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European Journal of Heart Failure 2006 8(6):555-563; doi:10.1016/j.ejheart.2005.11.006
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© 2005 European Society of Cardiology

Effect of aging on the pluripotential capacity of human CD105+ mesenchymal stem cells

Santiago Rouraa, Jordi Farréb, Carolina Soler-Botijaa, Anna Llacha, Leif Hove-Madsena, Jordi J. Cairób, Francesc Gòdiab, Juan Cincaa and Antoni Bayes-Genisa,*

a Laboratory of Cell Physiology, Cardiology Service, Hospital de la Santa Creu i Sant Pau, ICCC, Universitat Autònoma de Barcelona Barcelona, Spain
b Departament d'Enginyeria Química, Escola Tècnica Superior d'Enginyeria, E.T.S.E., Universitat Autònoma de Barcelona Barcelona, Spain

* Corresponding author. Cardiac Regeneration Program, Cardiology Service- ICCC, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain. Tel.: +34 93 5565765. E-mail address: abayesgenis{at}hsp.santpau.es


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Background: Whether aging modifies mesenchymal stem cell (MSC) properties is unknown.

Aim: To compare the differentiation capacity of human CD105+ MSCs obtained from young and elderly donors.

Methods and results: Cells were obtained from young (n = 10, 24 ± 6.4 years) and elderly (n = 9, 77 ± 8.4 years) donors. Cell senescence was assessed by telomere length assays and lipofuscin accumulation. Cell pluripotentiality was analysed by adipogenic and osteogenic induction media, and myocyte phenotype was attempted with 5-azacytidine (5-AZ). Immunofluorescence, Western blot, transmission electron microscopy and fluo-4 confocal imaging were used to analyse the sarcomere, gap junctions and Ca2+ dynamics. Cells obtained from young and elderly donors showed no significant differences in relative telomere length (40.1 ± 6.4% and 40.3 ± 3.6%, p = 0.9) and lipofuscin accumulation. Adipogenic and osteogenic potential of CD105+ MSCs was demonstrated. 5-AZ induced increased expression of sarcomeric proteins without complete sarcomere organization. Treated cells also showed increased presence of connexin-43 both in young and old donor-derived cells. Intercellular communications were verified by the observation of gap junctions and passage of Ca2+ between neighbouring cells. Spontaneous Ca2+ raises did not significantly increase after 5-AZ treatment in both age groups.

Conclusion: Age does not influence the adipogenic and myogenic differentiation potential of human CD105+ MSCs.

Key Words: Cardiomyogenic differentiation • Stem cells • Age • Senescence

Received November 23, 2004; Revised July 25, 2005; Accepted November 15, 2005


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Myocardial infarction, one of the leading causes of morbidity and mortality in developed countries, produces an irreplaceable loss of cardiomyocytes. Necrotic tissue cannot be reconstituted by the remaining cardiomyocytes, resulting in heart failure and/or death [1]. Several studies have cast doubt on the classic paradigm by which the human heart cannot be regenerated [2-5]. Whether resident or circulating stem cells may regenerate the heart remains under investigation. Studies on the adult murine heart indicate that mesenchymal stem cells (MSCs), the pluripotential progenitors of mesenchymal lineages, can be efficiently isolated, expanded ex vivo and directly applied to infarcted sites where they differentiate into cardiomyogenic lineage [6,7]. Thus, cell therapy focused on the use of MSCs may be an efficient treatment option for replacing excessive myocardial cell loss after heart injury. However, large inter-donor variations exist in the pluripotential capacity and growth of MSCs [8,9]. This functional heterogeneity may be caused in part by increasing age and might hamper MSC suitability for cardiac regeneration in elderly patients with acute myocardial infarction. Indeed, it is unknown whether autologous stem cells obtained from elderly patients have the same proliferative kinetics and phenotype plasticity.

