 2 animals died within 72 hours of MI and 3 animals died during the follow-up period, resulting in 7 animals being available for the imaging and histology protocols. In the MSC-treated group, 1 animal died after cell injection and 2 animals died during the follow-up period, resulting in 7 animals that were available for imaging and pathology examinations. Cine and tagging MRI assessment was performed at 4 time points after injection: 2 days and 1, 4, and 8 weeks. Also, MDCT was performed at 1 week and 8 weeks after MI. Detailed protocols are in the following sections. Partial data—ejection fraction and infarct size obtained from MRI, and histologically measured rim thickness—from a subset (n 6 MSC and n 6 control subjects) of these animals have previously been reported (12). MI creation and intramyocardial MSC injection. The protocol used here has been previously described (12). In brief, intravascular sheaths were placed in the right carotid artery (8-F), and a coronary angioplasty balloon (2.5 20 mm) was advanced into the proximal left anterior descending coronary artery. Myocardial infarction was induced by inflating the angioplasty balloon for 60 min, followed by artery reperfusion. After reperfusion, the catheter sheath in the carotid artery was removed, and the carotid artery was permanently closed. All animals were adequately heparinized during the surgical procedure. Three days after MI, animals randomized for MSC treatment received intramyocardial injections of allogeneic porcine MSCs (2.0 108 cells, Osiris Therapeutics, Baltimore, Maryland) under fluoroscopy, with a pistol-needle tip injection catheter advanced to the LV through a steerable guide catheter (Stiletto, Boston Scientific, Natick, Massachusetts). Hypokinetic, akinetic, and dyskinetic areas were identified during contrast ventriculography, and MSC injections were performed within and at the borders of the dysfunctional area, as defined by bi-plane ventriculography. A total of 15 injections were performed in each animal, each injection containing 1 cc, thereby delivering a total of 200 million cells/animal. Each injection was fluoroscopically guided to distribute cells evenly throughout the entire infarct and border zones. MRI protocol. The MRI images were acquired with a 1.5-T MR scanner (CV/i, GE Medical Systems, Waukesha, Wisconsin) at 4 time points after injection: 2 days and 1, 4, and 8 weeks. Global LV function was assessed with a steadystate free precession pulse sequence (18). Eight to 10 contiguous short-axis slices were prescribed to cover the entire heart from base to apex. Image parameters were the following: repetition time (TR) 4.2 ms and echo time (TE) 1.9 ms; flip angle 45°; 256 160 matrix; 8-mm slice thickness/no gap; 125 kHz; 28-cm field of view (FOV); and 1 number of signal average (NSA). To assess regional cardiac function, tagging MRI images were acquired with an electrocardiography-gated, segmented K-space, fast gradient recalled echo pulse sequence with spatial modulation of magnetization to generate a grid tag pattern (19,20). Images were obtained at the same Abbreviations and Acronyms Ecc systolic circumferential strain FOV field of view LV left ventricle/ventricular MDCT multi-detector computed tomography MI myocardial infarction MMP matrix metalloproteinase MRI magnetic resonance imaging MSC mesenchymal stem cell TIMP tissue inhibitor of matrix metalloproteinase JACC Vol. 48, No. 10, 2006 Amado et al. 2117 November 21, 2006:2116–24 Multimodality Imaging of MSC Therapy After MI location as the cine-MRI images, and image parameters were as follows: TR 6.7 ms and TE 3.2 ms; flip angle 12°; 256 160 matrix; views/s: 4; 8-mm slice thickness/no gap; 31.25 kHz; 28-cm FOV; 1 NSA; and 6 pixels tagging space. MRI analysis. Cine-MRI images were analyzed with a custom research software package (Cine Tool, GE Medical Systems). Endocardial and epicardial contours were done manually at end-diastolic and end-systolic phases of the cardiac cycle, and global cardiac function was assessed on the basis of Simpson’s rule method. The high resolution of current generation MRI scanners allows differential assessment of contractile function at various levels within the myocardium at any given circumferential point. We assessed contraction in 24 circumferential areas of the LV, and in each of theses areas we assessed contractile function from 3 different layers of the myocardial wall: endocardial, mid-myocardial, and epicardial layers. Tagged images were quantitatively analyzed with a custom software package (Diagnosoft HARP, Diagnosoft Inc., Palo