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Am J Physiol Heart Circ Physiol 292: H1095-H1104, 2007. First published October 20, 2006; doi:10.1152/ajpheart.01009.2005
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Pretreatment of adult bone marrow mesenchymal stem cells with cardiomyogenic growth factors and repair of the chronically infarcted myocardium

Jozef Bartunek,1 Jeffrey D. Croissant,2 William Wijns,1 Stephanie Gofflot,3 Aurore de Lavareille,4 Marc Vanderheyden,1 Yulia Kaluzhny,2 Naïma Mazouz,4 Philippe Willemsen,4 Martin Penicka,1 Myrielle Mathieu,5 Christian Homsy,4 Bernard De Bruyne,1 Kathleen McEntee,5 Ike W. Lee,4 and Guy R. Heyndrickx1,6

1Cardiovascular Center and Cardiovascular Research Center, Aalst, Belgium; 2Anterogen Research Laboratories, Boston, Massachusetts; 3Department of Pathology, Faculty of Medicine, University of Liège, Liège; 4Cardio3 NV, Braine l'Alleud; 5Department of Physiology, Faculty of Medicine, Free University of Brussels; and 6Department of Physiology, Louvain Medical School, Brussels, Belgium

Submitted 22 September 2005 ; accepted in final form 16 October 2006


   ABSTRACT
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 ABSTRACT
METHODS
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The in vivo cardiac differentiation and functional effects of unmodified adult bone marrow mesenchymal stem cells (MSCs) after myocardial infarction (MI) is controversial. We postulated that ex vivo pretreatment of autologous MSCs using cardiomyogenic growth factors will lead to cardiomyogenic specification and will result in superior biological and functional effects on cardiac regeneration of chronically infarcted myocardium. We used a chronic dog MI model generated by ligation of the coronary artery (n = 30). Autologous dog bone marrow MSCs were isolated, culture expanded, and specified into a cardiac lineage by adding growth factors, including basic FGF, IGF-1, and bone morphogenetic protein-2. Dogs underwent cell injection >8 wk after the infarction and were randomized into two groups. Group A dogs (n = 20) received MSCs specified with growth factors (147 ± 96 x 106), and group B (n = 10) received unmodified MSCs (168 ± 24 x 106). After the growth factor treatment, MSCs stained positive for the early muscle and cardiac markers desmin, antimyocyte enhancer factor-2, and Nkx2–5. In group A dogs, prespecified MSCs colocalized with troponin I and cardiac myosin. At 12 wk, group A dogs showed a significantly larger increase in regional wall thickening of the infarcted territory (from 22 ± 8 to 32 ± 6% in group A; P < 0.05 vs. baseline and group B, and from 19 ± 7 to 21 ± 7% in group B, respectively) and a decrease in the wall motion score index (from 1.60 ± 0.05 to 1.35 ± 0.03 in group A; P < 0.05 vs. baseline and group B, and from 1.58 ± 0.07 vs. 1.56 ± 0.08 in group B, respectively). The biological ex vivo cardiomyogenic specification of adult MSCs before their transplantation is feasible and appears to improve their in vivo cardiac differentiation as well as the functional recovery in a dog model of the chronically infarcted myocardium.