The isolation of human MSCs expressing CD105 [10] is reported in young and elderly donors. We further compared the CD105+ MSCs obtained from bone marrow stroma with increasing cell donor age in terms of senescence, pluripotentiality and Ca2+ dynamics.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Study subjects
Ten young donors with a mean age of 24±6.4 years and nine elderly donors with a mean of 77±8.4 years were included in the study. None of the young donors had cardiovascular risk factors and were only treated with diuretics and digoxin due to congenital heart defects. Four (44.4%) and five (55.5%) of the elderly patients suffered diabetes mellitus and dyslipidemia, respectively. Patients were treated with ACE inhibitors (67%), β blockers (55.5%) and aspirin (78%). Informed consent was obtained from all subjects and the study was approved by the hospital Ethics Committee.

2.2. Isolation and cell culture
5 to 15 ml of bone marrow aspirate were collected from human sternum or femur, respectively, with a syringe containing 6000 U heparin. Mononuclear cells were isolated according to minor modifications of a previously reported method [10]. Cells were resuspended in MACS buffer at 2-4x108 cells/ml and incubated with 50 µl of magnetic microbeads conjugated to human CD105 monoclonal antibody (Miltenyi Biotec., Auburn, CA, USA) for 15 min at 4 °C. Positive cells were separated on a MS+ magnetic column (Miltenyi Biotec.) and grown on regular culture plastic in PMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Gibco Invitrogen Corp., Grand Island, NY, USA) at 37 °C in 5% CO2 in air. Medium was changed after 72 h to select adherent cells and every 3 days thereafter. At day 14, cells pooled by trypsinization were designated as primary culture (passage 0) and replated for further expansion.

2.3. Flow cytometry
Flow cytometry was performed to assess the purity of the CD105+ cell populations. 2x105 cells were washed in staining buffer (PBS supplemented with 1% BSA and 0.1% sodium azide) into polypropylene FACS tubes and immunostained with 10 µl of a mouse monoclonal antibody against human CD105 conjugated with FITC (Serotec, Oxford, UK) for 30 min at 4 °C in the dark. Levels of CD105 expression were compared with that of IgG isotype control (Caltag Laboratories, Burlingame, CA, USA) and quantified using a Coulter EPICS XL flow cytometer (Beckman Coulter, Miami, FL, USA). All data acquisitions and analyses were carried out using the Expo32 software (Beckman Coulter).

2.4. Western blot
Total protein was extracted from cells according to minor modifications of a previously described protocol [11]. Membranes were incubated with mouse anti-human specific monoclonal antibodies against CD54 (1 µg/ml), CD45 and SH2 (0.5 µg/ml) (BD Biosciences, Palo Alto, CA, USA), connexin (Cx)-43 (1 µg/ml) (BD Transduction, Lexington, KY, USA), cardiac troponin (cTn)-I (10 µg/ml) (Chemicon, Temecula, CA, USA) and P-actinin (dilution 1:500) (Sigma, St. Louis, MO, USA), respectively. Proteins were detected on membranes with a peroxidase-conjugated secondary antibody (BD Biosciences) diluted 1:1000 in TBS containing 0.2% Triton X-100, followed by an enhanced chemiluminescence detection system (Amersham Biosciences, Buckinghamshire, England).

2.5. Immunocytochemistry
Immunostaining experiments were performed on cells grown on 13 mm glass coverslips (Marienfeld GmbH, Lauda-Koenigshofen, Germany) [11]. Cells were fixed, permeabilized and immunostained with mouse monoclonal antibodies against human CD106, CD117, SH2 (5 µg/ml), CD34 (10 µg/ml) (BD Biosciences), β-myosin heavy chain (β-MyHC) (10 µg/ml) (Chemicon) and Cx-43 (2.5 µg/ml) (BD Transduction) for 45 min at room temperature. After washing, coverslips were incubated with a goat anti-mouse IgG+IgM antibody conjugated with FITC (BD Biosciences) or Cy3 (Jackson ImmunoResearch, West Grove, PA, USA), diluted 1:200, for 40 min at room temperature. Finally, coverslips with immunostained cells were washed, mounted and examined under an immunofluorescence microscope. In some experiments, cell nuclei were stained with 0.25 µg/ml Hoechst for 15 min at room temperature.

