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European Journal of Heart Failure 2008 10(11):1065-1072; doi:10.1016/j.ejheart.2008.08.002
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© 2008 European Society of Cardiology

Characterization of the paracrine effects of human skeletal myoblasts transplanted in infarcted myocardium

Maitane Perez-Ilzarbea, Onnik Agbulutb, Beatriz Pelachoa, Cristina Ciorbaa, Edurne San Jose-Eneriza, Michel Desnosc, Albert A. Hagèged, Pablo Arandaa, Enrique J. Andreua, Philippe Menaschéb and Felipe Próspera,*

a Hematology, Cardiology and Cell Therapy, Clinica Universitaria and Division of Cancer, Foundation for Applied Medical Research, Division of Cancer, University of Navarra Pamplona, Spain
b University Paris 7, Department of Biochemistry EA 300, Paris, France
c AP-HP, Hôpital Européen Georges Pompidou, Department of Cardiology; University Paris Descartes, Faculty of Medicine INSERM U 633; Paris, France
d AP-HP, Hôpital Européen Georges Pompidou, Department of Cardiovascular Surgery; University Paris Descartes, Faculty of Medicine INSERM U 633; Paris, France

* Corresponding author. Hematology and Cell Therapy, Clinica Universitaria, Av Pio XII 36, Pamplona 31008, Spain. Tel.: +34 948 255 400. E-mail address: fprosper{at}unav.es (F. Prósper)


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 Appendix A. Supplementary data
 References
 
Background: The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms.

Methods and results: We analyzed gene expression and growth factors released by undifferentiated human SM (CD56+), myotubes (SM cultured until confluence) and fibroblasts-like cells (CD56–). Gene expression revealed up-regulation of pro-angiogenic (PGF), antiapoptotics (BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2, MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and cardiomyocytes (p<0.05) and a decrease in apoptosis of cardiomyocytes (p<0.05). Analysis of nude rats transplanted with human SM demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-β, and IGF-1 1 month after transplant.

Conclusions: Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of myogenic cell transplantation in infarcted myocardium.

Key Words: Skeletal myoblast • Paracrine • Cell therapy • Myocardial infarction

Received March 17, 2008; Revised June 26, 2008; Accepted August 18, 2008


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 Appendix A. Supplementary data
 References
 
Although cell therapy has emerged as an attractive new means of repairing infarcted myocardium [1-5] none of the currently investigated adult stem cell sources have convincingly demonstrated a potential for significant long-term engraftment and differentiation into functional cardiomyocytes [6-8]. Nevertheless, in the specific case of skeletal myoblasts (SM), the results of several preclinical and clinical studies have shown improved functional outcomes following transplantation of these myogenic progenitors into post-infarction scars [9-11] (review in Ref. [12]). As these benefits were usually observed when only a few cells were still present in the engrafted areas, the hypothesis has been raised that the improvement in cardiac function did not result from an increase in the number of contractile cells intended to replace those lost after damage, but from a paracrine action from the transplanted cells. So far, however, the pathways involved in the paracrine signalling triggered by skeletal myoblasts have remained largely elusive. The present study was therefore designed with three objectives (1) to determine the differential gene expression profile of SM and their progeny (myotubes) in comparison with fibroblast-like cells; (2) to characterize the cytokines and growth factors released by SM that could be implicated in the effects observed after their transplantation in infarcted myocardium, and (3) to correlate these in vitro findings with those occurring in vivo. For this specific purpose, we used tissue samples from a previous series (Ref. [13]) of rat experiments in which human skeletal myoblasts had been transplanted into infarcted areas.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Source of funding
 Appendix A. Supplementary data
 References
 
