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European Journal of Heart Failure 2005 7(5):730-738; doi:10.1016/j.ejheart.2004.09.019
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© 2004 European Society of Cardiology

Effects of bone marrow derived mesenchymal stem cells transplantation in acutely infarcting myocardium

Hainan Piaob,c,1, Tae-Jin Youna,1, Jin-Sook Kwonb,c, Young-Hwa Kimb,c, Jang-Whan Baea, Bora-Sohnc,d, Dong-Woon Kimb,c, Myeong-Chan Chob,c,*, Myoung-Mook Leea and Young-Bae Parka

a Department of Internal Medicine, Seoul National University College of Medicine and Cardiovascular Research Laboratory Clinical Research Institute, Seoul National University Hospital, Seoul, Korea
b Department of Internal Medicine, College of Medicine Chungbuk National University, Cheongju, Korea
c Medical Research Institute College of Medicine, Chungbuk National University, Cheongju, Korea
d Department of Clinical Pathology College of Medicine, Chungbuk National University, Cheongju, Korea

* Corresponding author. Department of Internal Medicine, College of Medicine, Chungbuk National University, # 62 Gaeshin-dong, Hungduk-gu, Cheongju, 361-711, Korea. Tel.: +82 43 269 6356 fax: +82 43 273 3252. E-mail address: mccho{at}chungbuk.ac.kr


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Background: Cellular cardiomyoplasty (CCM) is considered to be a novel therapeutic approach for post-myocardial infarction (MI) heart failure. In this study, the functional effects of cultured mesenchymal stem cells (MSCs) transplantation and the associated histopathologic changes were evaluated in a rat model of MI.

Methods: Rats were subjected to 5 h of coronary ligation followed by reperfusion and, 10 days after MI, animals were randomized into either the MSCs transplantation (MI-MSC, n=8) group or the control (n=8) group. Allogeneic MSCs (3x106 cells) or media were epicardially injected into the center and the border area of the infarct scar.

Results: Four weeks after the MSCs transplantation, the echocardiogram showed preserved anterior regional wall motion and increases in fractional shortening in the MI-MSC heart relative to the control heart. Left ventricular (LV) end-diastolic pressure was smaller in the MI-MSC than in the control group. Implanted MSCs formed islands of cell clusters on the border of the infarct scar, and the cells were positively immunostained by sarcomeric {alpha}-actinin and cardiac troponin T. In addition, the number of microvessels on the border area of the infarct scar was greater in the MI-MSC than in the control group.

Conclusion: Allogeneic MSCs transplanted into the MI scar formed clusters of cell grafts on the border of the infarct, expressed cardiac muscle proteins, increased microvessel formation, and improved regional and global LV function. Our data indicate that CCM using MSCs may have a significant role in the treatment of post-MI heart failure.

Key Words: Cells • Transplantation • Myocardial infarction • Heart failure

Received December 4, 2003; Revised August 2, 2004; Accepted September 23, 2004


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
It has been demonstrated that cellular cardiomyoplasty (CCM) using various cell types has promising favorable effects on damaged heart function [1–3]. Bone marrow derived mesenchymal stem cells (MSCs) are one of the stem cells in the bone marrow microenvironment that is not a hematopoietic cell. MSCs are considered to be one of the candidate cells for the CCM because MSCs have the potential to differentiate into myocytes or cardiomyocyte-like cells (cardiomyogenic cells) when treated with the DNA demethylating agent, 5-azacytidine (5-aza), in culture condition [4,5]. Tomita et al. demonstrated that 5-aza treated MSCs implantation could improve the damaged heart function with their myogenic and angiogenic potentials [6,7]. Meanwhile, some reports have shown that the untreated, cultured MSCs also exhibit myogenic or cardiomyogenic phenotypes in the myocardial environments and suggested their potentials for CCM [8,9]. Furthermore, it has been demonstrated that transplantation of the autologous or syngeneic MSCs, which were prepared before myocardial infarction (MI) and then injected 1 week after MI, increased microvessel formation and improved left ventricular systolic function after MI [10,11]. However, from the clinical point of view, transplanting the autologous MSCs during such a relatively early period after MI is not likely to be feasible, because it takes some times for the autologous MSCs to be enriched by plastic adherent cultures from bone marrow cells. In this case, CCM with prepared allogeneic or xenogeneic MSCs, considering that MSCs have the immunosuppressive effects on allogeneic T cell reaction [12,13], seems to be the alternative choice to the autologous cells. To date, however, the histopathologic changes and the functional effects of cultured allogeneic MSCs transplantation in the model of MI have not been fully elucidated. Thus, the purpose of this study was to evaluate the functional effects of cultured allogeneic MSCs transplantation and to assess the histopathologic changes associated with the MSCs transplantation in a rat model of MI.


