Copyright © 2006, European Society of Cardiology
Can bone marrow-derived multipotent adult progenitor cells regenerate infarcted myocardium?
aInserm U633, Paris, France, Assistance Publique-Hôpitaux de Paris, Ecole de Chirurgie, Paris, France
bHematology, Cardiology and Cell Therapy, Clínica Universitaria and Division of Cancer, Foundation for Applied Medical Research, Division of Cancer, University of Navarra, Pamplona, Spain
cUniversity Paris 7, Department of Biochemistry, Paris, France
dAssistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiology, University Paris-5, Faculty of Medicine, Paris, France, INSERUM U 633, France
eCRBM, CNRS FRE2593, Montpellier, France
fInserm and Clinical Investigation Center, Hôpital Européen Georges Pompidou, Paris, France
gAssistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Pathology, University of Paris 5, Faculty of Medicine, Paris, France, INSERUM U 430, France
hInserm U689, Paris, France
iInstituto Cavanilles, University of Valencia, Valencia, Spain
jAssistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery, University of Paris-5, Faculty of Medicine, INSERUM U633, Paris, France
* Correspondings author. Prósper is to be contacted at Hematology and Cell Therapy, Clinica Universitaria, Av Pio XII 36, Pamplona 31008, Spain. Tel.: +34 948 255 400. Agbulut, Inserm U633, Laboratoire d'étude des greffes et prothèses cardiaques, 96, rue Didot, 75014, Paris, France. Tel.: +33 1 4395 9360; fax: +33 1 4540 5049. Email address: onnik.agbulut{at}larib.inserm.fr fprosper{at}unav.es
Received 12 January 2006; revised 14 July 2006; accepted 14 July 2006
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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Objectives: To assess the functional effects of multipotent adult progenitor cells (MAPCs) transplanted in a rat model of chronic myocardial infarction.
Methods: Forty-four rats underwent coronary ligation and, 14 days later, were randomly allocated to receive in-scar injections (5x106 cells/150 µL) of green fluorescent protein (eGFP)-transduced allogeneic MAPCs (n=25) or culture medium (controls, n=19). Nine of the MAPC-treated hearts were employed for functional studies while the remaining 16 received cells co-labeled with ResovistTM and were only used for serial histological assessments. Left ventricular (LV) function was assessed echocardiographically before transplantation and 1 month thereafter in a blinded manner. Immunohistochemistry, electron microscopy and PCR were used to detect grafted cells. All data were compared by nonparametric tests.
Results: Baseline ejection fractions (EF, median;[interquartile range]) did not differ significantly among the groups: 30% [0.23;0.37] and 37% [0.32;0.38] in control and rMAPC-transplanted hearts, respectively. One month later, LV function of control hearts was found to have deteriorated, as reflected by a decline in EF to 24% [0.21;0.30], and although EF tended to remain more stable after cell transplantation (37% [0.27;0.41]), the difference between the two groups failed to achieve statistical significance (p=0.06). While MAPCs could be identified early post-transplant, no evidence of engraftment was further observed at 1 month by immunohistochemistry, electron microscopy or PCR.
Conclusions: In this model, MAPCs did not improve global pump function, and although some of these cells expressed endothelial markers during the early post-transplant period, we could not detect any evidence for differentiation into cardiomyocytes and no engraftment was further identified beyond 2 weeks after cell injections.
KEYWORDS Progenitor cells; Bone marrow; Transplantation; Myocardial infarction; MAPCs
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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Over the past decade, cell therapy has emerged as a potentially new means of repairing infarcted myocardium and both skeletal myoblasts [1–4] and bone marrow cells [5] are currently undergoing clinical trials. However, while there is compelling evidence that these two cell types can limit post-infarction remodeling and increase angiogenesis, there is no convincing evidence that they can transdifferentiate into cardiomyocytes [6–8]; hence, there is doubt that they will be capable of truly regenerating infarcted myocardial tissue. In this context, the characterization, in 2001, of a bone marrow-derived subpopulation of adult cells credited for a multilineage differentiation potential [9–13] and called multipotent adult progenitor cells (MAPCs) has raised the hope that these cells could be therapeutically used for regenerative purposes. While in vitro studies have clearly documented the ability of MAPCs to differentiate into cells of all three germ layers, little is known about their phenotypic fate following transplantation in infarcted myocardium. In addition, although MAPCs, when injected in the blastocyst, differentiate into cardiac cells contributing to the development of the heart [10], there is no published evidence that MAPCs differentiate into cardiomyocytes in vivo. All these unsettled issues prompted us to undertake the present study designed to examine the potential of MAPCs to contribute to cardiac regeneration in a model of chronic myocardial infarction.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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2.1. Experimental model
Ligation of the left coronary artery was performed in 8-week-old Sprague–Dawley female rats through a left thoracotomy (Harlan-France) [14]. Fourteen days later, rats were reoperated on by sternotomy and randomly allocated to receive an average of 3 in-scar injections (total volume of 150 µL), one in the core and two at the borders of the infarct region, of rat(r) MAPCs (5x106, n=25) or culture medium (n=19). Post-thawing viability, assessed by FACS using propidium-iodide exclusion, was consistently >87%. All rats were treated with cyclosporin A (10 mg/kg/d, i.p., Novartis) 2 days before transplantation and daily until sacrifice. All experiments were performed in accordance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources, Commission on Life Science, National Research Council, and published by the National Academy Press, revised 1996. All animal procedures were approved by the University of Navarra Institutional Committee on Care and Use of Laboratory Animals.
