EJCTS Click here to go to Edwards website
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ott, H.C.
Right arrow Articles by Hering, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ott, H.C.
Right arrow Articles by Hering, S.
Related Collections
Right arrow Cardiac - other
Right arrow Coronary disease
Right arrow Myocardial infarction

Eur J Cardiothorac Surg 2004;25:627-634
© 2004 Elsevier Science NL

Combined transplantation of skeletal myoblasts and bone marrow stem cells for myocardial repair in rats

H.C. Otta*, N. Bonarosa, R. Marksteinerb, D. Wolfc, E. Margreiterb, T. Schachnera, G. Laufera, S. Heringd

a Department of Cardiac Surgery, University of Innsbruck, Anichstrasse, A-6020 Innsbruck, Austria
b Biochemical Pharmacology, University of Innsbruck, Innsbruck, Austria
c Department of Hematology, University of Innsbruck, Innsbruck, Austria
d Pharmacology and Toxicology, University of Vienna, Vienna, Austria

Received 13 September 2003; received in revised form 3 December 2003; accepted 15 December 2003.

* Corresponding author. Tel.: +43-512-504-2501; fax: +43-512-504-2502
e-mail: h.c.ott{at}aon.at


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
Objectives: To prove whether intramyocardial transplantation of combined skeletal myoblasts (SM) and mononuclear bone marrow stem cells is superior to the isolated transplantation of these cell types after myocardial infarction in rats. Methods: In 67 male Fischer rats myocardial infarction was induced by direct ligature of the LAD. Seven days postinfarction baseline echocardiography and intramyocardial cell transplantation were performed. Via lateral thoracotomy 200 µl containing either 107 SMs or 107 bone marrow-derived mononuclear cells (BM-MNC) or a combination of 5x106 of both cell types (MB) were injected in 10–15 sites in and around the infarct zone. In controls (C) 200 µl of cell-free medium were injected in the same manner. Before injection both cell types were stained using a fluorescent cell linker kit (PKH, Sigma). In addition, SMs were transfected with green fluorescent protein. Nine weeks postinfarction follow-up echocardiography was performed and animals were sacrificed for further analysis. Results: At baseline echocardiography there was no difference in left ventricular ejection fraction (LVEF; C, SM, BM-MNC, MB: 60.1±3.2, 53.3±10.2, 53.1±8.7, 49±9.0%) and left ventricular end diastolic diameter (LVEDD; C, SM, BM-MNC, MB: 6.5±0.8, 5.17±0.8, 5.77±1.4, 6.25±0.8 mm) between the different therapeutic groups. Eight weeks after cell transplantation LVEDD was significantly increased in all animals except those that received a combination of myoblasts and bone marrow stem cells (MB; C, SM, BM-MNC, MB: 7.7±0.6 mm, P=0.001; 7.7±1.5 mm, P<0.001; 7.7±1.1 mm, P=0.005; 6.6±1.7 mm, P=0.397). At the same time LVEF decreased significantly in the control group (C), stayed unchanged in animals that received bone marrow stem cells (BM-MNC) and increased in animals that received myoblasts (SM) and a combination of both cell types (MB; C, SM, BM-MNC, MB: 45.3±7.0%, P=0.05; 63.9±15.4%, P=0.044; 54.3±6.3%, P=0.607; 63.0±11.5%, P=0.039). Conclusions: The present data show that the concept of combining SMs with bone marrow-derived stem cells may be of clinical relevance by merging the beneficial effects of each cell line and potentially reducing the required cell quantity. Further studies are required to identify the exact mechanisms underlying this synergy and to allow full exploitation of its therapeutic potential.

Key Words: Cellular cardiomyoplasty • Skeletal myoblast • Bone marrow • Cell transplantation • Cardiomyopathy

Abbreviations: BM-MNC, bone marrow-derived mononuclear cell • IVS, interventricular septum • LVEDD, left ventricular end diastolic diameter • LVEF, left ventricular ejection fraction • SM, skeletal myoblast • vWF, von Willebrand Factor


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
The adult heart lacks the potential of effective regeneration. Instead of tissue recovery the infarcted myocardium is transformed into a non-contractile fibrous scar. The following remodelling process leads to expansion of the initial infarct area and dilatation of the left ventricular lumen [1]. As one novel therapeutic option attempting to stop or even reverse the process of postinfarction remodelling and restoring left ventricular function, cellular cardiomyoplasty has been proposed and opened new perspectives for the treatment of ischemic heart disease [2]. In a number of myocardial injury models, different cell lines proved the potential to regenerate viable tissue after being transplanted into the infarcted heart [2–6]. Among those, skeletal myoblasts (SM) improved myocardial performance in vitro and in vivo, either delivered to the injured myocardium by intramural implantation or arterial injection [2,7–10]. There is evidence that the cardiac environment is permissive for myogenic differentiation and influences the developmental program of implanted myoblasts, improving their ability to assist myocardial performance [7,8]. These convincing preclinical data led to the first application in a clinical setting by Menasché and coworkers reported in 2001 [11] and the conductance of phase I clinical trials [12,13]. Corresponding to preclinical data, the results of these trials suggest a contribution of engrafted myotubes to the contractile function of treated areas.