2.1. Cell populations
Human muscle biopsies, obtained after written informed consent, from patients undergoing orthopaedic or cardiac surgery were minced and digested with trypsin-EDTA (0.5 mg/ml trypsin and 0.53 mM EDTA) and collagenase (1.5 mg/ml) as previously described [2]. Cells were grown in growth medium (GM) containing Ham-F12 media (GIBCO-BRL), 20% foetal calf serum (FCS) and 1% penicillin/streptomycin (GIBCO-BRL). By day 6 (50% confluence), CD56+ cells were isolated using CD56 labelled microbeads (Miltenyi Biotech, Germany) following the manufacturer's instructions. Cells were plated and maintained for 15 days in GM and three different populations of cells were obtained: C56+ cells (SM), CD56– cells (fibroblast-like cells), and myotubes (CD56+ cells allowed to fuse into myotubes by high cellular confluence). Two million cells of each fraction were harvested for RNA extraction. The study and all the procedures were approved by the Institutional Review Board for Human Studies and Ethics Committee and conform to the principles outlined in the Declaration of Helsinki for use of human tissue.

2.2. Oligonucleotide microarray analysis and validation
Total RNA from cells was isolated using the Trizol reagent (Life Technologies, Gaithersburg, MD) and purified with the Rneasy® Mini Kit (Qiagen, Valencia, CA) following the manufacture's instructions. RNA levels, quality and purity were assessed with the use of the RNA 6000 Nano assay on the Agilent 2100 bioanalyzer (Agilent, Palo Alto, CA). None of the samples showed RNA degradation or contamination with genomic DNA. After reverse transcription, samples were hybridized in U133A microarray (Affymetrix). Array data were normalized with MAS5.0 (Affymetrix Microarray Suite software). The following parameters were examined to ensure that arrays were well hybridized: all array Scale Factor (SF) were lower than 2.5, the number of presences was higher than 25% in all of the arrays and GAPDH values were ~1 and in any case greater than 2.5. Data were filtered to eliminate probes whose presence number is 0 and those whose intensity value is less than 100 in 90% of the samples (7933 probes). Unsupervised cluster analysis: hierarchical clustering based on the average-linkage method with the centered correlation metric was carried out using Cluster and Treeview software [14]. Supervised analysis: in order to identify genes with statistically significant changes in expression between groups, we used two different algorithms to increase the readability of the study: SAM (Significant Analysis of Microarrays) [15] and GARBAN. GARBAN analysis was based on genes where the differences between the 2 groups using a t-test showed a p value <0.05 or 0.01. In the case of SAM, all data were permuted over 100 cycles by using the two class (unpaired) format. Classification of the genes according to the Gene Ontology and matching of the gene products in the Boehringer Mannheim chart of Biological Pathways and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) were made using the GARBAN software.

2.3. Quantitative-real-time PCR (Q-RT-PCR)
Total RNA was extracted from cells or frozen heart sections using TrizolTM method (Invitrogen) following the manufacturer's instructions. Human Universal Reference Total RNA was purchased from BD Biosciences (USA) and used as a positive control. Reverse transcription was performed on 1 µg of total RNA, after heating at 70 °C for 5 min, with random hexamers as reaction primers. The reaction was carried out at 42 °C for 45 min in the presence of SuperScriptTM reverse transcriptase (Invitrogen). Q-RT-PCR was performed in a fluorescent thermal cycler (MJ Research), using 1 µl of cDNA in 20 µl reaction volume with 300 nM of each primer, 250 nM of probe (SIGMA) and TaqmanTM (Roche). Primers used for PCR are shown in Table 1. Amplification conditions were as follows: 95 °C for 3 min followed by 40 cycles consisting of 90 °C-60 s, 60 °C-60 s, 72 °C-60 s. All samples were also amplified to detect the GAPDH gene as control.


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  		Table 1 Primers use for PCR analysis of human cells

 
2.4. Cytokine arrays
To obtain culture supernatants, cells were cultured for 48 h under hypoxic (5% O2) conditions in the absence of serum to avoid the potentially confounding effect of serum-derived growth factors on outcome measurements. After 48 h, the supernatants of SM cultures were collected and frozen. Cytokine levels were measured by and ELISA cytokine antibody array (Ray Biotech) according to the manufacturer's instructions.

2.5. Proliferation/apoptosis studies
Human SM were cultured under normoxic (21% 02) or hypoxic (5% 02) conditions in F12 media supplemented with 2.5% FCS for the proliferation studies and with no serum for the apoptosis studies and collected after 48 h. Alternatively, F12 culture medium plus 2.5% serum was incubated without cells, during the same period of time at 37 °C and 5% CO2 as controls for the proliferation assays (non conditioned media (NCM).