2.6. Senescence assays
CD105+ MSC telomere length was measured by fluorescence in situ hybridization (FISH) with the Telomere PNA kit/FITC (Dako, Glostrup, Denmark) according to the manufacturer's instructions. Fluorescence intensity, directly correlated to telomere length, was quantified using a FACSVantage cytometer (BD Biosciences). After flow cytometry analysis, relative telomere length (RTL) was calculated as the ratio between telomere signal of each sample and the internal control (1301 cell line-T cell lymphoblastic leukaemia) [12].

Accumulation of the autofluorescent substance lipofuscin was assessed in cell cultures, prepared from young and elderly donors, at 488 nm with a laser scanning confocal microscope (Leica). Cells were fixed with 4% paraformaldehyde 30 min at 4 °C, rinsed with PBS and left unstained. One hundred cells from randomly selected fields were then examined. The area occupied by lipofuscin bodies was averaged within each cell using the NIH ImageJ software.

2.7. Adipogenic and osteogenic differentiation
Adipogenesis was induced as previously described [13]. Adipogenic induction medium (MDI+I) consisted of 1 µM dexamethasone, 0.5 mM isobutylmethylxanthine, 10 µg/ml insulin and 100 µM indomethacin (Sigma) added to the culture medium. MDI+I was replaced every 3-4 days. After the third MDI+I treatment, cells were examined for the presence of lipid vacuoles staining with Oil red O (Sigma) and electron microscopy.

Osteogenic differentiation was induced as previously described [14]. Cells were grown in presence of osteogenic supplements (100 nM dexamethasone, 10 mM sodium β-glycerophosphate and 0.05 mM ascorbic acid) for 14 days. Deposition of calcium matrix by differentiated cells was examined by von Kossa staining.

2.8. Myogenic differentiation
Subconfluent cells were differentiated by treatment with 10 µM of 5-azacytidine (5-AZ; Sigma) maintained in culture medium for 14 days. Myocyte lineage differentiation was examined for the presence of sarcomeric proteins, gap junctions and generation of spontaneous Ca2+ oscillations.

2.9. Electron microscopy
CD105+ MSCs, MSC-derived adipocytes and 5-AZ-treated MSCs were washed with PBS and prefixed with 2.5% glutaraldehyde in serum-free PMEM for 2 h at 4 °C. Cells were then washed with 0.1 M cacodylate buffer, pH 7.4 and postfixed in 1% osmium tetroxide in 0.1 M cacodylate for 2 h at 4 °C. Subsequently, samples were incubated in 2% uranyl acetate for 1 h, dehydrated with graded ethanols, embedded in Spurr's resin and cut into blocks. Finally, ultra-thin vertical sections stained with lead citrate and uranyl acetate were obtained. Images were acquired with an electron microscope Hitachi H-7000 (Hitachi, Tokyo, Japan) at 75 kV accelerating voltage and a charge-coupled camera.

2.10. Confocal imaging analysis
Free Ca2+ concentration was monitored in expanded CD105+ MSCs according to minor modifications of a previously described method [15]. Cells were plated in plastic dishes, the bottoms of which were replaced by glass coverslips, and incubated with 1 µM fluo-4AM, the calcium-specific probe (Molecular Probes, Eugene, OR, USA), for 30 min at 37 °C. Cells were then excited at 488 nm and fluorescence emission was detected between 500 and 650 nm under a laser scanning confocal microscope (Leica TCS SP2 AOBS) (Leica, Wetzlar, Germany). Fluo-4 signal (fluorescence) was recorded every 5 s and analysed using the LCS Lite software (Leica).