As a second potential candidate for cellular cardiomyoplasty bone marrow-derived stem cells (BMC) have been successfully transplanted in several experimental settings [14,15]. One major advantage of bone marrow stem cells over SMs may be their pluripotentiality. BMC are suggested to acquire the phenotypic characteristics of their host tissue and to differentiate into cardiomyocytes and endothelial cells following engraftment into myocardium [6,16–18]. In a recent experimental trial, the implantation of total unpurified bone marrow into injured myocardium was associated with cardiomyogenic and endothelial transdifferentiation [19]. Results of first clinical trials suggest that injected bone marrow-derived mononuclear cells (BM-MNC) cause an improvement of myocardial blood flow and an associated enhancement of regional and global left ventricular function in treated patients [20,21]. This effect has been seen in areas of hibernating myocardium and is therefore suggested to be primarily related to an induction of angiogenesis.

Corresponding to the different cell types, both therapeutic approaches have to deal with different limitations. In the case of SM transplantation the total myoblast survival in the human setting was calculated to be below 1%. This finding was discussed to be caused by the lack of a vascular bed allowing sufficient tissue perfusion [13]. In BM-MNC transplantation the number of cells transdifferentiated to cardiomyocytes was very small, raising concern over the functional efficacy of isolated BMC transplantation [19]. To overcome the individual limitations of both therapeutic options, the isolated transplantation of SMs and of bone marrow-derived cells, a combination of these two cell types may be a new therapeutic approach. The aim of the present study was therefore to prove whether intramyocardial transplantation of combined SMs and mononuclear bone marrow stem cells in a rat ischemic myocardial injury model is superior to the isolated transplantation of these cell types.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A. Conference...
 References
 
2.1. Isolation and expansion of skeletal myoblasts and bone marrow stem cells
Primary SMs were isolated from a male F344 Fischer rat (6 weeks of age). After intraperitoneal administration of ketamine (50 mg/100 g) and xylazine (1 mg/100 g) hind limb muscles (0.5–1.0 g) were dissected free from connective tissues, and minced into pieces of approximately 1 mm3. Muscle samples were enzymatically dissociated according to the cell dispersion technique described by Blau and Webster. Single satellite cells in suspension were manually collected with a micropipette under microscope control and transferred into the cell culture with 96-well plates. The cells were cultured in growth media and maintained in a proliferating state. Desmin was used as a marker to identify clones of myoblasts (Fig. 1A) . To confirm the ability to differentiate, desmin-positive mononucleated myoblasts were cultured in differentiation media. Under these conditions myoblasts fused into multinucleated myotubes (Fig. 1B). After 1 week in differentiation medium spontaneous contractions of the myotubes were observed. Electrophysiologic analysis revealed myoblast characteristic Ba2+ currents through T-type calcium channels in myoblasts (Fig. 1C, left panel) with corresponding current–voltage relationship (Fig. 1C, right panel).



View larger version (51K):
[in this window]
[in a new window]
  		Fig. 1. Characterisation of skeletal myoblasts before transplantation. Desmin was used as a marker to identify clones of myoblasts (A). Myoblasts fused into multinucleated myotubes if cultured in differentiation medium (B). Electrophysiological analysis revealed myoblast characteristic Ba2+ currents through T-type calcium channels in myoblasts (C, left panel) with the corresponding current–voltage relationship (C, right panel).

 
Before intramyocardial injection, cells were labelled using a fluorescent cell linker kit (PKH26-GL, Sigma-Aldrich Co., Vienna, Austria) following the instructions of the manufacturer.

Bone marrow stem cells were isolated from male F344 Fischer rats. After intraperitoneal administration of ketamine (50 mg/100 g) and xylazine (1 mg/100 g) both femurs were dissected and bone marrow was flushed with 5 ml medium using a 21 G needle. Cells were washed twice with RPMI Medium. Mononuclear cells were isolated by Percoll gradient (Lymphoprep, Nycomed). In rodents, bone marrow mononuclear cells comprise 4–17% CD34+ stem cells [23]. In humans, co-expression of VEGF-R2, CD34 and AC133 is a characteristic feature of endothelial progenitor cells (EPC) [24]. However, no data are currently available on the frequency of EPC in rat bone marrow mononuclear cells. Before transplantation, cells were labelled with a green fluorescent cell linker kit (PKH2-GL, Sigma-Aldrich Co., Vienna, Austria) following the instructions of the manufacturer.

2.2. Myocardial infarction and cell transplantation
Myocardial infarction was induced following a standardized protocol: 67 male F344 Fischer rats were anaesthetised (ketamine 10 mg/100 g and xylazine 1 mg/100 g intraperitoneally), placed in a supine position on a temperature-controlled plate (37 °C) and tracheally ventilated with a small animal Harvard respirator (Harvard Apparatus Rodent Ventilator Mod. 40-1003, Harvard Apparatus Inc., Millis, MA). The heart was exposed through a 2 cm left lateral thoracotomy and the left coronary artery was ligated with a Prolene® 7/0 suture under the distal portion of the left atrial appendix. Correct position of the ligation was assessed by colour change of the ischemic area, regional wall motion and electrocardiographic (ECG) recording. After haemostasis was achieved, the muscle layer and skin incision was closed with Vicryl® 3/0 running sutures after drainage of the left thoracic cavity with a 16 G silicon tube. All animals were monitored for 4 h postoperatively.