For proliferation studies, human umbilical cord blood venous endothelial cells (HUVEC), smooth muscle cells from cord blood or the rat derived cardiomyocyte cell line HL-1 were cultured in six well plates (5000 cells/well) in the presence of SM conditioned media (CM) (75% of the media) or control media (NCM) with 5% FCS and after 72 h, the number of viable cells was quantified by a nucleocounter (Chemometec, Denmark) following manufacturer's protocol. Three independent experiments were performed and every cell type grown in CM or NCM was seeded in triplicate.

For apoptosis assays, HL-1 cells (250.000 cells) were seeded in 96 well plates and cultured under hypoxic conditions (1% O2) and no serum in the presence of CM or NCM during 48 h. Apoptosis was measured using an ELISA kit (Roche Applied Science, Spain) in which oligonucleosomal fragments are quantified as we have previously described [16]. Three independent experiments were performed on cells seeded in triplicate.

2.6. In vivo transplantation study
Ligation of the left coronary artery was performed in seven-week-old immunodeficient nude mu/mu rats (Harlan France SARL, Gannat, France). Ten days later, rats underwent a sternotomy and then were allocated to receive in-scar injections (150 µl) of human SM (5x106, n=7), or culture medium (n=4). The viability of cells after thawing, assessed by fluorescence-activated cell sorting (FACS) using propidium iodide exclusion, was consistently >87%. 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. Animals were sacrificed 30 days after injection of the SM. Explanted hearts were cut into three segments and frozen in liquid nitrogen-cooled isopentane. Heart tissue specimens were then assessed for the expression of human-specific MMP-2, TNNI3, CNN3, PIGF, TNNT2, PAX7, TGF-beta, and IGF-1 by real time quantitative PCR (Q-RT-PCR). The functional results of this group of animals have been previously reported [13].

2.7. Statistical analysis
All data are expressed as mean±SEM and comparisons between two groups were analyzed by Mann Whitney U test. Comparisons among 3 or more groups were analyzed by Kruskall Wallis test. SPSS 12 software was used for statistical analyses and differences were considered statistically significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Source of funding
 Appendix A. Supplementary data
 References
 
3.1. Characterization of the gene expression profile of SM
The comparison between the gene expression profile of undifferentiated populations of SM (CD56+ cells), fibroblast-like cells (CD56– cells) and differentiated myotubes using the Affymetrix high-density oligonucleotide arrays U133A revealed that over 2093 were differentially expressed between SM and fibroblasts (p<0.05) (Supplementary Online Table S1). Following Gene Ontology classification, genes related to regulation of transcription, cell cycle and development were the most common genes differently expressed. The comparison between myotubes and fibroblast-like cells revealed that 889 genes were differentially expressed (p<0.05) (Supplementary Online Table S2), the most common genes being related to regulation of transcription and cell adhesion. The comparison between SM and differentiated myotubes showed 909 differentially expressed genes (Supplementary Online Table S3) predominantly related to regulation of transcription, cell adhesion and G-protein signalling. As expected, dendrogram analysis clustered the samples in three different groups according to the type of cells (data not shown).

As the goal of our study was to establish a link between SM and the mechanisms of their beneficial effect following in-scar transplantation, we first validated the expression of genes differentially expressed between the different populations and implicated in pathways potentially involved in cardiac repair (cardiac differentiation, angiogenesis, remodelling and apoptosis) by Q-RT-PCR (Fig. 1). Placental Growth Factor (PlGF), a VEGF homologue that signals through the VEGFR-1, was shown to be up-regulated in SM in comparison with either differentiated cells or fibroblast-like cells (Fig. 1). Genes implicated in heart development (TNNT2-cardiac specific troponin T, TNNC1-cardiac specific Troponin I) and extracellular matrix remodelling (metalloproteinases [MMPs] 2 and 7) were also found to be up-regulated in SM or in myotubes in comparison with fibroblast-like cells, whereas MMP-9 expression was only found in differentiated cells. Skeletal myoblasts also expressed transcription factors present in satellite cells such as Pax7 and higher levels of the antiapoptotic gene Bcl-2 and of calponin, a protein involved in smooth muscle contraction.