cardiac repair; myogenesis; chronic myocardial infarction; heart failure

SEVERAL CELL TYPES HAVE BEEN utilized in the regeneration of damaged heart tissue (2, 4, 9, 11, 13, 16–18, 23–25). After transplantation to the damaged heart, skeletal myoblasts differentiated into striated muscle cells in vivo and improved myocardial function (11). Hematopoietic stem cells were shown to induce neoangiogenesis associated with improved cardiac function in an experimental myocardial infarction (9, 18). However, increasing controversy exists regarding the ability of bone marrow-derived cells, including bone marrow mesenchymal stem cells (MSCs), to transdifferentiate into cardiac cells (4, 13, 15) with the risk for unwanted differentiation toward fibroblasts (31) or calcifications (32) after injections into the infarcted myocardium. Consequently, the search for novel strategies of coaxing the cells toward a cardiac lineage has been recommended (6, 16). Previous studies have used a DNA demethylating chemical compound, 5'-azacytidine, that induces uncontrolled myogenic specification by random demethylation (29, 30). Note that, during embryonic development, signals emanating from adjacent endoderm and ectoderm, such as bone morphogenetic protein (BMP)-2/4, FGF, and IGF, as well as direct cell-cell interactions through specific extracellular matrix (ECM) are required for the specification and stabilization of the mesoderm to form a cardiac field (1, 3, 7, 26). Likewise, the majority of experimental studies use small rodent models of myocardial infarction, and data in large animal models closer to the clinical situation are scarce. Therefore, we tested the primary hypothesis that growth factors involved in early cardiomyogenesis may drive adult MSCs toward the cardiomyogenic cell lineage in vitro. Second, using a dog model of chronic myocardial infarction, we explored the possibility that direct myocardial injection of growth factor-treated cells is associated with beneficial effects on biological and functional regeneration of chronically infarcted myocardium.


   METHODS
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Animals and bone marrow collection. A total of 30 dogs (15–45 kg) were used (Harlan). The study was approved by the Ethical Committee for the Animal Research of the Louvain Medical School, Brussels, and the Faculty of Medicine, Free University of Brussels, Belgium. The myocardial infarction was caused by ligation of the coronary artery (left circumflex, n = 25; left anterior descending artery n = 5). Care was taken to ligate all visible epicardial collaterals. Bone marrow (15–20 ml) was collected from the anterior iliac crest into a syringe flushed with heparin. The sample was treated with a mix (1:1) of DMSO and hydroxyethyl starch solution (6%/6% final concentration) and frozen at –80°C until further processing.

Isolation and treatment of human and dog MSCs. After being thawed, marrow was plated onto T75 collagen-treated culture flasks in standard growth media to remove nonadherent cells. The plastic-attached MSCs were culture expanded for 3 wk with media containing DMEM (Invitrogen), 20% fetal bovine serum (Hyclocene), 100 µM L-ascorbic acid (Sigma Aldrich), 5 mg/ml human leukemia inhibitory factor (LIF, Sigma Aldrich), and 20 nM dexamethasone (Sigma Aldrich). After expansion, in the differentiation medium, LIF was removed, FBS was reduced to 2%, and cardiac differentiation was induced by adding the following cardiomyogenic growth factors for 6 days: 50 ng/ml basic FGF (R&D systems), 2.5–25 ng/ml BMP-2 (R&D systems), and 2 ng/ml insulin-like growth factor 1 (R&D systems). Cardiomyogenic specification of human cells was confirmed by immunohistochemistry. After treatment, predifferentiated dog cells were labeled with 1 µM 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and frozen until injection. The karyotyping of the stem cells was performed before and after the growth factor treatment. Before injections, cells were washed using the culture medium and 1% PBS.

Experimental protocol. Pretreated autologous dog cells were injected directly into the infarcted myocardium at open thorax surgery. Before the cell injections, left ventricular (LV) dysfunction was documented as stable in all dogs at two consecutive echocardiography studies over the 3-wk period. Dogs were randomized into groups of animals receiving biologically treated (group A) and untreated (group B) MSCs on 2:1 ratio. In group A dogs, eight pilot animals receiving <20 x 106 cells were killed at 1, 2, and 4 wk for the histological analysis without functional follow-up. Seven animals received 147 ± 96 x 106 pretreated MSCs and were followed by echocardiography up to 12 wk. In control group B, all animals underwent myocardial injections of only culture-expanded MSCs without growth factor treatment. Two pilot dogs were killed at 2 wk after the cell injection for the histological analysis without functional follow-up. Of the five animals receiving culture-expanded MSCs without treatment with cardiomyogenic growth factors (168 ± 24 x 106 cells), one died within 24 h after the cell injection and four underwent follow-up at 12 wk with echocardiography similar to that of group A animals. Five additional dogs in group A and three in group B underwent injections for quantitative histological analyses of cardiomyogenic conversion 1 mo after cell injection. To inhibit apoptotic cell death, a caspase-8 inhibitor, zVAD (5 µM), was added to the cell suspension before cell injections in all animals of both groups. At 12-wk follow-up, animals were killed, the hearts were removed, and the infarct area was dissected into several samples and snap frozen in optimal cutting temperature (OCT) compound (Tissue-Tek, Sakura, Japan) until further histological analyses.