Alto, California) (21). Regional strain magnitude was determined from the 24 radially displaced segments for each short-axis section covering the entire LV and averaged among slices for each region and each time point, as previously described (22), generating a strain map for each point over the cardiac cycle. The peak systolic circumferential strain (Ecc) was determined from the strain map for each point. This approach is most similar to many commonly used indexes based on tissue Doppler functional imaging. Negative Ecc values represent myocardial contraction, whereas values of increasing strain (toward positive values) reflect worsening contractile function in that region. Less negative Ecc values represent hypokinetic myocardium. A value of 0 represents akinetic non-contractile myocardium, and a positive value represents dyskinetic myocardial segments. Peak systolic circumferential strain (peak Ecc) was calculated for each layer as previous described (21). Also, peak Ecc was separated from infarcted and non-infarcted (remote) areas for each animal. MDCT imaging protocol. The MDCT images were obtained in the first week after MI (baseline) and 8 weeks after MSC injection in a subset of animals (n 4 in each group) with a 32-detector scanner (Aquilion 32 Toshiba Medical Systems Corporation, Otawara, Japan). The specific protocol used has been previously described and validated by Lardo et al. (23). In brief, a stack of axial slices (0.5-mm slice thickness) was prescribed to cover the entire heart. Images were acquired 5 min after contrast injection (iodixanol 150 ml, 5 cc/s), the ideal time for infarct visualization. Imaging acquisition parameters were as follows: gantry rotation time 400 ms; detector collimation 0.5 mm 32; pitch 7.2; tube voltage 135 kV; tube current 420 mAs; and scanning FOV 13.2 mm. MDCT imaging analysis. Axial images were reconstructed at 75% of the cardiac cycle and at 2-mm slice thickness. Image analysis was performed with a custom research software package (Cine Tool, GE Medical Systems), and areas of necrosis were identified as hyperenhanced areas. The multi-detector slice CT technique was chosen owing to its high spatial resolution and ability to avoid the partial volume effect, allowing for better characterization of scar region (24). In the infarct zone, thickness measurements of the entire infarct zone, the scar, and the endocardial rim were made at 4 points in each slice and 4 slices for each animal, and the average was taken. Stem cell harvest and isolation. Male swine MSCs (Osiris Therapeutics) were obtained, isolated, and expanded as previously described (12,25). All used cells were harvested when they reached 80% to 90% confluence at passage 3, and then cells were placed in cryo bags at a concentration of 10 to 15 million MSCs/ml and frozen in a control rate freezer to 180°C until the day of implantation. Trypan blue staining was performed to attest viability of thawed MSC lots before injection. Only MSC lots containing 85% or more of viable cells were used in the study. Postmortem analysis. Quantification of viable myocardium in the subendocardial border of the MI was confirmed by gross pathology. For this purpose, at the end of the 8-week period, animals were humanely killed and hearts were excised and sectioned into 8-mm-thick short-axis slices. Each slice was digitally photographed. For each slice, non-viable infarcted areas and viable subendocardial border thickness were manually measured with a custom research software package (Image J 1.34s; NIH, Bethesda, Maryland), and an average of 6 measurements were taken for each specimen that was used. The amount of viable subendocardial tissue is expressed in absolute terms and as a ratio of viable tissue (mm width) normalized by infarct transmurality (mm width) of each slice. The subendocardial rim thickness data from a subset of these animals have previously been reported (12). The LV circumference was measured at the endocardium and epicardium, as was the circumference of the infarct zone, and the infarct zone was expressed as the percentage of the LV circumference average of the endocardial and epicardial measurements. After photographs were taken, myocardial tissue samples were obtained from 2 regions: infarcted and remote (normal) areas. Hematoxylin and eosin (H&E) stain was performed, and myocyte length was measured from the remote and infarcted areas (viable tissue at subendocardial border), with a custom research software package (Scion Image Analysis program; Beta version 4.0.2; Scion Corporation, Frederick, Maryland). One hundred