2.11. Data analysis
Descriptive statistics include mean±S.D. Student's t-test was used for comparison between groups. A p value ≤0.05 was considered statistically significant. Pearson's correlation coefficient was used to assess the association between MSC differentiation potentials and cell donor age.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Isolation and phenotyping of CD105+ MSCs
A mesenchymal stem cell population originating from human bone marrow obtained from young and elderly donors was successfully isolated, expanded in monolayer culture and characterized. After the initial selection process by the superficial expression of CD105 using magnetic beads, cells adhered to plastic culture dish and cultured for 14 days. Different growth media were tested to establish the optimum culture conditions for attachment and expansion. The PMEM supplemented with 10% FBS and 1% antibiotics showed better cell growth than DMEM, and no difference was observed between media supplemented with 10% or 20% FBS (not shown).

A few elongated, fibroblast-like cells appeared attached under the conditions described at day 3. These initial MSCs were further enriched by repeated culture media changes every 3 days to remove floating non-attached cells and were uninterruptedly maintained in culture over 4 months and 10 passages. Cultures showed a time-dependent transition from the earliest multipotential progenitor cells, extremely rounded and small, to thin spindle-shaped cells (Fig. 1). Purity of expanded cells was demonstrated by expression of superficial CD105 at the beginning of in vitro culture by flow cytometry (Fig. 2A), which remained highly stable and well preserved during repetitive passaging of cells (not shown).


Figure 1
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  		Fig. 1 Establishment of primary CD105+ MSCs cultures. (A-B) Round and small self-renewing cells (arrows) from femoral and sternal aspirates. (C-D) Spindle-shaped CD105+ MSCs at pre-confluence and confluence, respectively.

 


Figure 2
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  		Fig. 2 Phenotypic characterization of human CD105+ MSCs. (A) Purity of the CD105+ cell population at the beginning of culture. Representative distributions of cell fluorescence against CD105 in cells obtained from a young (20-year-old) and an older (74-year-old) donors. (B) Western blot and immunofluorescence analysis. Cells expressed SH2, CD117 and CD106, and were negative for CD45, CD54 and CD34. F=femur origin, S=sternum origin, –=negative control, +=positive control.

 
For CD105+ MSCs phenotyping, expression of CD54, SH2, CD106, CD117, CD45 and CD34 were analysed by Western blot and immunocytochemistry. As previously reported, expression or absence of some of these markers characterizes human bone marrow-derived MSCs [13]. Cultured cells were strongly positive for SH2, CD117 and CD106, and negative for CD45, CD54 and CD34 (Fig. 2B).

3.2. Senescence characteristics of CD105+ MSCs
Telomere length (RTL), a marker of cell senescence, did not appear related to donor age in CD105+ MSCs. Mean RTL was 40.1±6.4% in cells from young subjects and 40.3±3.6% in cells from elderly donors, respectively (p=0.9). A representative example of RTL for young and old donor cells is shown in Fig. 3A.


Figure 3
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  		Fig. 3 Cell senescence analysis. (A) RTL analysis of CD105+ MSCs from a 20- and a 74-year-old donors. 1301 cell line was used as internal control. (B) Representative examples of lipofuscin accumulation in cells prepared from a young and an older donors after 3 and 14 days in culture. (C) Quantification of lipofuscin accumulation over time in cultures prepared from young and elderly donors.

 
Cell senescence dynamics were also studied as cultures progressed over 10 passages. Accumulation of the cell-aging biomarker lipofuscin [16] within cellular lysosomal apparatus was studied. At day 3, CD105+ MSCs obtained from young and elderly donors showed no significant differences in lipofuscin accumulation. Small autofluorescent and perinuclear lipofuscin bodies gradually increased in cultured cells over time (Fig. 3B). At day 14, lipofuscin accumulation was significantly higher both in young and elderly donor-derived cells as compared with day 3 (p=0.02 and p=0.04, respectively) (Fig. 3C).