   2. Materials and methods
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1. Experimental animals
Male Sprague–Dawley rats weighing 240 to 290 g were used in this study. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996), and all protocols were approved by the Chungbuk National University Animal Care and Use Committee (ACUC).

2.2. Preparation of MSCs
Bone marrow cells were obtained by flushing the femurs and tibias with 10 ml of Dulbecco's Modified Eable's Medium (GibcoBRL, USA) supplemented with 10% fetal bovine serum and 50 IU/ml penicillin–streptomycin. The bone marrow cells were fractionated over 1.073 g/ml Percoll solution (Sigma, USA) and the mononuclear cells were obtained. The cells were plated in tissue culture dishes and were incubated. The non-adherent cells were discarded with the media changes, which were done every 3 days. When the cultures became near confluence, adherent cells were detached with trypsin-EDTA and subsequently passaged. The twice-passaged cells were harvested and were used for cell transplantation.

2.3. MI models
Induction of MI was performed as previously described with minor modification [14]. The rats were anesthetized with a mixture of ketamine hydrochloride (100 mg/kg body weight i.m.) and xylazine (10 mg/kg i.m.). Rats were intubated and ventilated with a rodent ventilator (model 683, Harvard Apparatus, USA). A left thoracotomy was performed in the fourth intercostal space and the pericardium was opened. The left coronary artery was occluded using the ‘shoe-string— tie with a curved needle and 6–0 silk suture. Then, the chest was closed in layers and the pneumothorax was evacuated, leaving both ends of the coronary suture protruding from the chest. Reperfusion was performed 5 h later by pulling on the exteriorized suture to release the knot [15].

2.4. MSCs implantation
Ten days after MI, the rats were randomly assigned into either the MSCs transplantation (MI-MSC, n=8) group or the control (n=8) group. The rats were anesthetized, ventilated and the chest was opened as described above in initial surgery. MSCs (3x106 cells) in 50 µl medium or medium were injected into the border and the center of the infarct scar using a 30 G needle.

2.5. In vivo functional assessment
Four weeks after the MSCs transplantation, the rats were anesthetized, and echocardiography was performed as described in a previous study [14]. Initially, using a 7.0 MHz transducer connected to a conventional echocardiographic system (Acouson, 128XP, USA), a two-dimensional short-axis view of the left ventricle (LV) was obtained at the level of the papillary muscles. After optimizing the gain setting and confirming both anterior and anterolateral wall akinesia, two-dimensional targeted M-mode tracings were recorded at a paper speed of 100 mm/s. End-systolic and end-diastolic LV internal dimensions were measured from at least three consecutive cardiac cycles on the M-mode tracings, based upon the American Society for Echocardiology leading-edge method. The LV percent fractional shortening (FS), as a measure of systolic function, was calculated as FS (%)=[(LVDd–LVDs)/LVDd]x100, where LVD indicates LV internal dimension, s is systole and d is diastole. All measurements were performed by an investigator who was blinded to the treatment group.

After the echocardiographic examination, the hemodynamic variables were measured. 1.4F micromanometer catheter (Millar Instruments, USA) was placed in the isolated right carotid artery, and advanced to the LV, and the pressure was recorded.

2.6. Histologic preparations and morphometric analysis
The heart was arrested in diastole by directly injecting 2–3 ml of 2 M KCl into the LV. The heart was retrogradely perfused with normal saline from the abdominal aorta at a constant pressure of 100 mm Hg and then was fixed with 10% phosphate-buffered formalin. The heart was excised, immersed into 10% buffered formalin solution for 24 h, and then cut into four transverse slices. The slices were embedded in paraffin and histologic sections were prepared.

Infarct size and wall thickness of the explanted hearts were measured in the masson-trichrome stained, two mid-ventricular sections with the use of computerized planimetry (Scion Image, Scion, USA), and were averaged. Infarct size was defined by the sum of the epicardial and endocardial circumferences of the fibrous scar tissues divided by the sum of the LV epicardial and endocardial circumferences [16]. Wall thickness was measured in the center of infarct, at the border of the infarct (right and left lateral), and in between (right and left mid-lateral), and then these five values were averaged [17].