myocytes were measured at each site from 2 control and 2 MSC animals with similar initial infarct size and morphology. Statistical analysis. All values are expressed as mean SEM. Indexes of cardiac function as well as amount of viable tissue at day 2 and week 8 were compared between groups with non-paired independent samples Student t test. The change in sub-endocardial rim thickness and scar thickness within groups from baseline to 8 weeks were 2118 Amado et al. JACC Vol. 48, No. 10, 2006 Multimodality Imaging of MSC Therapy After MI November 21, 2006:2116–24 analyzed with a paired Student t test. Differences in ejection fraction and infarct size during the 8-week period were compared within the groups with repeated measurements analysis of variance (ANOVA) and between groups with 2-way ANOVA with an interaction term. Simple linear regression analysis was performed to compare peak Ecc between groups at different time points, and the Huber/ White Sandwich estimator of variance was used to take correlation between the animals into account. A level of p 0.05 was considered statistically significant. RESULTS Serial imaging demonstrates reappearance of myocardial tissue. There were no baseline differences between groups (Table 1). We examined the tissue characteristics of the infarcted regions in vivo with high definition MDCT imaging at several time points and compared this with histological analysis. The CT images offered important details regarding changing scar morphology and tissue characteristics due to MSC treatment. The MDCT images in the first week after MI (Fig. 1) demonstrated the presence of a rim of tissue at the endocardial layer of the MI scar, which had a tissue density similar to remote myocardium (130.9 31.6 Hounsfield units). This rim increased in thickness during an 8-week interval, consistent with the development of potentially viable myocardial tissue. Additionally, there was a distinct difference in tissue density between viable myocardium and the infarct scar (235.948.6 Hounsfield units) and this viable sub-endocardial tissue. Image-based quantification of this sub-endocardial region over 8 weeks revealed no change in the overall thickness of the infarct zone but an increase in thickness of the viable sub-endocardial tissue in the MSC-treated group, from 1.02 0.16 mm to 2.02 0.28 mm (p 0.028, n 4). There was a corresponding reduction in the thickness of the scar in this region from 5.1 0.5 mm to 3.6 0.2 mm (p 0.044, n 4) (Fig. 2). However, in the untreated group, the total infarct zone thickness, the scar thickness, and the rim thickness remained unchanged from week 1 to week 8 (p NS, n 4). The generation of a sub-endocardial rim of myocardium was confirmed by pathologic analysis. The MSC-treated animals had a 2.9 0.4-mm sub-endocardial rim of tissue, representing 35 3% of the wall thickness in the infarct zone (n 6). Untreated animals (n 7), however, had a thinner sub-endocardial rim, 1.4 0.1 mm (p 0.02 vs. MSC group), accounting for 21 2% of the wall thickness (p 0.004 vs. MSC-treated) (Fig. 3). Additionally, volumetric analysis of the infarct scar in 2 animals from each group demonstrated that MSC injection significantly reduced the volume of myocardial infarct scar tissue over the course of the study period (49.5 9.9% decrease in infarct size at 8 weeks after MI; p 0.05 vs. baseline), whereas no change in infarct size was observed in non-treated animals (p 0.05 vs. MSC-treated animals). The circumferential extent of the infarcted zone was not different between groups (22 3% vs. 24 4% of the LV circumference in MSC-treated and untreated animals, respectively, p 0.56). Thus, regeneration of the infarct zone Table 1. Baseline Characteristics Control MSC-Treated p Value MI size (% of LV volume) 18.3 3.4 20.2 2.3 NS
LVED mass* 48.2 2.9 56.7 4.7 NS
LVEF (%)* 29.8 1.9 29.1 2.6 NS
MI zone thickness (mm)† 6.2 0.3 6.3 0.6 NS
Baseline characteristics were not different between groups. *Measured by cine
magnetic resonance imaging. †Measured by Multi-slice detector computed tomography.
LVED left ventricular end-diastolic; LVEF left ventricular ejection fraction;
MI myocardial infarction; MSC mesenchymal stem cell.
Figure 1. Multi-detector computed tomography scan of a pig 1 week after myocardial infarction. The different tissue densities of the infarct scar and the
sub-endocardial rim of viable tissue are clearly apparent. The sub-endocardial rim has a density similar to the myocardium outside the infarct zone. The
relative densities in Hounsfield units (HU) are also represented. MSC mesenchymal stem cell.