One week after myocardial infarction, all animals (n=52) underwent a second thoracotomy following the same protocol as described above. The heart was exposed via the same intercostal space as in the first operation and the infarcted area of the lateral left ventricular wall was clearly identified. Animals were now divided into four subgroups: the myoblast group (n=14), the bone marrow group (n=14), the combined therapy group (n=14) and the control group (n=10). In the myoblast group 107 homologous SMs were injected into 15 microdepots of 10 µl each in the centre and border zone of myocardial infarction. Injections were performed using a modified 24 G needle (Becton Dickinson, Helsingborg, Sweden) covered with a rubber tube allowing only a 1.5 mm puncture to avoid intracavitary injection. The needle was mounted on a modified 1 ml syringe that was connected via a polyethylene tube (outer diameter, 2.0 mm; inner diameter, 1.0 mm; Becton Dickinson, Helsingborg, Sweden) to a time gated pneumatic pump (PV 830, Pneumatic PicoPump, World Precision Instruments, Sarasota, FL, USA). The picopump was adjusted to a pressure of 30 psi at a gated impulse duration of 500–700 ms. Under these conditions a volume of 10 µl was injected intramyocardially with each pressure impulse. Cell death related to this procedure (shear force in needle and syringe) was calculated to be below 5% in a separated experiment. After accurate haemostasis the chest was closed and drained. In the bone marrow group 15 depots of 10 µl each containing 107 homologous bone marrow mononuclear cells were injected. In the combined treatment group, 15 depots of 10 µl containing 5x106 homologous SMs as well as 5x106 homologous bone marrow stem cells (therefore the sum of 107 cells, corresponding to the other cell-treated study groups) were injected following the same protocol as in the myoblast group. In the control group 15 depots of 10 µl empty culture medium (DMEM 31885-023, GIBCO) were injected following the same protocol. After closure of the chest all animals were monitored for 4 h and underwent the same postoperative routine.

2.3. Functional assessment
Left ventricular function was assessed by two-dimensional echocardiography 1 week after myocardial infarction (at baseline, before cell transplantation) and 8 weeks after cell transplantation. Under general anaesthesia with ketamine (0.1 mg/100 g) the chest was shaved and a layer of acoustic coupling gel was applied. Two-dimensional and M-mode measurements were performed with a commercially available 15 MHz linear-array transducer system (AcuNav, Acuson Corp., Mountain View, CA). Parasternal long axis views were recorded and left ventricular dimensions and volumes were calculated according to standard formulas. All measurements were performed by two experienced investigators who were blinded to the treatment group.

2.4. Histology and immune histochemistry
Within 3 days after follow-up echocardiography all animals were sacrificed with an overdose of ketamine and xylazine and hearts were harvested and cryopreserved for further histological analysis. The ventricles were cross-sectioned into three sections. From each section 8 µm slides were prepared using a cryostat and standard histological studies were performed with haematoxylin and eosin staining. The transplanted myoblasts were identified by fluorescence. For von Willebrand Factor (vWF) staining cryosections were mounted on glass slides, rinsed in PBS and fixed in 100% methanol at -20 °C for 5 min. All further incubations were carried out at room temperature and all rinses and dilutions were performed with a blocking solution consisting of 2% BSA and 0.5% Triton X-100 in PBS. An initial blocking step was performed with this solution for 30 min. An antibody to anti-vWF (F3520, Sigma) with a dilution of 1:200 was applied for 1 h. After rinsing a peroxidase-conjugated anti-rabbit IgG (A0545, Sigma) with a working dilution of 1:500 was applied for 30 min. The sections were rinsed and stained with AEC chromogen (3-amino-9-ethylcarbazole in N,N-dimethylformamide; IMMH5, Sigma). Endogenous peroxide was quenched with 3% hydrogen peroxide. The number of capillaries was counted in the scar tissue of all four animal groups using a standard light microscope at a 400x magnification. Ten high-power fields in each scar were randomly selected, and the number of capillaries in each was averaged and expressed as the number of capillary vessels per high power field (0.2 mm2). Myoblast graft size was measured in five randomly selected areas within each infarction scar of myoblast-treated animals at 40x magnification. Assuming an elliptical graft shape, the graft area was calculated as area=length/2xwidth/2x.

2.5. Data analysis
Statistical analysis was performed using SPSS 9.0 for windows. In the following, data are expressed as mean±standard deviation. Comparisons of continuous variables among animal groups were studied by a one-way ANOVA. Longitudinal studies comparing data within each group were achieved by the use of paired t-tests. A value of P<0.05 after Bonferroni correction was considered significant.