2.7. Immunohistochemistry
Immunostainings for {alpha}-actinin, cardiac Troponin T (cTnT) and von Willebrand factor (vWf) were used to identify the differentiation of MSCs implants and the microvessel formation. Different slides were prepared for immunohistochemistry and were incubated with mouse monoclonal anti-{alpha}-actinin antibody (1:400, Sigma, USA), mouse monoclonal anti-cTnT antibody (1:200, NeoMarkers, USA), or rabbit polyclonal anti-Factor VIII related antigen/vWf antibody (1:80, NeoMarkers, USA) for 30 min in room temperature. In the cases of {alpha}-actinin and cTnT, slides were incubated with rhodamine red dye-conjugated goat anti-mouse IgG (1: 50, Jackson Immunoresearch, USA) for 30 min, and fluorescence was detected and photographed with a confocal microscope. In the case of vWf, slides were incubated with texas red dye-conjugated goat anti-rabbit IgG (1:50, Jackson Immunoresearch, USA) and were examined under a light microscope. Quantitative assessment of microvessels was performed on the border of the MI scar as well as in the center of the MI scar at a x400 magnification. Four high power fields (HPF) per each area were scanned for analysis, and the number of microvessels in each was averaged and was then expressed as the number of microvessels per HPF.

2.8. Statistical analysis
Results are expressed as mean±standard error of mean (S.E.M.). Statistical significance was determined using the two-tailed Student's t-test (SPSS for window, version 7.5). Differences were considered statistically significant when P<0.05.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1. Effects of allogeneic MSCs transplantation on in vivo LV function
Two-dimensional echocardiographic examination of the control hearts revealed akinetic anterior, anterolateral and lateral walls and a remarkable dilatation of the LV. In the MI-MSC hearts, however, the anterior wall–the anterior border of infarct area–motion was relatively preserved compared to the control hearts, although the anterolateral and lateral walls were also akinetic. Therefore, calculated fractional shortening was significantly larger in the MI-MSC than in the control group (27.1±1.4% vs. 19.8±2.6%, P<0.05), although LV end-systolic dimension and end-diastolic dimension did not significantly differ between two groups (Fig. 1). In addition, the LV end-diastolic pressure was smaller in the MI-MSC than in the control group and the difference showed a borderline significance (6.4±0.7 vs. 10.8±1.8 mm Hg, P=0.05) (Fig. 2).


Figure 1
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  		Fig. 1 Echocardiographic data 4 weeks after cell implantation or medium injection. (A) M-mode echocardiograms showed that left ventricular (LV) anterior wall motion was preserved in the MI-MSC hearts (c and d), but not in the control hearts (a and b). (B) LV dimensions. LV end-systolic dimension (ESD) and LV end-diastolic dimension (EDD) were not significantly different between the control and the MI-MSC group (n=8 in each group). (C) LV systolic function. Fractional shortening (FS) was significantly higher in the MI-MSC compared to the control group (n=8 in each group).

 


Figure 2
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  		Fig. 2 Hemodynamic data 4 weeks after cell implantation or medium injection. LV systolic pressure (LVSP) (A) was higher and LV end-diastolic pressure (LVEDP) (B) was lower in the MI-MSC than in the control group (n=8 in each group).

 
3.2. Histologic changes with MSCs transplantation
On histologic analysis, extensive anterior infarction with fibrosis of anterior, anterolateral and lateral walls was observed in the hearts of all animals. Masson's trichrome stain showed the transmural nature of the infarction. The infarct size was similar in the MI-MSC (41.0±4.1%) and the control hearts (43.2±2.0%). However, the scar thickness of the MI-MSC hearts was thicker than that of the control hearts (1.54±0.05 vs. 1.14±0.1 mm, P<0.05) (Fig. 3).


Figure 3
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  		Fig. 3 Morphometric data 4 weeks after cell implantation or medium injection. MI size (A) was not significantly different between the control and the MI-MSC group. However, scar thickness (B) was greater in MI-MSC than in the control hearts (n=8 in each group).

 
The implanted MSCs, cytoplasm-rich mononuclear cells, were found in the heart of the MI-MSC. The implanted MSCs were observed as islands of cell clusters preferentially located in the anterior border area of the MI scar. There was no cell cluster in the center of the MI scar. The immunohistochemical stain demonstrated that the cells in the cluster expressed sarcomeric {alpha}-actinin and cTnT (Fig. 4).