2.2. Isolation, preparation and characterization of grafted rMAPCs
rMAPCs were obtained from adult Sprague–Dawley bone marrow. Cells were isolated and characterized as previously reported [9–13] with minor modifications. Briefly, bone marrow-derived mononuclear cells were plated at a concentration of 1x106 cells/cm2 in expansion medium supplemented with 10 ng/mL of epidermal growth factor (Sigma), 10 ng/mL of platelet derived growth factor (R and D Systems) and 10 ng/mL leukaemia inhibitory factor in wells coated with 10 ng/mL fibronectin (Sigma). Expansion medium consisted of 58% low-glucose DMEM (Gibco BRL), 40% MCDB-201 (Sigma), 2% fetal calf serum (Biochrom), 1X insulin transferrin selenium, 1X linoleic acid bovine serum albumin (Sigma), 10–8 M dexamethasone (Sigma), 10–8 M ascorbic acid 2-phosphate (Sigma), 100 U penicillin, and 1000 U streptomycin (Gibco). Media was changed every 3–4 days and cultures maintained until clusters of adherent cells were more than 50% confluent. They were detached with 0.25% trypsin-EDTA (BioWitthaker) and replated at a concentration of 5x102/cm2 under the same culture conditions. Cells were maintained at the same confluence throughout the life of the culture. rMAPCs were characterized by FACS after staining with monoclonal antibodies against CD44, MHC Class I, SSEA1, SSEA4, CD45, CD90, CD73, CD106, CD31 and CD49b. rMAPCs were transduced with a third-generation lentivirus vector expressing eGFP cDNA using a MOI of 50 in the presence of polybrene (4 µg/mL). Transduction of the cells was performed after 50 population doublings. More than 50% of MAPCs were eGFP positive, as determined by flow cytometry.
In a separate group, rats were transplanted with eGFP-rMAPCs labeled with ferucarbutran (ResovistTM, Schering AG, Germany) at a concentration of 25 µg iron/mL for 24 h as described [15,16]. Efficiency of labeling with superparamagnetic iron oxide particles was greater that 98%. All cells were kept frozen until implantation.
2.3. Functional assessment
Immediately before and 1 month after transplantation, LV function was studied by 2D echocardiography in isoflurane-anesthetized animals. Transthoracic 2D echocardiography was performed with the use of a commercially available 13- to 15-MHz linear-array probe (15L8) specially designed for cardiac ultrasonic studies in rat models and connected to a numeric ultrasound device (Sequoia 512®, Acuson Corp, Mountain View, CA, USA). The animals were placed in the supine or lateral position on a warming pad. Parasternal 2D echocardiographic views of the heart were then obtained with the following machine settings: space time T1, contour 3, and delta 5. End-diastolic and end-systolic long-axis views of the LV were standardized as follows: inclusion of the apex, the posterior papillary muscle, the mitral valve, and the aortic root. Two-dimensional echocardiographic measurements were performed with the cine-loop feature to retrospectively obtain adequate visualization of these fast-beating hearts. This device allows a 160-Hz maximal frame rate with high axial and lateral resolutions. Numeric images were then reviewed at a slower frame rate to catch true end-diastolic and end-systolic phases. End-diastolic and end-systolic areas (A) were obtained by hand-tracings of the LV endocardial contours on the frame showing the largest (and the smallest) LV cavity size by using the cine-loop acquisition, according to the American Society of Echocardiography leading edge method. On these frames, end-diastolic (or end-systolic) lengths (L) of the LV were obtained by tracing a line connecting the more distal part of the apex and the center of a line connecting the mitral annular hinge points. End-diastolic and end-systolic volumes (LVEDVs and LVESVs, respectively) were then calculated by means of the single-plane area-length method (volume=8xA2/3x
xL). Left ventricular ejection fraction (LVEF, %) was calculated as ((LVEDV–LVESV)/LVEDV)x100. All measurements were averaged on 3 consecutive cardiac cycles and analyzed by a single observer who was blinded to the treatment status of the animals. Intraobserver variability in echocardiographic assessment was assessed from 2 sets of baseline measurements in 10 randomly selected rats with the use of Bland and Altman analysis: Measurements were very close for linear (r>0.95, SEE<0.1 mm), volume (r>0.93, SEE<0.08 mL), and EF (r>0.95, SEE=4%) measurements. Only rats with infarct size of such magnitude that LVEF was below 40% at baseline were included in the study so as to maximize possible treatment effects.