2.6. Animal care
This study was approved by the Animal Care Commission of the University of Innsbruck and by the Ministry of Science, Republic of Austria. Care of animals was in accordance with the ‘Guide for the Care and use of Laboratory Animals’ (NIH publication 85-123, revised 1985).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A. Conference...
 References
 
3.1. Functional assessment
Major functional results are summarized in Fig. 2 . Baseline echocardiography was performed 1 week after myocardial infarction, the day before cell transplantation. There was no significant difference among the different therapeutic groups. Thus left ventricular ejection fraction (LVEF) was 60.1±3.2% in control rats, 53.3±10.2% in the myoblast group, 53.1±8.7% in the bone marrow group and 49.0±9.0% in the group receiving a combination of myoblasts and bone marrow stem cells (P=0.259). At the 8 weeks time point after cell transplantation, there was a significant improvement of ejection fraction in the myoblast group (63.9±15.4%, P=0.044) and in the group that received the combined therapy (63.0±11.5%, P=0.039), whereas LVEF stayed unchanged in the bone marrow group (54.3±6.3%, P=0.607) compared to the corresponding values of baseline echocardiography (Fig. 2A). Rats of the combined therapy group, receiving both SMs and bone marrow stem cells improved to a greater extent than those receiving SMs alone, however this difference did not reach the level of significance. In the control group a functional decline was noticed at the 8 weeks time point (45.3±7.0%, P=0.05).



View larger version (34K):
[in this window]
[in a new window]
  		Fig. 2. Effects of skeletal myoblast (SM), bone marrow mononuclear cell (BM-MNC) transplantation and of the combined treatment on cardiac function. SMs (n=12), BM-MNCs (n=9), combined SMs and BM-MNCs (n=10) and medium alone (n=7) was injected into ischemic myocardium. EF, LVEDD and IVS were calculated 1 week (baseline) after myocardial infarction and 8 weeks after cell transplantation. Results shown are mean±SD. Percentage changes indicate differences between before and after values (baseline and 8 weeks). Results shown are mean±SEM. *P<0.05, **P<0.01 vs. baseline or control values.

 
As a marker of left ventricular remodelling, the left ventricular end diastolic diameter (LVEDD) was assessed. At baseline, the LVEDD was 6.5±0.8 mm in control rats, 5.2±0.8 mm in the myoblast group, 5.8±1.4 mm in the bone marrow group and 6.3±0.8 mm in the group receiving a combination of myoblasts and bone marrow stem cells (P=0.112). Eight weeks after cell transplantation LVEDD was significantly increased in all animal groups (control group: 7.7±0.6 mm, P=0.001; myoblast group: 7.7±1.5 mm, P<0.001; and bone marrow group: 7.7±1.1 mm, P=0.005) except in the group that received a combination of myoblasts and bone marrow stem cells (6.6±1.7 mm, P=0.397; Fig. 2B). Left ventricular end diastolic volume LVEDV was 0.94±0.27 in the control group, 0.57±0.26 in the myoblast group, 0.41±0.24 in the bone marrow group and 0.63±0.26 in the combined treatment group (P=0.148). At follow-up, LVEDV was significantly increased in the control group (1.74±0.30, P=0.003), the myoblast-treated group (1.61±0.65, P=0.04) and the bone marrow group (1.80±0.98, P=0.008), but not in the combined treatment group (1.02±0.63, P=249). Diastolic and systolic ventricular septal thicknesses were quantified to measure compensatory ventricular hypertrophy and were found to be 0.7±0.1 and 0.9±0.2 mm in control rats, 0.8±0.3 and 0.9±0.3 mm in the myoblast group, 0.7±0.2 and 0.9±0.1 mm in the bone marrow group and 0.9±0.3 and 1.0±0.2 mm in the group receiving a combination of myoblasts and bone marrow stem cells at baseline (P=0.710 and 0.105, respectively). At the 8 weeks time point a significant increase of diastolic and systolic ventricular septal thickness was found in the control group (1.2±0.2 mm, P=0.034 and 1.3±0.1 mm, P=0.006), the bone marrow group (1.1±0.2 mm, P=0.004 and 1.4±0.3 mm, P=0.012) and in a more decent form also in the myoblast group (1.1±0.3 mm, P=0.019 and 1.2±0.3 mm, P=0.064). In contrast, the group treated with a combination of myoblast and bone marrow stem cells showed no significant change in septal thicknesses (1.0±0.2 mm, P=0.452 and 1.1±0.2 mm, P=0.566; Fig. 2C).

3.2. Histology and immune histochemistry
Representative histological slides of transplanted cell depots in the infarcted region of the left ventricular wall 8 weeks after cell transplantation are shown in Fig. 3 . SMs formed multinucleated myotubes in both myoblast-treated groups (Fig. 3A and C). These myotubes had aligned with the fibre axis within the host myocardium. Examination of random areas on light microscopy indicated that the diameter of myoblast grafts was larger in the combined treatment group than in the group that received only myoblasts (0.239±0.158 vs. 0.090±0.050 mm2, P<0.001). In the bone marrow group, one part of the transplanted mononuclear cells scattered over the infarcted area of the left ventricle, whereas the second fraction remained at the implantation site (Fig. 3B). The formation of homogenous grafts as in myoblast-treated animals was not seen in the bone marrow group. In the combined treatment group myotubes and scattered BM-MNCs were found (Fig. 3C).