Figure 4
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  		Fig. 4 Photomicrographs of implanted MSCs 4 weeks after cell transplantation. (A) MT staining showed a cluster of implanted cells at the anterior border area of MI scar (blue stain denotes the fibrous tissues in MI scar) (x100 magnification). (B) HE staining of an adjacent tissue section (x100 magnification). (C) Higher-power view (x400 magnification) showed cytoplasm-rich mononuclear transplanted MSCs. Immunohistochemical staining revealed sarcomeric {alpha}-actinin (D) and cardiac troponin T (cTnT) (E) expressions in implanted MSCs (x100 magnification).

 
The immunohistochemical stain for vWf revealed that there were numerous microvessels around the implanted cell clusters. Accordingly, the measured microvessel density at the border area of the MI scar was significantly higher in the MI-MSC than in the control group (9.5±1.0 vs. 5.3±0.5 vessels/HPF, P<0.05) (Fig. 5).


Figure 5
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  		Fig. 5 Microvessel formation after MSCs transplantation. Numerous blood vessels expressing vWf were observed near the implanted cell clusters in the MI-MSC heart (B) relative to the control heart (A) (x100 magnification). (C) vWf positive vessel cells in higher (x400) magnification. (D) Microvessel density. Vessel density was increased mainly in the border of infarct scar where the implanted cell clusters were observed (n=8 in each group).

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
MSCs are considered to have great advantages for CCM because MSCs can differentiate into myogenic or cardiomyogenic cells in proper culture conditions and with 5-aza treatments [4,5]. However, the ideal in vitro conditions or methods for MSCs to differentiate into cardiomyogenic cells have not been determined. Furthermore, there is some concern with clinical safety problems because 5-aza treatments might demethylate a wide variety of genes [18,19]. Meanwhile, MSCs not pre-exposed to 5-aza before transplantation also exhibited cardiomyogenic phenotypes in myocardial environments, such as in the normal myocardium [8] or cryo-injured myocardial scar tissues [6]. However, differentiation and functional effects of implanted MSCs not pre-exposed to 5-aza have not been fully elucidated in the model of MI. Therefore, we evaluated the functional effects of cultured MSCs transplantation in a rat model of coronary ligation followed by reperfusion, and we also assessed the histopathologic changes associated with the MSCs transplantation.

The main findings of our study are as follows: (1) allogeneic MSCs can be successfully engrafted and are able to survive and form the islands of cell clusters in the reperfused but, transmurally infarcted myocardial scar. (2) MSCs transplantation attenuates the LV systolic dysfunction after MI. (3) Implanted MSCs, which were not pre-treated with 5-aza, express the cardiac muscle proteins in the MI scar. (4) MSCs transplantation attenuates the scar thinning and increases microvessel formation after MI.

4.1. MI model
With the advances of coronary angioplasty or coronary bypass surgical techniques, an increasing number of patients receive revascularization therapy early or late after MI in order to improve regional blood supply and cardiac function. Therefore, it is likely that the majority of patients who will require the CCM would be the ones whose myocardium has been severely damaged despite reperfusion therapy. Therefore, from the current clinical point of view, analyzing the effects of CCM in the model of reperfused, but severely damaged heart seems to be very important and practical. However, most previous studies have examined the effects of CCM in the cryo-injured damaged heart that is not relevant to the clinical myocardial ischemia or in the MI heart created by permanent coronary artery occlusion. It has been demonstrated that reperfusion of 1–2 h or 6–8 h after the coronary ligation in rats did not reduce the infarct size or extent of transmural necrosis compared with permanent ligation [20]. Therefore, the present 5-h coronary occlusion followed by reperfusion model in the rat was developed in order to mimic the reperfused, but severely damaged heart in clinical settings of MI.

4.2. Timing of cell transplantation
It has been demonstrated that infarct expansion starts early after MI and cardiac remodeling is continued chronically in a rat model of moderate to large MI [21]. It has also been shown the infarct expansion plateaus about a week after MI in rats [22]. Therefore, logic suggests that in order to effectively attenuate infarct expansion and improve cardiac function, one can transplant cells any time within 1 week of MI. However, transplanting the cells immediately after MI is not likely to be clinically feasible. In addition, since it has been shown that inflammatory process after MI peaks at 1 week [23] and that cell transplantation is most successful after the inflammatory reaction resolved [24], we decided to inject the cell after this period of intense inflammation. Actually, Pouzet et al. reported in their study that they chose a time point at which the acute post-MI inflammatory response has been terminated to inject skeletal myoblasts [25].