JACC Vol. 48, No. 10, 2006 Amado et al. 2119
November 21, 2006:2116–24 Multimodality Imaging of MSC Therapy After MI
was not due to regeneration from the border zone inward
into the scar, but due to regeneration of the sub-endocardial
rim. Histological analysis confirmed that this rim at the
endocardial region of the infarct zone contained properly
aligned cardiac myocytes (Fig. 4A). These myocytes were
smaller than normal, with myocyte width of 11.6 0.2 m
vs. 12.6 0.2 m (p 0.05) (Fig. 4B) for cardiomyocytes
in the region remote from the infarct. This finding excludes
myocyte hypertrophy as a cause for the increase in subendocardial
rim thickness and is consistent with myocardial
regeneration in this region (3,4,8,12).
Regenerated myocardium has coordinated contraction.
Next we examined whether the reduction in infarct size and
its replacement with new myocardium resulted in recovery
of contractile function in this region. For this purpose, we
used MRI tagging to calculate peak Ecc. Representative
tagged imaging with color mapping to demonstrate normal
and abnormal contraction is shown in Figure 5.
As depicted in Figure 6, both groups of animals had
hypokinetic wall motion in the endocardial layers of the
infarct zone at baseline, 5.5 0.3% and 5.5 0.5%,
respectively, for MSC-treated and non-treated groups (p
NS). However, whereas this area remained hypokinetic in
control animals for the 8-week duration of the study, the
subendocardial area of MSC-treated pigs where regeneration
of myocardium occurred began to increase peak negative
strain by 4 weeks, followed by near-full peak Ecc
recovery, comparable to that of remote or non-infarcted
myocardium, by 8 weeks after injection (peak Ecc at week 8:
11.8 0.9 vs. 5.1 0.5 in MSC-treated vs. control
subjects, p 0.05).
The epicardial layer of myocardium exhibited a somewhat
different course over the 8-week study period. As shown in
Figure 6, a depressed regional function was observed in both
groups with peak Ecc 4.2 0.3% and 5.5 0.5% for
MSC-treated and control groups, respectively (p NS).
However, improvement in cardiac function was observed in
both groups by the end of the 8-week study period (9.4
0.3 and 6.8 0.9 in MSC-treated and control groups,
respectively; p 0.05 vs. baseline for both and p NS
between groups). This improvement in regional function is
expected, because the region most likely contains viable
myocardium, as confirmed previously by histological analysis
(26).
To complete the analysis of regional function of the
whole wall, we examined the temporal course of peak Ecc in
the mid-wall region. Interestingly, despite the improvement
observed at the epicardial layer in the non-treated animals,
no improvement or movement gathering occurred in the
mid-wall layer. Initial peak Ecc was depressed at 4.1
0.4% at baseline and did not improve in the course of the
8 weeks in the non-treated animals, with a peak Ecc of
4.7 0.6% (p NS vs. baseline). However, MSCtreated
animals demonstrated an important improvement in
regional peak Ecc shortening at the mid-wall region, from
4.6 0.3% to 10.8 0.6%, leading to near normal
levels at 8 weeks after injection (p 0.05 vs. non-treated).
These data are consistent with restoration of contractile
function in the endocardial and mid-wall areas of the
infarcted myocardium in animals treated with MSC injection,
not evident in untreated animals. It is important to
note that the entire infarct zone of both groups remained
hypokinetic at the time of the week-1 MRI, demonstrating
that the development of contractility in the MSC-treated
group developed over several weeks. The timescale of
improvement coupled with the CT findings of scar replacement
with myocardium in this area strongly suggests that
regeneration of functional myocardium rather than just
Figure 2. Multi-detector computed tomography scan of the same mesenchymal stem cell (MSC)-treated animal at 5 days (A), 4 weeks (B), and 8 weeks
(C) after MSC injection. There is a time-dependent reduction in the thickness of the infarct scar and a corresponding increase in the thickness of the
sub-endocardial rim of tissue over the 8-week follow-up.
2120 Amado et al. JACC Vol. 48, No. 10, 2006
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recovery of stunned myocardium is the mechanism of
recovery.
Regenerated myocardium improves global cardiac function.
Enhancement of regional cardiac function in the
infarct zone leads to improvement in global cardiac function
(Fig. 7). As previously reported, the LV ejection fraction
improved in a time-dependent manner in the MSC-treated
group but not in the control group (12). Importantly, this
improvement followed a time course similar to that of the
observed myocardial regeneration and the improvement in
regional contraction.