View larger version (122K):
[in this window]
[in a new window]
  		Fig. 3. Grafted cells 8 weeks after transplantation. Haematoxylin–eosin stainings are shown on the left, the corresponding fluorescent images on the right. SMs and BM-MNCs were prelabelled with red and green fluorescence. In the myoblast group (A) surviving SM differentiated into multinucleated myotubes that had aligned with the cardiac fibre axis in the host myocardium. In the bone marrow group (B) implanted BM-MNCs were found to be partly scattered over the infarcted area and partly staying at the implantation site. In the combined treatment group (C) myotubes and scattered BM-MNCs were found.

 
Tissue sections were stained for anti-factor VIII to detect endothelial cells (Fig. 4) . Enhanced angiogenesis with a large number of mature capillaries was found in the area surrounding grafted SMs of the myoblast group (Fig. 4A; 41.5±14.0; P<0.001 vs. control group; P=0.011 vs. bone marrow group) and the combined treatment group (Fig. 3C; 41.7±7.9; P<0.001 vs. control group; P=0.19 vs. bone marrow group). In rats that were only treated with bone marrow stem cells fewer capillaries were counted (Fig. 4B; 26.3±11.7; P=0.042 vs. control group), the control group showed no obvious angiogenesis (Fig. 3D; 13.2±5.3). Comparison of fluorescence slides to corresponding sections that have been stained for anti-factor VIII revealed that BM-MNCs partly corresponded to factor VIII-positive cells (36.38±10.28% of fluorescent cells corresponded to VIII positive cells).



View larger version (108K):
[in this window]
[in a new window]
  		Fig. 4. Effects of SMs and BM-MNCs on neovascularisation. Endothelial cells were stained with anti-factor VIII antibody, and vessel numbers were counted. Total numbers of vessels in 10 areas were evaluated in each animal (SM, n=12; BM-MNCs, n=9; combined SM and BM-MNC, n=10; controls, n=7) as described. Results shown are mean±SEM. *P<0.05, **P<0.01.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A. Conference...
 References
 
SMs as well as bone marrow-derived mononuclear stem cells are promising candidates for cell therapy of the injured heart. Both cell types have been successfully transplanted to infarcted myocardium [2,7–10]. BM-MNCs and SMs have been shown to undergo adaptive changes induced by their host environment and have proven the potential to improve left ventricular function and postinfarction remodelling [6–8,16–18]. In recent trials, both cell types have already reached experimental clinical application [12,13,20,21]. However, the mechanisms underlying the positive effects on myocardial performance seem to be as different as the phenotypes of these cell lines. With the engraftment process, SMs are found to differentiate into multinucleated myotubes, providing mechanical support as fatigue-resistant, slow twitch fibres [2,8]. BM-MNCs containing various kinds of cell lineages, are regarded to regenerate both cardiomyocytes and endothelial cells, adding contractile function and raising myocardial blood flow [17,19]. The present preclinical study in rats was designed to test the therapeutic effectiveness of a combined transplantation of SMs and BM-MNCs compared to the isolated application of these cell types.

The main findings of this study include that (1) the isolated transplantation of SMs is more effective than the isolated transplantation of BM-MNCs in regard to improving left ventricular function; (2) but not regarding the postinfarction remodelling; (3) the combined transplantation of SMs and BM-MNCs is more effective than the isolated transplantation of these cell types in preventing postinfarction left ventricular dilatation and compensatory left ventricular hypertrophy; (4) the combined transplantation of SMs and BM-MNCs as well as the isolated transplantation of SMs are more effective in inducing angiogenesis than the isolated transplantation of BM-MNCs; (5) in combination with BM-MNCs half of the number of transplanted SMs was as effective in restoring left ventricular function and inducing angiogenesis as in isolated transplantation of SMs (5x106 vs. 107); (6) the engraftment of SMs is more effective in combined transplantation of SMs and BM-MNCs compared to the isolated transplantation of SMs. Thus this study demonstrates that a combination of SMs and BM-MNCs is superior to the isolated transplantation of these cell types. Considering the fact that BM-MNCs cause a dramatically favourable effect on regional blood flow [17], it is conceivable that myoblast differentiation and engraftment is improved by a combined treatment. The increase in tissue perfusion and salvage may be one mechanism underlying the clear benefit of BM-MNCs on the postinfarction remodelling if combined with SMs. In this study, isolated BM-MNC transplantation lead to an increased angiogenesis compared to the control group and incorporated into capillary vessel walls. However, BM-MNCs were not as effective as isolated and combined SMs in inducing angiogenesis. Native myoblasts are known to secrete vascular endothelial growth factor (VEGF) and to hold the potential to induce angiogenesis and to increase coronary arterial blood flow [25]. As a second potential factor underlying the beneficial effect of BM-MNC transplantation, the broad spectrum of inflammatory cytokines secreted by BM-MNCs such as IL-1ß and TNF-{alpha} may increase myoblast differentiation and direct the migration within the infarcted area [17,22]. The finding that the transplantation of isolated BM-MNC without myoblasts had only little effect on the contractile function is likely due to the fact that the pluripotent bone marrow cells able to transdifferentiate into contractile cells comprise only a small fraction of the total bone marrow pool [19]. Prior enrichment of stem cells by means of FACS-sorting or magnetic beads before cell transfer might improve the results in terms of a significant improvement of the contractile function, as far higher concentrations of pluripotent stem/progenitor cells within the microenvironment of the infarcted region become achievable.