Immunohistochemistry. Isolated MSCs were plated onto collagen-coated four-well glass chamber slides in differentiation media. Cells were fixed in ice-cold methanol, blocked in 2% BSA/PBS, and analyzed by immunofluorescence. Heart tissue sections were examined for DiI-positive cells under the fluorescent microscope. The following antibodies and dilutions were used: MF-20 anti-myosin heavy chain (MHC) (mouse IgG1 anti-human 1:200, Developmental Studies Hybridoma Bank, Univ. of Iowa, Iowa City), anti-GATA-4 (mouse IgG2a anti-human 1:200, Santa Cruz), antimyocyte enhancer factor (MEF)-2 (goat IgG anti-human 1:100, Santa Cruz), anti-Nkx-2.5 (rabbit IgG anti-human 1:100, Santa Cruz), anticardiac MHC (mouse IgG1 anti-human 1:100, Chemicon), and troponin I (goat IgG anti-human 1:100, Santa Cruz). Appropriate secondary antibodies matching the primary antibodies were used in each staining. Alexa Fluor 488 secondary antibody and DiI were purchased from Molecular Probes. Confocal microscopy was performed to confirm colocalization of the DiI and myocardial markers. The proportion of DiI-labeled cells colocalizing with cardiac MHC was expressed as a percentage of cells per square millimeter in analyzed tissue sections.

Echocardiography. Echocardiography was performed in awake animals from the parasternal short axis. Images were obtained at the basal LV just beyond the mitral valve, at the level of the midpapillary muscle and at the apical level. M-mode echocardiograms were recorded at the level of the infarcted myocardium. Images were digitally stored and analyzed off-line by an experienced blinded echocardiographer. Area (from two-dimensional) and fractional (from M-mode) shortenings were calculated from these data. The %wall thickening of the dysfunctional myocardium was calculated from the average of three consecutive beats. Wall motion score index was calculated using the 16-segment model. Baseline and follow-up echocardiograms were reviewed by a panel of blinded echocardiographers.

Statistical analysis. Data are expressed as means ± SE. ANOVA for repeated measurements was used to test the differences in LV function over the follow-up period when appropriate. Student’s paired and unpaired t-tests or Mann-Whitney test were used for appropriate comparisons. Statistical significance was set at <0.05 level.


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Ex vivo cardiac specification of human bone marrow MSCs with cardiomyogenic growth factors. We first tested the hypothesis that biological growth factors can induce MSCs to express cardiomyogenic-specific markers ex vivo. Canine MSCs exhibited a fibroblastic morphology similar to that of MSCs isolated from other species, such as mouse, rat, and human. Human MSCs isolated from bone marrow in a similar manner were positive for c-kit and CD13 and negative for CD34, CD45, and CD49 (Fig. 1), suggesting the presence of a homogenous MSC population.


Figure 1
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  		Fig. 1. Flow cytometry analysis of the culture expanded mesenchymal stem cells (MSCs) before treatment with growth factors. Positivity of the given marker is given by the rightward shift against the background (shaded curves). Note that culture-expanded cells were CD34 and CD45 negative. In contrast, presence of other stem cell markers including CD117, CD90, CD44, and CD13 suggests the presence of homogenous MSC population. PDGFR, PDGF receptor.

 
Following growth factor addition, pretreated MSCs showed a uniform expression of MEF-2, GATA-4, and Nkx-2.5 and cardiac transcription factors (Fig. 2). Expression of MEF-2 and GATA-4 were nuclear localized, whereas Nkx-2.5 expression was found perinuclear and also in the cytoplasm. Growth factor treatment also induced the expression of desmin, an intermediate filament expressed in cardiac and skeletal muscle progenitor cells. Treated MSCs did not fully differentiate into contracting cardiomyocyte, nor did they express MF-20, a marker for MHC in fully differentiated muscle cells. Treated MSCs were also expressing in a low proportion early endothelial cell marker CD14 (12%). Expression of the leukocyte alkaline phosphatase, used as an osteogenic marker, was very faint or not induced at all after growth factor treatment. No expression of early cardiac markers has been observed in untreated, culture-expanded MSCs. Taken together, these results indicate that treatment of MSCs with biological growth factors induced predominantly the expression of genes specific for cardiomyogenic progenitor cells. Of note, culture expansion and differentiation resulted in no changes in the karyotypes of the MSCs (data not shown).