Infarct healing is usually completed by 3 weeks after MI in rats [23], suggesting that cells may be given late after MI, when the infarct expansion and inflammation is completed and the infarct scar is fully maturated. However, the purpose of the present study was not to evaluate this delayed chronic period but rather to investigate the effects of MSCs transplantation during relatively early period after MI. In addition, we thought that the hard and paper-thin nature of the infarct scar with progressive fibrosis and thinning that results in rats late after MI makes it difficult to inject cells precisely into the scar and would not be a good environment for implanted cells to survive.

Therefore, we decided to inject the MSCs 10 days after MI, when the inflammation was diminished, but the scar was not fully maturated, to evaluate the effects of MSCs transplantation during relatively early period after MI. The optimal time for transplanting cells after MI is a controversial issue, which needs to be clarified in further studies.

4.3. Effects of the MSCs implantation on LV function
In this study, the exact pathophysiologic mechanisms for echocardiographic and hemodynamic improvement observed in the MI-MSC were not clear. The implanted MSCs maintained their morphologies as round mononuclear cells and the islands of implanted cells did not make contact with the host myocardium. Therefore, it is unlikely that synchronous contractions of implanted MSCs contributed for the functional improvement. Actually, it has not been proved whether the transplanted MSCs could contract in vivo, although Makino et al. had demonstrated that the 5-aza treated immortalized MSCs beat spontaneously in vitro condition [5]. It is also unlikely that MSCs implantation attenuated LV remodeling such as scar expansion or LV dilation after MI, since LV end-diastolic dimension or infarct size were not different between MI-MSC and the control group in this study.

However, there are some possible hypotheses that support the beneficial effects of MSCs transplantation. First, The clusters of implanted MSCs were mainly located in the anterior border of the infarct scar in which the abnormal wall motions were attenuated and the cells expressed sarcomeric {alpha}-actinin and cTnT, which have been considered to be the markers of cardiomyogenic differentiation of transplanted cells [5–7,9]. It is likely that, with their differentiation into cardiomyocyte-like cells, implanted MSCs contained more elastic properties than before and that the MSCs might improve the regional function through altering the scar elasticity [6]. It has been demonstrated that the elastic properties of transplanted cells are important for functional improvement after cell transplantation because the elasticity might limit the scar from overstretching and/or improve the diastolic performance [26–28]. With their elastic properties, the tethering effects of the adjacent myocardium also might contribute to the regional wall motion improvement in the infarct border [7]. A second mechanism seems to be associated with the altered scar geometry. The scar thickness in the MI-MSC was greater than that of the control group. Increased scar thickness seems to reduce the wall stress, and this salutary effect of cell implantation might contribute to the functional improvement [26]. It seems that reduced scar thinning in the MI-MSC was not due to the implanted cell volume, since the entire scar was generally thickened compared to the control heart despite the implanted cells were mainly located in the anterior border of the scar. Previously, others also reported similar observations in the model of fetal cardiomyocyte or MSCs transplantation and suggested that CCM might alter the extracellular matrix remodeling [9,29]. In addition, there were more microvessels in the MI-MSC than in the control heart. Improvement of the blood flow with angiogenesis would improve the contractile function of hibernating myocardium. Previous investigators have demonstrated that engrafted MSCs differentiate into endothelial cells and improve cryo-injured or infarcted cardiac function [6,7,10]. Furthermore, using radioactive microsphere techniques, recent study have confirmed that MSCs transplantation after MI increases vascular densities and regional blood flow in the infarct scar [11]. These reports and our observation support the functional benefit of MSCs transplantation with their angiogeneic potential.

Taken together, it is likely that by altering the local elasticity and scar geometry, and also by increasing the local perfusion, the MSCs implantation improved regional and global LV functions in this study.