3.3. Pluripotentiality of CD105+ MSCs
Morphologic changes induced by adipogenic differentiation were clearly apparent within the first adipogenic induction medium treatment. As adipogenesis progressed, cell cytoplasm was filled with lipid droplets, which stained with Oil red O, and were visualised under phase-contrast microscopy (Fig. 4A,B) and electron microscopy (Fig. 4C,D). The percentage of Oil red O-positive cells was 71.1±28.1% and 55.5±28.4% in young and elderly donor-derived cultures after adipogenic treatment, respectively (p=0.18) (Fig. 4G). No significant correlation was observed between in vitro adipogenic activity and cell donor age.


Figure 4
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  		Fig. 4 Adipogenic and osteogenic differentiation of CD105+ MSCs. (A-B) Oil red O-staining from CD105+ MSC-derived adipocytes from a 74-year-old (A) and a 27-year-old (B) donors. (C-D) Lipid droplets of varying size within the cytoplasm. (E-F) von Kossa staining of CD105+ MSC-derived osteocytes from a 74-year-old (E) and a 27-year-old (F) donors. (G, H) Correlations between cell donor age and CD105+ MSC adipogenic and osteogenic potential, respectively.

 
CD105+ MSC osteogenic differentiation was also examined. During osteogenic stimulation, undifferentiated CD105+ MSCs changed from their characteristic spindle-shaped morphology to a cuboidal appearance. Deposition of calcified matrix on culture dishes was assessed by the amount of von Kossa staining (Fig. 4E,F). The percentage of von Kossa-positive cells was 55±20% and 12.8±17 in young and elderly donor-derived cultures after osteogenic treatment, respectively (p=0.002). A strong negative correlation was observed between in vitro osteogenic activity and cell donor age (Fig. 4H).

3.4. Myogenic differentiation of CD105+ MSCs
CD105+ MSCs were treated with 5-AZ to attempt the acquisition of a myocyte phenotype in culture. 5-AZ induced the expression of myocyte-specific markers but failed to promote the formation of sarcomere-like structures or beating cells within treated cultures. Treated cells showed increased cardiac β-MyHC expression in both young (Fig. 5A,B) and elderly donors (Fig. 5C,D). cTnI was not detected in either control or 5-AZ-treated cells (Fig. 5E). Electron microscopy of treated cells revealed presence of non cross-striated myofibrils, suggesting incomplete sarcomere organization (Fig. 5F).


Figure 5
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  		Fig. 5 Myogenic differentiation of CD105+ MSCs: sarcomeric proteins. (A-D) β-MyHC expression is significantly increased after 5-AZ treatment in a young (27-year-old) and an older (74-year-old) donors. (E) Absence of cTnI expression in treated cells. (F) Electron microscopy showing intracellular non cross-striated myofibrils (arrowheads) in treated cells. Bar=5 µm.

 
Formation of intercellular functional gap junctions was also evaluated in young and old donor-derived cells by expression of Cx-43, electron microscopy and Ca2+ wave propagation. Increased Cx-43 expression was found by immunofluorescence (Fig. 6A-D) and Western blot (Fig. 6E) in treated cells from both young and elderly donors, and electron microscopy identified the presence of intercellular gap junctions (Fig. 6F). Identification of spontaneous rhythmic Ca2+ oscillations by fluo-4 confocal imaging between adjacent cells confirmed active intercellular communications (Fig. 7A). Furthermore, as one wave dispersed, captured images frequently revealed another wave forming and travelling within another area of the same cell. However, increasing donor age and 5-AZ treatment did not significantly modify the generation and frequency of spontaneous Ca2+ raises. The percentage of elderly donor-derived cells with spontaneous Ca2+ events was 42±13.2% in control conditions and 46.5±17.4% after 5-AZ treatment (p=0.7). In young donor-derived cultures, 44.6±11.3% of cells showed spontaneous Ca2+ events in control conditions compared with 70±23% after treatment (p=0.2) (Fig. 7B).