Figure 2
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  		Fig. 2. Cardiac and myogenic markers in bone marrow MSCs (BMSCs) before and after growth factors. Top panels show presence of desmin, an early muscle specific marker, and myosin heavy chain (MHC), a muscle-specific marker of differentiated cells. Bottom panels show expression of antimyocyte enhancer factor 2 (MEF-2), GATA-4, and Nkx-2.5, muscle- and cardiac-specific transcription factors in treated cells and untreated controls.

 
Histological analysis after cell injection. Next, we examined the potential of the specified autologous MSCs to undergo further cardiac differentiation after injection in a chronic dog model of infarcted myocardium due to permanent coronary ligation. Injections were performed at least 8 wk after myocardial infarction when stable LV regional dysfunction was documented by echocardiography. Heart sections that contained DiI-labeled cells were analyzed for the expression of cardiac and muscle specific markers at 2 wk, 4 wk, and 12 wk after injection (Fig. 3). At 2 wk after injection (Fig. 3, panel I), DiI-labeled cells were positive for adult sarcomeric myosin. At 4 and 12 wk after myocardial injections of pretreated MSCs (Fig. 3, panels II–IV), DiI-labeled prespecified cells colocalized with cardiac-specific myosin or troponin I consistent with further differentiation into the cardiac myocyte-like cells. The presence of newly differentiated cardiomyocyte-like cells was observed within the fibrotic area of the infarction and parallel to the neighboring myocytes. Confocal microscopy (Figs. 4 and 5) corroborated colocalization of injected cells with troponin I or cardiac myosin and documented that morphology of transformed cells was similar to neighboring cardiac myocytes including the presence of striations (Fig. 5, top). Furthermore, injected cells expressed connexin 43, being consistent with the integration in the host tissue (Fig. 5, bottom). No calcifications or osteogenic formation was noted in all analyzed sections. To address cardiomyogenic conversion rates, a separate quantitative histological analysis was performed in five group A dogs and three group B dogs (Fig. 6). Total surface tissue area analyzed for colocalization of DiI- and MHC-positive cells was similar in both groups (2.502 ± 0.504 cm2 in group A vs. 2.249 ± 0.872 cm2 in group B, respectively; not significant). Injection of modified cells was associated with a fivefold increase in the density of DiI-labeled cells per square millimeter as compared with injection of untreated bone marrow MSCs (3.61 ± 0.99% cells/mm2 in group A dogs vs. 0.72 ± 0.29% cells/mm2 in group B dogs, P = 0.037).


Figure 3
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  		Fig. 3. Histological tissue analysis of the specified cells at 2 wk (panel I), 8 wk (panel II), and 12 wk (panels III and IV) after injection. Panel I shows staining for sarcomeric MHC. Panels II and IV show staining for cardiac troponin I. Panel III shows staining for the cardiac-specific MHC. In each panel, A shows injected cells labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (red color), and B shows staining for the cardiac-specific marker (green color). In panel I, C shows 4,6-diamidino-2-phenylindole (DAPI) staining for nuclei. In panels II–IV, C shows the phase contrast image. In all panels, D is a merged image. Note a colocalization of the DiI-labeled, treated cells with muscle or cardiac specific markers. In panel I, colocalization between DiI-labeled cells and muscle marker is corroborated by nuclear DAPI staining.

 

Figure 4
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  		Fig. 4. Confocal microscopy image of the tissue analysis 12 wk after the injection. A: DiI labeled. B: staining for troponin I. C: merged image. Bottom: the staining in Fig. 3, panel I, is shown at various view angles to document the colocalization between DiI-labeled cells and cardiac markers.