DISCUSSION
Here we show that multimodality imaging with MRI and
MDCT allows precise characterization of tissue morphology
and regional and sub-regional cardiac function
that demonstrates the structural and functional response
to cell-based therapy in a large animal model. Together,
our results demonstrate that transplantation of allogeneic
MSCs in pigs 3 days after MI stimulates cardiac repair by
leading to the reappearance and/or growth of new tissue
along the sub-endocardial rim of the MI that partially
Figure 3. (A) Multi-detector computed tomography scan of representative myocardial infarcts at weeks 1 and 8 after stem cell injection. As indicated by
the arrows, both infarct zones are characterized by the presence of a rim of myocardial tissue on their endocardial surface. However, the thickness of this
rim progressively increases in mesenchymal stem cell (MSC)-treated animals and remains unchanged in control subjects. (B) Representative pathology
specimens from control and MSC-treated animals confirm increased sub-endocardial rim thickness in the treated animals.
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replaces the infarct scar. This tissue is contractile and
mechanically coupled with the existing myocardium,
demonstrating that cellular cardiomyoplasty with MSCs
likely stimulates regeneration of myocytes as the basis for
the improvement in regional and global function and the
reduction in infarct size. Importantly, these insights were
gleaned from imaging modalities that did not require
labeling of injected cells.
Figure 6. Plot of peak circumferential strain (peak Ecc) over time comparing
mesenchymal stem cell (MSC)-treated and non-treated animals for
3 myocardial layers (endocardial, epicardial, mid-wall layers). Peak negative
Ecc values represent myocardial shortening, whereas increasingly positive
values reflect relative myocardial dysfunction. As can be appreciated, peak
negative Ecc values at the endocardial and mid-wall layers for MSCtreated
infarct regions decrease (i.e., improve) over the 8-week follow-up
period compared with the control group, which remained dysfunctional.
†p 0.05 difference within the group; ‡p 0.05 difference between
groups; n 6 in each group. MI myocardial infarction.
Figure 4. Hematoxylin and eosin (H&E) stain of tissue obtained from the
subendocardial rim (A) and the remote zone (B) of mesenchymal stem cell
(MSC)-treated animals. As shown, the rim of tissue is made up of cardiomyocytes
with normal ultrastructural architecture. (C) There is no difference in
the size of myocytes taken from the remote myocardium of both control and
MSC hearts. However, the myocytes from the infarct rim are significantly
smaller in MSC-treated animals versus control animals (n 2 each, 100
cells/heart), suggesting that the enhanced rim size in MSC animals might be
due to new cardiomyocyte regeneration. *p 0.001 MSC rim versus control rim.
Figure 5. Color map depicting non-contractile and dysfunctional areas of myocardium detected by tagging magnetic resonance imaging. Bar color
demonstrates the spectrum in change of systolic cardiac function: blue and green colors identify normal contractile areas and red colors represent
dysfunctional areas. Non-treated animals exhibited worsening regional systolic function during the 8-week period, whereas a visual improvement was
noticed in the mesenchymal stem cell (MSC)-treated animals.
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There is growing evidence that cellular cardiomyoplasty is
feasible and capable of restoring global cardiac function
(2–6,8,12,27). Studies using MSCs (8,12) and MSC-like
(28) cells have demonstrated a beneficial effect in post-MI
hearts, and these reports together have encouraged the
application of this novel therapy in phase I clinical trials.
However, there is ongoing controversy regarding the mechanistic
underpinnings by which stem cell therapy improves
cardiac function (11). There is strong evidence that MSCs
participate in the regulation of collagen formation and that,
in so doing, alter the passive characteristics of the scar,
rendering it less susceptible to stretch and contributing to
overall improvement in global chamber function. In this
regard, Xu et al. (29) have reported that MSCs in a rat
model of MI positively influence the composition of extracellular
matrix at the scar, altering the expression of matrix
metalloproteinase (MMP-1) and tissue inhibitor of matrix
metalloproteinases (TIMPS), among other components.
Others have demonstrated improved ventricular compliance
after MSC therapy and suggested that this, in concert with
reduced scar formation, might contribute to the improvements
in LV function (17). Although the current study does
not refute this mechanism, it strongly supports the notion
that cardiac tissue reappears in an endomyocardial rim
corresponding to the location of cell administration. The
demonstration of a reduction in scar volume in concert with
the increase in myocardium in the infarct zone supports the
idea that effects on MMPs and TIMPs and myocyte
regeneration are both operative mechanisms.