In conclusion, the present data show that the concept of combining SMs with BMCs may be of clinical relevance by merging the beneficial effects of each cell line and potentially reducing the required cell quantity. Further studies are required to identify the exact mechanisms underlying this synergy and to allow full exploitation of its therapeutic potential.


   Acknowledgments
 
We thank S. Mueller, Department of Cardiology, University of Innsbruck for diligent and expert assistance in the performance of the echocardiograms and R. Kroess for assistance with surgical procedures, histology and immune histochemistry. This work was supported by FWF grants P12828 (S.H.) and P15527 (S.H.).


   Footnotes
 
Presented at the Joint 17th Annual Meeting of the European Association for Cardio-thoracic Surgery and the 11th Annual Meeting of the European Society of Thoracic Surgeons, Vienna, Austria, October 12–15, 2003.


   Appendix A. Conference discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
References
 
Dr B. Walpoth (Geneva, Switzerland): Why did you choose 7 days after the infarction? And how long did you have your rat survive to show these results?

Some people think that the skeletal myoblasts you need enormous numbers in order to have them survive, and what is the relation between the numbers you injected and the numbers you more or less found on the histology?

Dr Ott: Well, to the first of your questions, we chose this timeframe (cell injection 7 days after infarction) to be in line with other protocols that have been performed earlier, in order to obtain comparable data.

To answer the second question, we did not quantify the surviving myocytes; however, we had the impression that the myocyte survival was significantly higher in the combined treatment group. To quantify the statistical relevance of this finding way was not possible in this case.

Our working hypothesis was that the addition of bone marrow stem cells would increase the survival of myoblasts, as we know, that graft survival is very low. I think that this is what happened in this study.

Dr Walpoth: So you think it's probably mediator-induced due to the transplantation of the bone marrow. That probably is the mechanism.

Dr Ott: I think so. Also because in the combined treatment group, only the half number of myoblasts was transplanted.

Dr Walpoth: And how long did they survive?

Dr Ott: We sacrificed animals 9 weeks after myocardial infarction.

Dr R. Tam (Brisbane, Australia): Do you think the increase in ventricular function is due to the increase in angiogenesis rather than the survival of the transplanted cells?

Dr Ott: As we saw, the increase in left ventricular function was found in skeletal myoblast-treated animals and even more in the combined treatment group. I do think that the increase in left ventricular function in the combined treatment group is more decent because more myoblasts had survived.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Conference...
 References
 