Previously, Tomita et al. examined the effects of MSCs implantation in the cryo-injured rat myocardium [6]. They reported that, although both the 5-aza pre-treated and untreated cultured MSCs were stained positively for the troponin I and induced angiogenesis in the scar tissues, only the 5-aza pre-treated MSCs reduced scar expansion and LV dilation, and improved heart function. The speculated reason for the unique benefit of 5-aza pre-treated MSCs was that they might contain more contractile structures and elastic properties than the untreated MSCs. However, in our study using a rat model of MI induced by coronary ligation and reperfusion, MSCs implantation improved the LV systolic function in the absence of the 5-aza pre-treatment. Discrepancies with Tomita's study may be related to the differences in implantation cell numbers (3x106 vs. 106 cells), transplantation sites (center and border of the scar vs. center of the scar) and the model of myocardial injury (coronary ligation followed by reperfusion vs. cryo-injury). In this study, it is possible that relatively larger numbers of MSCs could survive in the border area because sufficient numbers of MSCs were injected into the border area as well as into the center of the infarct scar that was not cryo-injured but reperfused. The implanted MSCs adjacent to the normal myocardium seem to have more favorable conditions not only for their survival but also for their differentiation into cardiomyogenic cells. In the recent study using a swine model of MI, Shake et al. reported that 5-aza untreated MSCs which were transplanted into the MI scar tissue expressed the muscle specific proteins such as {alpha}-actinin, troponin T, phosphpholamban and tropomyosin and attenuated the systolic paradoxical wall motion [9]. These data support our results about milieu-influenced cardiomyogenic differentiation of untreated, cultured MSCs and clinical potentials of the MSCs transplantation.

4.4. Allogeneic MSCs transplantation
The allogeneic, not the autologous, nature of implanted MSCs in our study seems to give a new insight for the CCM. The allogeneic MSCs could lessen the beneficial effects of cell implantation because of possible rejection problems. However, previous reports have demonstrated that the MSCs have immunosuppressive effects on allogeneic T cell reaction, and even the effect of a single intravenous dose of MSCs can be comparable to that of the potent immunosuppressives currently being used clinically [12,13]. It also has been demonstrated that the allogeneic MSCs implantation elicits no immunologic reactions in other animal study [30] and the xenogeneic MSCs transplantation is even possible [31]. We also have confirmed the successful engraftment of allogeneic MSCs transplantation up to 2 weeks in the pilot study. Meanwhile, it has been demonstrated that daily injection of immunosuppressive agents, such as cyclosporin A, that inhibit the calcineurin mediated pathway, could affect the cardiac remodeling after MI [16,32]. Therefore, we performed this experiment using the allogeneic MSCs without immunosuppressive regimens and confirmed that the MSCs implantation, although allogeneic, attenuated the cardiac dysfunction after MI. The possibility of allogeneic MSCs transplantation, although it is likely that we can obtain the autologous MSCs with ease in clinical settings, gives us another therapeutic consideration in case when we transplant the MSCs relatively early after MI. or in case when we cannot get a sufficient amount of autologous MSCs from the patients because of old age, other hematologic diseases or bone diseases.

4.5. Study limitation
Since the implanted MSCs were not labeled with markers, such as bromodeoxyuridine or 4',6-diamidino-2'-phenylindole (DAPI), there were limitations in detecting the implanted MSCs and in elucidating their differentiation in cardiac milieu. We had transplanted DAPI-labeled MSCs in our pilot study to elucidate the feasibility of cell implantation and to detect the implanted cell survival in myocardial scar tissues. We had found the clusters of DAPI-positive implanted MSCs in the infarct scar and the morphologies of DAPI-positive cells were assessed with HE stained adjacent tissue section. The cells were cytoplasm-rich mononuclear cells as described in this study. Therefore, the typical morphologies of the transplanted MSCs made the easier recognition of the implanted cells possible in this study. Unfortunately, however, it is not possible to say definitively whether the cells in microvessels were from the implanted MSCs or host. Although previous studies have demonstrated that the implanted MSCs could differentiate into endothelial cells [6,7,26], the possible paracrine effects of implanted cells on microvessel formation should be clarified later.


   5. Conclusion
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
Implanted allogeneic MSCs formed clusters of cell grafts in the border area of the infarct, expressed cardiac muscle proteins, attenuated the scar thinning, increased microvessel formation and improved regional and global LV function in a rat model of MI. Our data demonstrated that CCM using MSCs may have a significant role in the treatment of post-MI heart failure.


   Acknowledgements
 
This work was supported by a grant (R05-2002-000-00828-0) from the Korea Science and Engineering Foundation and a grant (SC13121) from Stem Cell Research Center of the 21st Century Frontier Research Program, Korea.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
1 T.J. Youn and H. Piao contributed equally to this work. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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