Mechanistic basis for tissue regeneration. Evidence for
MSC differentiation into myocytes remains controversial.
Whereas in vitro observations support differentiation (30),
in vivo studies demonstrate incomplete differentiation with
MSCs harboring myocyte-specific proteins but lacking full
cardiomyocyte phenotypes (12,31). Mesenchymal stem cells
are reported to produce cardiac connexin 43 (14) and could in
theory electromechanically couple to host myocardium (32).
The demonstration of smaller but seemingly mature
cardiomyocytes in the sub-endocardial rim of the infarct
zone confirms that the increase in tissue seen in this region
is not due to hypertrophy of the cardiomyocytes but rather
suggests growth of new tissue. It has previously been shown
that the cardiomyocytes in this region are ki67 positive, also
suggesting growth of new tissue (12). However, whether
this occurs due to MSC differentiation into cardiomyocytes,
to proliferation of endogenous cardiomyocytes or their
precursors, or to other currently unappreciated mechanisms
remains to be determined. Studies aimed at elucidating the
relative contributions of these mechanisms are currently
ongoing.
There are other postulated potential mechanisms of
action. First, MSCs likely stimulate homing of endogenous
precursor cells (33,34). Systemically injected MSCs home to
the site of tissue damage after MI (35,36), demonstrating
their participation in cellular trafficking. Additionally,
MSCs themselves secrete stromal derived factor 1 (SDF-1),
a stem cell chemoattractant cytokine (37) and might therefore
promote trafficking and/or differentiation of other stem
cells, such as endothelial progenitor cells (EPCs) or endogenous
cardiac stem cells. It is attractive to speculate that
MSCs might act as an amplifier to enhance the function of
endogenous cardiac stem cells in effecting myocardial regeneration.
Indeed, it has been shown that exogenous
signals introduced after MI can cause proliferation and
differentiation of endogenous cardiac stem cells, resulting in
myocardial regeneration and improved LV function (38).
Mesenchymal stem cell transplantation might act in a
paracrine or cell– cell signaling fashion to effect cardiac
repair via cytokine-induced enhancement of endogenous
cardiac stem cell function.
Another proposed mechanisms for new tissue regeneration
is cell fusion (39) of the implanted MSCs with host
cells. However, fusion seems increasingly unlikely given the
accumulation of abundant data revealing diploidy of regenerated
cells (40–42). Our study has not specifically addressed
the issue of how myocyte regeneration occurs, but
we have confirmed that it does occur, that it happens in a
time-dependent manner, that it develops along the subendocardial
surface of the scar, and that it results in functional
contractile improvement. Further studies are underway to
more fully elucidate the mechanism by which MSC therapy
induces myocardial regeneration.
In summary, we have used multimodality state-of-the-art
imaging to serially follow the response of a large-animal
model of MI to MSC cell therapy and demonstrate the
presence of myocardial regeneration over several time
points. In addition, we demonstrate that this new tissue
enhances regional systolic function, which in turn leads to
an improvement in global chamber performance. These data
demonstrate the efficacy of intramyocardial MSC injection
after MI and highlights the value of noninvasive multimodality
imaging not only in elucidating mechanisms underlying
new tissue regenerative therapies but also in serially
assessing these tissue changes and their functional consequences.
These results have implications for the design of
clinical trials of stem cell therapy.
Figure 7. Impact of mesenchymal stem cell (MSC) therapy on global
ventricular function. Ejection fraction assessed by cine magnetic resonance
imaging (MRI). Ejection fraction is markedly reduced in both groups at
baseline and remains depressed in non-treated animals. However, a
progressive improvement in the course of 8 weeks is observed in MSCtreated
animals. *p 0.05 versus day 2 after injection (time 0); †p 0.05
versus non-treated (n 6).
JACC Vol. 48, No. 10, 2006 Amado et al. 2123
November 21, 2006:2116–24 Multimodality Imaging of MSC Therapy After MI
Reprint requests and correspondence: Dr. Joshua M. Hare,
Johns Hopkins Medical Institutions and Institute for Cell Engineering
(ICE), Division of Cardiology, BRB 651, 733 North
Broadway, Baltimore, Maryland, 21205. E-mail: jhare@med.
miami.edu or jhare@mail.jhmi.edu.
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