2.4. Histological and immunohistochemical assessment
Explanted hearts were cut into 3 apical, mid-ventricular and basal blocks. Blocks were either frozen in liquid nitrogen-cooled isopentane or paraffin-embedded. Twenty-five heart transverse 7-µm slides, each containing 3 cryosections, were prepared from each frozen block (10 from the apical and mid-ventricular blocks, 5 from the most basal one) for histological (hematoxylin and eosin) and immunohistological assessment. Paraffin-embedded blocks were cut in 3 µm sections, deparaffinized and stained with the appropriate antibodies. The detection of transplanted cells was primarily based on the presence of eGFP positive signals (1:400, rabbit polyclonal [pAb], Interchim.; 1:300 pAb Molecular Probes) while the presence of iron oxide particles in the cells was detected by Prussian blue staining as described [16]. Thereafter, immunolabeling was performed with antibodies directed against fast myosin-heavy chain (MyHC) (1:10, mouse monoclonal antibody [mAb], Novo-Castra), myosin-binding protein-C (1:200, pAb, provided by L. Carrier, Inserm U582, France), caveolin-1
(1:50, pAb, Santa-Cruz), smooth muscle actin (undiluted, mAb, Dako), connexin 43 (1:200, mAb, Sigma), calponin (1:200, mAb, Sigma), tropomyosin (1:100, mAb, Sigma) and BSL-1 lectin (10 µg/mL, Sigma).
To quantify capillary density, 9 sections per heart were randomly selected in the border zone and stained with an anti-caveolin-1 antibody. Arteriolar density was quantified after staining with antibodies against SM-actin. A total of 9 sections per heart were randomly selected and the number of SM-actin-positive arterioles counted. Images were processed by a DMRB Leica microscope equipped with epifluorescence optics. Digital images were analyzed using Vision explorer software (Graftek Imaging). Data are expressed as the number of capillaries or arterioles per mm2.
2.5. Ultrastructural analysis
For electron microscopy studies, the heart was fixed with 2% glutaraldehyde. Sections were post-fixed with 0.5% osmium, rinsed, dehydrated and embedded in araldite (Durcupan, Fluka). Semi-thin sections (1.5 µm) were cut with a diamond knife and stained lightly with 1% toluidine blue. Semi-thin sections were re-embedded in an araldite block and detached from the glass slide by repeated freezing (liquid nitrogen) and thawing. The block with semi-thin sections was cut in ultra-thin (0.05 µm) sections with a diamond knife, stained with lead citrate and examined under a Jeol 100CX electron microscope.
2.6. PCR
DNA was extracted from the whole heart (n=9 for each group) by proteinase K digestion and phenol extraction. PCR was performed using an eGFP primer (upstream-primer-5'-TAC GGC AAG CTG ACC CTG AA-3'; downstream-primer-5'-ATG CCG TTC TTC TGC TTG TC-3'). Amplification conditions were as follows: 95 °C for 3 min followed by 30 cycles consisting of 90 °C–60 s, 62 °C–60 s, 72 °C–60 s, followed by an extension at 72 °C for 10 min. All samples were also amplified to detect the GAPDH gene as a control for the presence of amplifiable DNA.
2.7. Statistics
Because of the small sample sizes, it could not be assumed that the data would fit a normal distribution and, consequently, data were compared by nonparametric Wilcoxon tests. Results are thus expressed as medians and confidence intervals. A p value<0.05 was considered significant (Table 1).
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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Among the 44 rats available for the study, 20 (eGFP-transduced rMAPCs: 9; controls: 11) underwent pre-and post-transplantation functional studies while the remaining 24 (eGFP-transduced iron-labeled rMAPCs: 16; controls: 8) were exclusively used for serially tracking the fate of the grafted cells by immunohistochemistry and electron microscopy (at 48 h, 1 week, 2 weeks and 4 weeks after transplantation; for each time point: 4 rMAPCs-treated hearts and 2 controls).