 

Figure 5
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  		Fig. 5. Confocal microscopy shows colocalization of DiI-labeled cells with cardiac myosin together with presence of striations (top). The presence of connexin 43 is documented by the presence of punctuated staining at the site of cell-to-cell contact (bottom; arrows indicate colocalization with connexin 43). Blue color indicates DAPI staining for nuclei and red color indicates DiI dye.

 

Figure 6
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  		Fig. 6. A: example of tissue section comparing the density of DiI-labeled cells (red) colocalizing with cardiac myosin (green) after injection of modified BMSCs pretreated with cardiomyogenic growth factors (top) and nonmodified BMSCs (bottom). B: bar graph showing proportion of DiI-labeled cells/mm2 colocalizing with cardiac myosin after injection of modified and nonmodified BMSCs.

 
Hemodynamics and functional effects. The secondary objective was to investigate the effects of the transplanted cells on LV function. Table 1 shows baseline LV morphology and function before and after cell injections. In noninfarcted hearts, LV area shortening was 72 ± 4% and regional wall thickening was 76 ± 5%. After coronary ligation and before cell injections, LV dysfunction was confirmed by the stable reduction of percent area shortening and reduced percent wall thickening of the infarcted region (Fig. 7). The reduction in LV function as compared with baseline was similar in both groups (Table 1). Injections of untreated MSCs were not associated with any significant changes in both area shortening and regional wall thickening. In contrast, in dogs with pretreated MSCs, cell injections were associated with a significant increase in both area shortening and regional wall thickening observed at 4 wk and persisted up to 12 wk after the injection, suggesting a higher degree of functional recovery after the injection of cardiac-specified MSCs. Finally, wall motion score index decreased significantly in group A as compared with group B dogs (Table 1). No relationship was noted between the functional improvement and proportion of cells expressing cardiac myosin in tissue sections.


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  		Table 1. LV morphology and function before and after cell injections

 

Figure 7
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  		Fig. 7. The percent area shortening and left ventricular (LV) wall thickening of the infarcted tissue before and at 4, 8, and 12 wk after the cell injection. bullet, Dogs with treated MSCs; {circ}, dogs with untreated cells. *P < 0.05 vs. baseline (BL) and vs. untreated cells; not significant (NS) vs. BL for untreated MSCs.

 

   DISCUSSION
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GRANTS
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The results of the present study can be summarized as follows: 1) pretransplantational treatment of adult autologous MSCs cells using appropriate biological growth factors is feasible and leads to cardiomyogenic specification and 2) biological and functional effects upon cardiac regeneration of chronically infarcted myocardium were superior after myocardial injections of prespecified MSCs as compared with unmodified cells.

Cardiomyogenic specification of bone marrow MSCs. An immediate challenge in cardiac regeneration is to devise a strategy that leads to a reproducible degree of cardiac differentiation. MSCs have pluripotent characteristics, can contribute to the development of most somatic tissues, and may represent an optimal cell type for this purpose. They can undergo cardiac differentiation after injection into the normal myocardium (8). However, the use of unmodified bone marrow cells in experimental myocardial infarction models is controversial (5, 10, 21, 22, 31, 32). In the swine model of myocardial ischemia-reperfusion injury, allogenic mesenchymal bone marrow cells appeared to contribute to the neovascularization and myogenesis and led only transiently to improved cardiac performance (21). On the other hand, in a dog model of myocardial infarction, injection of the same cells led to a functional improvement in parallel to increased capillary density despite the absence of cardiac markers in DiI-labeled cells (22). In addition, in this study, follow-up was limited only to 30 days. Other investigators (5, 10, 31, 32) suggested that unmodified mononuclear or MSCs injected into the chronically infarcted areas do not differentiate into the cardiomyocyte lineage and may differentiate into fibroblasts (31) or form intramyocardial calcifications (32). Increasing controversy regarding the ability of bone marrow progenitors to transdifferentiate in vivo into cardiomyocyte-like cells led to the suggestion that alternative strategies with coaxing the cells toward the cardiac lineage or addressing critical molecular pathways of cardiac differentiation should be investigated as an approach to increase the reproducibility of cardiac differentiation (6, 16). The concept of ex vivo pretransplantational myogenic specification was previously attempted using 5'-azacytidine (29, 30). However, 5'-azacytidine as an inductive chemical compound may not be suitable for clinical use. It acts randomly on the genome such that myogenic differentiation is observed only in 30% of the treated cells (30) and the possibility of generating undesirable cell type remains high.