  1. Pfeffer J.M., Pfeffer M.A., Fletcher P.F., Braunwald E. Progressive ventricular remodelling in rat with myocardial infarction. Am J Physiol 1991;260:1406-1414.
  2. Taylor D.A., Atkins B.Z., Hungspreugs P., Jones T.R., Reedy M.C., Hutcheson K.A., Glower D.D., Kraus W.E. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat Med 1998;4(8):929-933.[CrossRef][Medline]
  3. Koh G.Y., Soonpa M.H., Klug M.G., Field L.J. Long-term survival of AT-1 cardiomyocyte grafts in syngeneic myocardium. Am J Physiol 1993;264:1727-1733.
  4. Li R.K., Jia Z.Q., Weisel R.D., Merante F., Mickle D.A. Smooth muscle cell transplantation into myocardial scar tissue improves heart function. J Mol Cell Cardiol 1999;31:513-522.[CrossRef][Medline]
  5. Hutcheson K.A., Atkins B.Z., Hueman M.T., Hopkins M.B., Glover D.D., Taylor D.A. Comparison of benefits on myocardial performance of cellular cardiomyoplasty with skeletal myoblasts and fibroblasts. Cell Transplant 2000;9:359-368.[Medline]
  6. Kocher A.A., Schuster M.D., Szabolcs M.J., Takuma S., Burkhoff D., Wang J., Homma S., Edwards N.M., Itescu S. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis. Reduces remodelling and improves cardiac function. Nat Med 2001;7:430-436.[CrossRef][Medline]
  7. Kessler P.D., Byrne B.J. Myoblast cell grafting into heart muscle: cellular biology and potential applications. Annu Rev Physiol 1999;61:219-242.[CrossRef][Medline]
  8. Murry C.E., Wiseman R.W., Schwartz S.M., Hauschka S.D. Skeletal myoblast transplantation for repair of myocardial necrosis. J Clin Invest 1996;98:2512-2523.[Medline]
  9. Robinson S.W., Cho P.W., Levitsky H.I., Olson J.L., Hruban R.H., Acker M.A., Kessler P.D. Arterial delivery of genetically labelled skeletal myoblasts to the murine heart: long term survival and phenotypic modification of implanted myoblasts. Cell Transplant 1996;5:77-91.[Medline]
  10. Menasche P. Cell transplantation for the treatment of heart failure. Semin Thorac Cardiovasc Surg 2002;14:157-166.[CrossRef][Medline]
  11. Menasché P., Hagege A.A., Scorsin M., Pouzet B., Desnos M., Duboc D., Schwartz K., Vilquin J.T., Marolleau J.P. Myoblast transplantation for heart failure. Lancet 2001;357:279-280.[CrossRef][Medline]
  12. Menasche P., Hagege A.A., Vilquin J.T., Desnos M., Abergel E., Pouzet B., Bel A., Sarateanu S., Scorsin M., Schwartz K., Bruneval P., Benbunan M., Marolleau J.P., Duboc D. Autologous skeletal myoblast transplantation for severe postinfarction left ventricular dysfunction. J Am Coll Cardiol 2003;41(7):1078-1083.[Abstract/Free Full Text]
  13. Pagani F.D., DerSimonian H., Zawadzka A., Wetzel K., Edge A.S., Jacoby D.B., Dinsmore J.H., Wright S., Aretz T.H., Eisen H.J., Aaronson K.D. Autologous skeletal myoblasts transplanted to ischemia-damaged myocardium in humans. Histological analysis of cell survival and differentiation. J Am Coll Cardiol 2003;41(5):879-888.[Abstract/Free Full Text]
  14. Orlic D., Kajstura J., Chimenti S., Bodine D.M., Leri A., Anversa P. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701-705.[CrossRef][Medline]
  15. Kawamoto A., Gwon H.C., Iwaguro H., Yamaguchi J.I., Uchida S., Masuda H., Silver M., Ma H., Kearney M., Isner J.M., Asahara T. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001;103:634-637.[Abstract/Free Full Text]
  16. Tomita S., Li R.K., Weisel R.D., Mickle D.A., Kim E.J., Sakai T., Jia Z.Q. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 1999;100:II247-II256.
  17. Kamihata H., Matsubara H., Nishiue T., Fujiyama S., Tsutsumi Y., Ozono R., Masaki H., Mori Y., Iba O., Tateishi E., Kosaki A., Shintani S., Murohara T., Imaizumi T., Iwasaka T. Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines. Circulation 2001;104:1046-1052.[Abstract/Free Full Text]
  18. Shake J.G., Gruber P.J., Baumgartner W.A., Senechal G., Meyers J., Redmond J.M., Pittenger M.F., Martin B.J. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg 2002;73:1919-1925.[Abstract/Free Full Text]
  19. Agbulut O., Menot M.L., Zhelin L., Marotte F., Paulin D., Hagege A.A., Chomienne C., Samuel J.L., Menasche P. Temporal patterns of bone marrow cell differentiation following transplantation in doxorubicin-induced cardiomyopathy. Cardiovasc Res 2003;58:451-459.[Abstract/Free Full Text]
  20. Perin E.C., Dohmann H.F., Borojevic R., Silva S.A., Sousa A.L., Mesquita C.T., Rossi M.I., Carvalho A.C., Dutra H.S., Dohmann H.J., Silva G.V., Belem L., Vivacqua R., Rangel F.O., Esporcatte R., Geng Y.J., Vaughn W.K., Assad J.A., Mesquita E.T., Willerson J.T. Transendocardial, autologous bone marrow cell transplantation for severe, chronic ischemic heart failure. Circulation 2003;107:2294-2302.[Abstract/Free Full Text]
  21. Tse H.F., Kwong Y.L., Chan J.K., Lo G., Ho C.L., Lau C.P. Angiogenesis in ischaemic myocardium by intramyocardial autologous bone marrow mononuclear cell implantation. Lancet 2003;361(9351):47-49.[CrossRef][Medline]
  22. Torrente Y., El Fahime E., Caron N.J., Del Bo R., Belicchi M., Pisati F., Tremblay J.P., Bresolin N. Tumor necrosis factor-alpha (TNF-alpha) stimulates chemotactic response in mouse myogenic cells. Cell Transplant 2003;12(1):91-100.[Medline]
  23. Morel F., Szilvassy S.J., Travis M., Chen B., Galy A. Primitive hematopoietic cells in murine bone marrow express the CD34 antigen. Blood 1996;88(10):3774-3784.[Abstract/Free Full Text]
  24. Peichev M., Naiyer A.J., Pereira D., Zhu Z., Lane W.J., Williams M., Oz M.C., Hicklin D.J., Witte L., Moore M.A., Rafii S. Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. Blood 2000;95(3):952-958.[Abstract/Free Full Text]
  25. Suzuki K., Murtuza B., Smolenski R.T., Sammut I.A., Suzuki N., Kaneda Y., Yacoub M.H. Cell transplantation for the treatment of acute myocardial infarction using vascular endothelial growth factor-expressing skeletal myoblasts. Circulation 2001;104(12 Suppl 1):I207-I212.