3.1. Characterization of rMAPC
rMAPCs were characterized after 50 population doublings by flow cytometry, RT-PCR and in vitro differentiation (Fig. 1). Animals were transplanted with CD44 low and MHC class I-negative, CD45 negative and CD90 positive cytogenetically normal rMAPCs derived from the same bone marrow. rMAPCs were shown to differentiate in vitro into cells expressing phenotypic markers of mesoderm-(endothelial and skeletal muscle cells), endoderm-(hepatocyte) and ectoderm-(neuron) derived tissues (Fig. 1).
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3.2. Functional assessment
Baseline EFs did not differ significantly between the 2 groups (controls: 30% [0.23;0.37]; rMAPC: 37% [0.32;0.38], p=0.15), thereby suggesting initial ischemic injuries of similar extent. One month later, EF had declined in the control group to 24% [0.21;0.30] whereas it tended to be better preserved following rMAPC transplantation (37% [0.27;0.41]). However, the difference between the two groups failed to reach the threshold of statistical significance (p=0.06). Individual values are depicted in Fig. 2.
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3.3. Phenotypical characterization of transplanted cells
In the hearts transplanted with rMAPCs, grafted cells were detected at 48 h, 1 week and 2 weeks but could not be detected by either immunofluorescence, immunohistochemistry or PCR using anti-eGFP antibodies and eGFP primers, respectively, 1 month after transplantation (Fig. 3A–D).
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In the set of experiments involving ResovistTM, rMAPCs labeling with ResovistTM at different concentrations did not affect cell proliferation or viability (data not shown). In those animals transplanted with rMAPCs labeled with iron particles, iron positive cells were detected at the different time intervals for up to 1 month post-transplant (Fig. 3E–H). These cells were not stained with antibodies against cardiac-and smooth muscle-specific antigens (not shown). Furthermore, no co-localization between GFP positive cells and smooth muscle actin was detected at any time (not shown). However, 2 weeks after transplantation a number of iron-labeled cells were positive for endothelial markers (BSL-1) (Fig. 4). Interestingly, at 4 weeks, iron loaded cells were positive for the macrophage marker, CD68 whereas no GFP or lectin staining was detected (Fig. 3I–L). Control animals transplanted with a solution containing iron particles in media or with a lysate of iron-labeled rMAPCs did not show positive staining for Perls (data not shown).
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Ultrastructural analysis demonstrated the presence of iron in the lysosomes of the transplanted cells at 48 h, 1 week (Fig. 5A, C) and 2 weeks after transplantation with labeled cells showing initial formation of fibers. Interestingly, accumulation of macrophages loaded with iron particles was detected at 4 weeks after transplantation with disappearance of other types of iron-labeled cells (Fig. 5B, D).
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Angiogenesis did not differ among the 2 groups, 1 month after transplantation, with a median number of capillaries in the border zone of 1754 [1445;1891] and 1700 [1636; 1881] per mm2 in controls and rMAPCs-transplanted hearts, respectively. Also, arteriolar density measured as the number of SM-actin positive vessels per mm2 was also not different between control animals (58.8 [36.76; 88.24]) and rMAPCs-transplanted animals (59.27 [38.38; 114.71]).
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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The major finding of the present study is that, in a rat model of chronic myocardial infarction, transplantation of rMAPCs failed to increase EF beyond that seen in control placebo-injected hearts, which correlated with a lack of sustained engraftment of the injected cells.