Biological cardiomyogenic signals and specification of adult bone marrow progenitors. We have taken clues from early embryonic heart development to test whether growth factors involved in early cardiomyogenesis have the ability to drive MSCs toward the cardiomyogenic cell lineage. During chick, amphibian, or mammalian embryonic development, signals emanating from the overlying anterior endoderm are required for the specification and stabilization of the underlying mesoderm to a cardiogenic cell lineage (1, 3, 7, 26). Moreover, recent reports indicate that coculture of human embryonic stem cells with mouse visceral endoderm-like cells resulted in a high degree of conversion of human embryonic stem cells into beating cardiomyocytes (20). While the ideal composition of the growth factor "cocktail" secreted by the endoderm to induce cells to a cardiogenic lineage is yet to be determined, several factors secreted by anterior endodermal cells, including BMP-2 and basic FGF, have been implicated as critical components of this early cardiomyogenic inductive signaling (14, 20, 26). Nevertheless, it remains a challenge to apply knowledge obtained from embryonic cells to adult stem cells. Therefore, in the present study, we tested the hypothesis that cardiomyogenic growth factors may induce cardiac specification of adult bone marrow MSCs ex vivo. The pretreated cells retained their morphological appearance of typical MSCs, but successful cardiac myogenic specification was confirmed by the presence of the early myogenic and cardiac markers desmin, MEF-2, and Nkx-2.5. Yet, to enhance the engraftment of the specified cells after injections, the course of cardiac differentiation was controlled such that specified cells expressed early cardiac muscle markers and remained negative for the MHC protein, an adult marker of fully differentiated muscle cells.

To mimic the clinical setting of chronically dysfunctional myocardium, further cardiac myogenic differentiation was tested in a dog model of myocardial infarction with cell injections performed >8 wk after ligation. Myocyte-like differentiation of specified MSCs was observed as early as 2 wk after the injections. The new expression of cardiac myocyte specific markers was observed in the later stages and persisted until the end of the 12-wk follow-up. Of note, injected cells formed new islands of cardiac-like cells even within the infarcted tissue and showed morphology similar to myocytes in the surrounding area, including presence of striations, parallel orientation, and expression of connexin 43. It should be noted that despite the faint expression of alkaline phosphatase in vitro, no calcifications were observed in vivo after injection of pretreated cells. In contrast, density of DiI-labeled cells colocalizing with cardiac myosin was higher after pretreatment with growth factors as compared with nonmodified bone marrow MSCs. Recent data of Mangi et al. (10) suggested that cardiac differentiation and repair mediated by MSCs in acutely injured myocardium can be enhanced by enhancing Akt-mediated survival signaling. Accordingly, to inhibit pharmacological apoptosis, caspase-8 inhibitor was added to the cell suspension before myocardial injections in all animals. Nevertheless, in our large animal chronic model of the myocardial infarction, the extent of cardiomyogenic differentiation as evidenced from proportion of cardiac myosin in DiI-labeled cells was lower in tissue sections of hearts from dogs who received nonmodified MSCs despite the similar extent of pharmacological inhibition of apoptosis. Taken together, these data show for the first time that ex vivo specification of bone marrow mesenchymal progenitors using cardiomyogenic growth factors is feasible and may lead to a greater colocalization with cardiac markers as compared with injection of nonmodified MSCs.

As to the secondary objective, regional contractility was improved at the site of injections with specified MSCs. Though it is attractive to hypothesize that the improved regional function was a direct result of the regeneration of cardiomyocytes, the conversion rates remain low and the exact mechanism responsible for the functional improvement remains to be investigated. Alternative mechanisms may include paracrine effects of specified cells on the neighboring myocytes and the positive effects on angiogenesis or cell fusion. Furthermore, other, yet unknown effects related to the pharmacological pretreatment should be considered. Invasive monitoring of global and regional LV performance and/or MRI and perfusion studies in parallel to cell tracking may provide additional insights into the functional effects of the specified MSCs.