Figure 01
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  		Fig. 1 Expression of genes implicated in cardiac repair. Q-RT-PCR for genes involved in angiogenesis (PIGF), heart development (TNNT2, TNNC1), extracellular matrix remodelling (MMP-2, MMP-7 and MMP-9), SM specific transcription factors (Pax7), apoptosis (Bcl-2) and smooth muscle (calponin) on SM, fibroblasts and differentiated myofibers. Expression levels are presented as percentages in comparison with SM expression and were normalized by using GAPDH as housekeeping gene. The mean (±SEM) of 4 different experiments in triplicate is shown. * (p<0.05).

 
3.2. Characterization of cytokines and growth factors released by SM
To establish a link between expression and release of certain growth factors implicated in cardiac repair, we analyzed the profile of cytokines secreted by SM by an ELISA array. In line with the gene expression profile analysis, a number of growth factors implicated in angiogenesis were released by SM including VEGF, PlGF, angiogenin, angiopoietin, HGF and PDGF-BB (Table 2). Interestingly, proteases involved in matrix remodelling (MMPs) and their inhibitors (TIMPs) were also detected in the supernatants of SM cultures albeit at variable levels (Table 2).


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  		Table 2 Secretion of growth factors and cytokines by SM under hypoxic conditions

 
To determine the functional effect of these cytokines on cell proliferation and apoptosis we examined the cellular effects of SM-derived conditioned media obtained under both normoxic (21% O2) and hypoxic (5% O2) conditions to more closely match the milieu in which the SM are transplanted (scar tissue with low oxygen supply). Proliferation of HUVECs was found to be increased in the presence of conditioned media obtained both under normoxia (3.1±0.9 fold increase p<0.05) and hypoxia (5.0±2.0 fold increase; p<0.05) in comparison with standard media. Similarly, conditioned media induced an increase in smooth muscle cell proliferation (1.9±0.3 and 1.7±0.2 fold increase in hypoxia and normoxia respectively; p<0.05) and HL-1 proliferation (1.7±0.3 p<0.05 and 1.4±0.2 fold increase, in hypoxia and normoxia respectively) (Fig. 2). Although there was a trend to higher cytokine secretion in hypoxic conditions, these differences did not reach statistical significance. In addition, culture of cardiomyocytes with myoblast-derived conditioned media prevented apoptosis of HL-1 cells (40±3% reduction; p<0.05) in comparison with control media (Fig. 3).


Figure 02
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  		Fig. 2 Effect of conditioned media on proliferation of HUVEC, smooth muscle cells and cardiomyocytes. Endothelial cells (HUVEC) (A), smooth muscle cells (B) and cardiomyocytes (HL-1) were cultured for 48 h in either standard media (control) or conditioned media from SkM obtained under hypoxic or normoxic conditions and the number of cells analyzed. Cell proliferation in conditioned media is represented as a percentage in comparison with standard media (100%). Microphotographs in A, B and C show a representative experiment of 3 different studies while graphs represent the mean±SEM of 3 different experiments in triplicate. * (p<0.05).

 


Figure 03
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  		Fig. 3 Effect of conditioned media on apoptosis of HL-1 cardiomyocytes. Cardiomyocytes (HL-1) were cultured for 48 h in either standard media (control) or conditioned media from SkM obtained under hypoxic or normoxic conditions after which apoptosis was measured as described in the Materials and methods. The mean±SEM of 3 different experiments in triplicate is shown. * (p<0.05).