This article has been cited by other articles:


Home page
 ICVTSHome page
N. Bonaros, R. Rauf, E. Werner, B. Schlechta, E. Rohde, A. Kocher, J. Bonatti, and G. Laufer
Neoangiogenesis after combined transplantation of skeletal myoblasts and angiopoietic progenitors leads to increased cell engraftment and lower apoptosis rates in ischemic heart failure
Interactive CardioVascular and Thoracic Surgery, April 1, 2008; 7(2): 249 - 255.
[Abstract] [Full Text] [PDF]

Home page
 ICVTSHome page
S. Chanseaume, K. Azarnoush, A. Maurel, V. Bellamy, S. Peyrard, P. Bruneval, A. A. Hagege, and P. Menasche
Can erythropoietin improve skeletal myoblast engraftment in infarcted myocardium?
Interactive CardioVascular and Thoracic Surgery, June 1, 2007; 6(3): 293 - 297.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
A. D. Moelker, T. Baks, E.J. van den Bos, R.J. van Geuns, P.J. de Feyter, D. J. Duncker, and W.J. van der Giessen
Reduction in infarct size, but no functional improvement after bone marrow cell administration in a porcine model of reperfused myocardial infarction
Eur. Heart J., December 2, 2006; 27(24): 3057 - 3064.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
N. Bonaros, R. Rauf, D. Wolf, E. Margreiter, A. Tzankov, B. Schlechta, A. Kocher, H. Ott, T. Schachner, S. Hering, et al.
Combined transplantation of skeletal myoblasts and angiopoietic progenitor cells reduces infarct size and apoptosis and improves cardiac function in chronic ischemic heart failure
J. Thorac. Cardiovasc. Surg., December 1, 2006; 132(6): 1321 - 1328.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart J SupplHome page
P. Menasche, M. Desnos, and A. A. Hagege
Myoblast transplantation during cardiac surgery
Eur. Heart J. Suppl., December 1, 2006; 8(suppl_H): H52 - H56.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
H. C. Ott, J. Brechtken, C. Swingen, T. M. Feldberg, T. S. Matthiesen, S. A. Barnes, W. Nelson, and D. A. Taylor
Robotic minimally invasive cell transplantation for heart failure
J. Thorac. Cardiovasc. Surg., July 1, 2006; 132(1): 170 - 173.
[Full Text] [PDF]

Home page
 Exp Biol MedHome page
L. Ye, H. K. Haider, and E. K. W. Sim
Adult Stem Cells for Cardiac Repair: A Choice Between Skeletal Myoblasts and Bone Marrow Stem Cells
Exp Biol Med, January 1, 2006; 231(1): 8 - 19.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
I. Elmadbouh, Y. Chen, L. Louedec, S. Silberman, B. Pouzet, O. Meilhac, and J.-B. Michel
Mesothelial cell transplantation in the infarct scar induces neovascularization and improves heart function
Cardiovasc Res, November 1, 2005; 68(2): 307 - 317.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
I. A. Memon, Y. Sawa, S. Miyagawa, S. Taketani, and H. Matsuda
Combined autologous cellular cardiomyoplasty with skeletal myoblasts and bone marrow cells in canine hearts for ischemic cardiomyopathy
J. Thorac. Cardiovasc. Surg., September 1, 2005; 130(3): 646 - 653.
[Abstract] [Full Text] [PDF]

Home page
 Asian Cardiovasc. Thorac. Ann.Home page
J. C Chachques, C. Salanson-Lajos, P. Lajos, A. Shafy, A. Alshamry, and A. Carpentier
Cellular Cardiomyoplasty for Myocardial Regeneration
Asian Cardiovasc Thorac Ann, September 1, 2005; 13(3): 287 - 296.
[Abstract] [Full Text] [PDF]

Home page
 Eur. J. Cardiothorac. Surg.Home page
M. Siepe, C. Heilmann, P. von Samson, P. Menasche, and F. Beyersdorf
Stem cell research and cell transplantation for myocardial regeneration
Eur. J. Cardiothorac. Surg., August 1, 2005; 28(2): 318 - 324.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
K. Azarnoush, A. Maurel, L. Sebbah, C. Carrion, A. Bissery, C. Mandet, J. Pouly, P. Bruneval, A. A. Hagege, and P. Menasche
Enhancement of the functional benefits of skeletal myoblast transplantation by means of coadministration of hypoxia-inducible factor 1{alpha}
J. Thorac. Cardiovasc. Surg., July 1, 2005; 130(1): 173 - 179.
[Abstract] [Full Text] [PDF]

Home page
 Eur. J. Cardiothorac. Surg.Home page
H. C. Ott, R. Kroess, N. Bonaros, R. Marksteiner, E. Margreiter, T. Schachner, G. Laufer, and S. Hering
Intramyocardial microdepot injection increases the efficacy of skeletal myoblast transplantation
Eur. J. Cardiothorac. Surg., June 1, 2005; 27(6): 1017 - 1021.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Personal Folders
Right arrow Download to citation manager
Right arrow Permission Requests
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ott, H.C.
Right arrow Articles by Hering, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ott, H.C.
Right arrow Articles by Hering, S.
Related Collections
Right arrow Cardiac - other
Right arrow Coronary disease
Right arrow Myocardial infarction

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ANN THORAC SURG ASIAN CARDIOVASC THORAC ANN EUR J CARDIOTHORAC SURG
J THORAC CARDIOVASC SURG ICVTS ALL CTSNet JOURNALS