Limitations. Several limitations should be acknowledged. First, limited functional improvement was seen after injection of nonmodified MSCs. Besides the controversy related to biological and functional effects of nonmodified MSCs (21, 22, 31, 32), this can be related to differences in the design of the current and previous studies, animal models, extent of LV dysfunction, and cell source. In previous studies, functional improvement was associated with allogenic source in larger infarctions and cells were injected earlier after myocardial infarction as compared with injections >8 wk after the coronary ligation in our study. Note also that in our study, caspase-8 inhibitor was added to both modified and nonmodified MSCs to prevent apoptotic cell death. Nevertheless, it was added at low doses and did not modify the functional response in the chronically infracted myocardium. Second, despite the functional improvement after injections of modified MSCs paralleled by colocalization with cardiac markers and striations, paracrine effects of modified MSCs ought to be considered. Likewise, despite the presence of gap junction proteins, full functional integration and excitation-contraction coupling of the implanted cells within the hosting myocardium need to be demonstrated. Thus passive changes in the wall composition and/or paracrine mechanisms cannot be excluded as potential mechanisms underlying the functional improvement. Third, only one cell type was injected, which may be suboptimal to achieve long-term sustainable cardiac repair. However, it should be noted that treatment with growth factors led to cardiac lineage cell specification typically found in early cardiac embryogenesis. In analogy to cardiac embryogenesis, such cells may hypothetically recruit other cell types needed for myocardial repair. In fact, injected modified cells were identified, albeit at very low frequency, in vascular-like structures, suggesting the potential of these cells to contribute to repair of other myocardial structural components. On the other hand, further studies are needed to address whether preconditioning of MSCs toward multiple tissue lineages may promote more complete regeneration of cardiac tissue. Fourth, in our study, the fate and conversion rates of injected cells were followed by histochemistry. DiI is characterized by a "punctuated" cytoplasmic staining and is widely used to track the fate of cells. However, this technique is limited to a single time point, and cell counting from tissue section is exposed to a number of limiting factors like section orientations compromising the quantitative histological analysis. These factors include DiI turnover in dying or multiplying cells, feasibility to analyze number of section in a large animal model, etc. Thus additional studies are needed to quantify the engraftment rates and survival of modified cells using dedicated cell tracking imaging techniques with MRI-compatible labeling or molecular tagging. Metabolic and electromechanical characteristics of the newly formed cardiac myocyte-like cells were not addressed in this study. Finally, the major limitation of any cell transplantation techniques is cell loss after injection. In addition to using antiapoptotic treatment, improved delivery strategies, for instance with the use of matrix-based delivery (33), should be explored to maximize engraftment and in vivo differentiation.

Summary. In the present study, we succeeded to demonstrate that ex vivo treatment with inductive growth factors is feasible in a large animal model and leads to cardiac specification of adult MSCs and their cardiac myocyte-like differentiation after injections into the chronically infarcted myocardium associated with superior functional effects. Thus the pretransplantational cardiac specification of autologous MSCs can be tested as a new strategy to achieve a reproducible degree of cardiac differentiation of the MSCs.


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The study has been partially supported by grants from the Direction Générale de Technologie de la Recherche et de L’Energie, department of the Walloon Regional Government, and by the Grant Action de Recherche Concertée Card 36CI-0106-271 (to G. H. Heyndrickx).


   DISCLOSURES
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Drs. J. Bartunek, W. Wijns, B. De Bruyne, and G. R. Heyndrickx are members of a nonprofit organization that is a founder of Cardio3. Drs. I. Lee and C. Homsy are shareholders of Cardio3.


   ACKNOWLEDGMENTS
 
We appreciate secretarial assistance of J. Cano in preparing the manuscript.

Present addresses: J. D. Croissant, Invitrogen Corp., Eugene, OR 97405; Y. Kaluzhny, MatTek Corp., Ashland MA 01721.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Bartunek, Cardiovascular Center and Cardiovascular Research Center, Aalst, Moorselbaan 164, 9300 Aalst, Belgium (e-mail: jozef.bartunek{at}olvz-aalst.be)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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