The initial in vitro observation that MAPCs differentiated, at the single-cell level, into cells with characteristics of the 3 embryonic layers [10] raised the hope that they could be therapeutically useful for repairing degenerated tissues despite the technical challenges associated with their identification (primarily based on negative surface markers) and the complexity of their culture. However, in the current experiments, we failed to detect GFP-labeled rMAPCs by immunohistochemistry in any heart section 1 month after transplantation even though cell engraftment could be documented during the first 2 weeks post-transplant based on iron labeling of the transplanted cells as well as on GFP tagging. Finding no PCR products for GFP at 1 month after injection suggested that there were no viable rMAPC in the hearts at this time point. In line with these data, proliferation of rMAPCs was not found by PCNA staining (not shown). The observation that transplanted bone marrow cells disappear over time has previously been made in other studies [7,17] and may reflect an intrinsic sensitivity of these cells to scar-associated hypoxia or the lack, in these fibrotic areas, of the signals required for promoting cell survival and/or committing these undifferentiated cue-sensitive cells towards a given lineage. The assumption about an already advanced stage of healing and fibrosis is actually consistent with our finding that apoptosis events that characterize the early post-infarction period were not detected by the TUNEL assay (not shown). The fact that iron-labeled macrophages were readily detected 1 month after transplant also suggests an inflammatory reaction possibly due to graft rejection that might have occurred in spite of cyclosporine. Indeed, recent studies have demonstrated that the lack of HLA class I antigen expression in MAPCs is associated with NK-mediated rejection when transplanted in NOD-SCID mice [18]. In the current experiments, the contrast between the trend for rMAPC-transplanted hearts to prevent a deterioration of EF over time and the absence of their sustained engraftment suggests the possible involvement of paracrine mechanisms whereby cytokines and/or growth factors released by the cells [19] present during the early post-transplantation period would initiate cardioprotective effects that may then remain effective even if the source cells have gone. These effects might involve favorable changes in extracellular matrix composition, in keeping with the recent finding that transplantation of mesenchymal stem cells in infarcted rat myocardium reduces collagen content and mRNA levels of transforming growth factor-β-1 [20]. Along with a possible "girdling" effects of the grafted cells, these biochemical changes of the extracellular matrix could contribute to some mechanical stabilization of the ventricular wall. An effect on angiogenesis or arteriogenesis, as documented for endothelial progenitors [21], is not supported by the present results despite the finding of a small number of iron-labeled cells positive for endothelial markers at different time points after transplant. Whether this phenotype results from differentiation, as it has been described with human MAPCs, or from a fusion event cannot be distinguished by our experiments [12,22]. Finally, the paracrine recruitment of cardiac resident stem cells [23] is another possible mechanism but it remains speculative thus far and is actually not supported by the failure of rMAPC transplantation to significantly improve EF. From this standpoint, a possible confounding role of eGFP can be excluded, as although GFP may impair electromechanical coupling through blockade of actin–myosin bridges by the sarcomeric deposits of the fluorescent protein [24], such a phenomenon is irrelevant to the present experiments where none of the transplanted rMAPCs showed a cardiomyocytic differentiation involving the development of an organized contractile apparatus.
This study has some limitations that need to be acknowledged. First, the follow-up period was limited to 1 month so that no conclusions can be made regarding the maintenance of treatment effects beyond this time point. Second, we used a permanent coronary artery occlusion model that may not reflect contemporary medical practice where most patients with acute myocardial infarction undergo early reperfusion of the culprit vessel. The hypoxic sensitivity of rMAPCs could explain why they did not survive after 2 weeks. Thus, one cannot exclude that their fate would have been different in a revascularized milieu. Third, cells were injected 2 weeks after infarction at a time where healing is in the granulation phase and this environment may not be favorable to graft survival. It is therefore possible that earlier injections could have allowed cells to find more appropriate signals for survival and differentiation. Fourth, immune NK-mediated rejection may account for lack of engraftment, thereby raising the possibility of enhanced cell survival by more appropriate immunosuppressive regimens. Finally, no attempt was made to commit the rMAPCs toward a cardiac lineage prior to implantation; indeed, even pluripotent embryonic stem cells have a limited rate of in vitro spontaneous differentiation into cardiomyocytes, and driving them along this pathway requires different combinations of growth factors and culture conditions [25,26]. Thus, it is likely that pre-differentiation of rMAPCs towards a cardiac phenotype may be necessary to improve engraftment and terminal differentiation into mature cardiomyocytes. Finally, we cannot exclude that the small sample sizes may have led us to miss significant differences in post-transplantation EFs which actually approached the .05 threshold.
In spite of these limitations, the present data allow us to reasonably conclude that the multi-lineage differentiation potential demonstrated in vitro by MAPCs does not result in an in vivo long term cardiac engraftment and differentiation when using undifferentiated rMAPCs transplanted in a post-infarction scar. Although this finding was paralleled by the lack of significant improvement in LVEF, additional studies appear warranted to determine whether changes in MAPC processing, their local microenvironment or the timing of their delivery could optimise the use of these cells and lead to a potential therapeutic benefit.
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Acknowledgments |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionAcknowledgmentsReferences |
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This work was supported by Inserm, la Fondation de France and the Government of Navarra, Spanish Ministerio de Ciencia y Tecnología (SAF 2002-04574-C02), FEDER (INTERREG IIIA) and the "UTE project CIMA". CB had a fellowship from "la Fondation pour la Recherche Médicale" during the course of this study. We thank the Production and Control Departement of Genethon which is supported by the Association Française contre les Myopathies (AFM), in the frame of the GVPN network (http://www.gvpn.org) for providing us lentiviral vectors.
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Notes |
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1 OA and MM contribute equally to this manuscript.
Time for primary review 26 days
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