 
3.3. In vivo findings
To examine whether cytokines found by ELISA could also be detected in an in vivo model, we analyzed sections of rat hearts subjected to a chronic myocardial infarction and transplanted with human derived SM. As previously described [13], these hearts had demonstrated a better functional recovery than medium-injected control hearts, as evidenced by higher ejection fractions, smaller increases in endsystolic volumes and a leftward shift in preload-recruitable stroke work. Three samples of each heart were obtained from the infarcted zone where the myoblasts had previously been immunohistochemically detected. Twenty-five serial cryostat sections of 25 µm thickness were obtained from each sample for human RNA detection. In 6 out of 7 SM-transplanted animals, Q-RT-PCR detected a positive signal for MMP-2, TNNI3, CNN3, PIGF, TNNT2, TGF-β and IGF-1 that ranged between 0.02% and 88% in comparison with a universal human mRNA control (Table 3). In contrast, expression of human-specific mRNA was not detected in animals injected with control media suggesting that these growth factors are released in vivo after transplantation of SM, can be detected up to 1 month after transplant and thus might have contributed to the functional effects associated with SM treatment of MI.


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  		Table 3 Expression of cytokines in rat hearts as a percentage in comparison with universal human mRNA

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Source of funding
 Appendix A. Supplementary data
 References
 
It is now commonly accepted that regardless of their origin, transplanted cells do not survive well in the host myocardium, with studies reporting 70% to 93% of cell death in only the first 3 days and with an increased cell death over time [17-20]. Despite this, most preclinical studies have reported that cell transplantation improves the functional outcome in both ischaemic and nonischaemic cardiomyopathy models [1,10,21]. To account for this discrepancy, it has been hypothesised that the benefit of the procedure is not related to the addition of new donor-derived contractile elements but rather to indirect effects like mechanical limitation of remodelling ("girdling" effect) or paracrine actions of the graft on the host tissue. So far, this paracrine paradigm has been primarily investigated in bone marrow cells and analysis of the secretome of these cells tends to confirm that the pathways that they activate involve increased angiogenesis, limitation of apoptosis, changes in extracellular matrix composition effecting greater scar elasticity and, more hypothetically, recruitment of endogenous cardiac stem cells [22-25]. In contrast, the putative paracrine effects of SM have been less well deciphered [26]. Nevertheless, with the exception of the recently described skeletal precursors of cardiomyocyte cells (Spoc) isolated from mouse muscle, which have demonstrated their potential to differentiate into beating and functional cardiomyocytes both in vitro an in vivo [27], the general consensus is that SM do not differentiate into cardiomyocytes [6]. Therefore, the paracrine hypothesis looks like a sound mechanism to explain the experimentally-demonstrated improvement of function in myoblast-grafted failing hearts independently of true cardiac regeneration [9,26,28-32]. Overall, our microarray data, more directly validated by Q-RT-PCR measurements, suggests that injected SM, differentiated myotubes expected to result from their in vivo fusion and fibroblast-like cells that contaminate myoblast injections, release important factors involved in angiogenesis, remodelling and apoptosis inhibition pathways that can ultimately lead to enhanced cardiac tissue protection.

Thus, the profile of genes expressed by SM in comparison with the other two sources of cells supports the potential of SM to produce proteins that can induce angiogenesis and vasculogenesis. An increase in PIGF may amplify the responsiveness of endothelial cells to VEGF and thus contribute to increase angiogenesis in the ischaemic myocardium [33] as we have demonstrated in a swine model of chronic myocardial infarction treated with autologous SM [9] while up-regulation of calponins (CNN3), a family of proteins involved in smooth muscle contraction, supports the potential of transplanted cells to differentiate into smooth muscle, as we have previously reported [9]. A relation between VEGF, angiogenesis, and cardiac repair after muscle stem cell transplantation into ischaemic hearts has been recently demonstrated in a murine model of MI [34].

Cell transplantation has also been associated with prevention of cardiomyocyte apoptosis [35]. The up-regulation of antiapoptotic genes like BAG-1 and Bcl-2, previously linked to enhanced cell survival in ischaemia-reperfusion models [36,37] as well as the ability of SM-derived supernatants to decrease HL-1 cardiomyocyte apoptosis in vitro indicate that SM could also contribute to mitigate post-infarction cell death. Although SM are not endowed with the potential to differentiate into cardiomyocytes, [6] expression of cardiac specific genes was surprisingly detected in SM and differentiated cells such as troponin I (TNNC1) [38], cardiac specific troponin T (TNNT2) [39], cardiac troponin I (TNNI3) and β-myosin heavy chain (MYH7) albeit at low levels (0.3-1% of cardiomyocytes in comparison with cardiomyocytes). These findings suggest that SM could contain a genetic program that, under specific circumstances, would allow them to partially differentiate into cardiac muscle or at least that a population of cells within the myoblast pool could have this potential [27].

Cardiac remodelling ensues after myocardial infarction and contributes to the development of fibrosis and eventually heart failure [40]. While changes in the balance between MMPs and their natural inhibitors (TIMPs) participate in the derangement of the heart after infarction [41], transplantation of stem cells has been associated with potentially beneficial changes in the matrix remodelling process [9]. In our study, matrix MMPs such as MMP-7 (involved in the early stage of tissue remodelling [42]) and the gelatinase MMP-2 were up-regulated in SM. MMP2 may be an important player in regulating SMC phenotype, proliferation, migration and in maintaining the integrity of the vascular wall through governing proteolysis during arteriogenesis [43]. Moreover, activated MMP-2 could bind to {alpha}vβ3 on the surface of angiogenic endothelial cells, and may influence angiogenesis by inducing vascular migration [44].

The results of the ELISA on SM culture supernatant establish a link between gene expression and secretion of growth factors by SM, which is further supported by the functional effect of the culture supernatants on proliferation and apoptosis of smooth muscle, endothelial and cardiac cells. The ultimate link between the paracrine effects of SM demonstrated in vitro and the functional benefits associated with their intramyocardial injection was best provided by the results of our xeno-myoblast transplantation study showing that the engrafted cells acted as cytokine sinks releasing some of the factors involved in the key events (particularly, increased angiogenesis and decreased fibrosis) that contribute to tissue salvage. Of note, these cytokines could be detected for as long as 1 month after cell transplantation in spite of the low percentage of SM that was still engrafted at this remote time point. Of note, the functional results of this group of animals, which have been reported previously [13], showed a preservation of left ventricular function, compared with controls; this finding is consistent with the paracrine paradigm in that, in a rabbit model of occlusion/reperfusion subjected to bone marrow cell transplantation, levels of TGF-β and MMP-1 were negatively correlated with post-treatment ejection fractions [24].

Put together, these data might, at least partly, explain the findings of the randomised controlled MAGIC trial in which left ventricular enddiastolic and endsystolic volumes of patients injected with a high dose of SM were significantly reduced compared with those receiving a low dose of cells or the placebo medium [45]. At the end, the trophic effects of SM were best evidenced by the ability of SM-derived conditioned media to enhance the proliferation of the major cell types that constitute myocardial tissue, particularly under hypoxic conditions simulating the scarred environment in which cells are usually transplanted.

In conclusion, our study provides new insights into some of the mechanisms that participate in the putative benefit of SM-induced cardiac repair. However, apart from this cognitive information, the present results may also have more practical implications. Namely, if a predominant mode of action of SM is the release of cardioprotective factors, then one could consider delivering these cells encapsulated in microparticles that allow an inward flux of oxygen and nutrients ensuring graft viability and an outward flux of cell-released products while the size of the external membrane pores is sized in a way that prevents invasion by immune cells. Such a delivery system should limit the inflammatory component of cell death while allowing transplantation of allogeneic cells, thereby circumventing some of the hurdles associated with autologous cell therapy products (variable functionality, delayed availability, cost of customized quality controls). The successful control of diabetes by transplantation of encapsulated insulin-producing allogeneic and even xenogeneic cells suggests that this option is clinically realistic [46].


   Source of funding
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
Appendix A. Supplementary data
 References
 
Supported in part by grants from Fondo de Investigaciones Sanitarias PI042125, ISCIII-RETIC RD06/0014, FEDER (INTERREG IIIA), European Union FWP7 (INELPLY) and the "UTE project CIMA" to FP and the Foundation LeDucq (CaPTAA network) to PM.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ejheart.2008.08.002.


   Acknowledgements
 
HL-1 cardiomyocyte cell line was kindly provided by Dr. Claycomb (Louisiana State University Medical Center, USA).


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Source of funding
 Appendix A. Supplementary data
 References
 

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