Figure 6
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  		Fig. 6 Myogenic differentiation of CD105+ MSCs: intercellular connections. (A-D) Cx-43 expression assessed by inmmunofluorescence (A-D) and Western blot (E) is increased after 5-AZ treatment in a young (27-year-old) and an older (74-year-old) donors. (F) Electron microscopy showing intercellular gap junction (arrow). Bar=5 µm.

 


Figure 7
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  		Fig. 7 Ca2+ oscillations in CD105+ MSCs. (A) Propagation of a Ca2+ wave between two neighbouring cells. Images were taken at times indicated at the bottom of each panel after beginning of the recording. (B) Percentage of cells with Ca2+ events before and after treatment in the two age groups. C=control, AZ=5-azacytidine.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
The great clinical impact of cardiovascular diseases in Western countries has prompted the search for alternative sources of cardiomyocyte precursors for therapeutic purposes. Evidence of cardiac chimerism after transplantation has been described [2,17], which suggests the existence of non-cardiac cells able to adopt a cardiomyocyte fate. The origin of these cells remains unclear; however, recent research points to cardiac resident stem cells and/or circulating bone marrow-derived stem cells as the more likely sources. The potential of haematopoetic bone marrow-derived progenitors for cardiac regeneration is controversial. Although experimental cell transdifferentiation towards a cardiovascular lineage has been reported in some studies [4], other well-designed gene-targeted studies failed to demonstrate this phenomenon [18,19]. MSCs have shown ability for both cell fusion with differentiated adult cells [7,20] and differentiation in situ to cardiomyocyte lineage when injected into the murine heart [6]. Therefore, MSCs have very promising potential for cardiac regeneration.

The hypothesis that aging could curtail MSC ability to differentiate into adipogenic, osteogenic and myogenic lineages was tested. We isolated a subset of undifferentiated MSCs expressing CD105 and demonstrated their in vitro versatility when these cells were obtained from both young and elderly donors. CD105+ MSCs were capable of differentiating towards two main mesenchymal pathways: adipogenic and osteogenic lineages. No significant differences were found in the adipogenic activity observed in cells from young and elderly donors. Interestingly, a reduction in osteogenic potential was found in old donor-derived cell cultures. Whether this reduced MSC osteogenic activity may be related to disruption of bone tissue homeostasis, characteristic of elderly patients, deserves further research.

The molecular mechanisms that regulate MSC myogenic differentiation are complex and not fully understood. Strategies to trigger this process in vitro have been developed. These include exposure to synthetic chemical compounds, exogenous cytokines, growth factors and co-culture with mature cardiomyocytes [21]. We decided to attempt CD105+ MSC differentiation by exposure to 5-AZ, which is able to induce cardiomyogenic differentiation in both embryonic [22] and adult stem cell cultures [23]. The exact mechanism of action of 5-AZ is still unknown, although it has been suggested that activates silent genes by inhibiting DNA methylation processes [24]. The data presented indicates that treatment with 5-AZA induced the expression of some myogenic proteins (such as β-MyHC) but failed to promote the formation of sarcomere-like structures or beating cells. Treated CD105+ MSCs abundantly expressed the integral gap junction protein Cx-43 necessary for electrochemical interaction of cardiac muscle cells. Coupling between neighbouring CD105+MSCs was confirmed by the observation of spontaneous Ca2+ wave propagation in fluo-4 confocal imaging experiments. More comprehensive experiments including electrophysiological measurements may allow the unequivocal identification of cardiac phenotype in these stem cell-derived myocytes.

Cell senescence applies to loss of the replication ability of normal somatic cells and has usually been attributed to the progressive telomere shortening, which accompanies each cell proliferation event [25,26]. Here, we found short but not significantly different telomere lengths in CD105+ MSCs from both young and elderly donors expanded in vitro. This observation is in agreement with similar MSC replicative capacity studies [27-29]. Moreover, we further characterized senescence of these cells by lipofuscin, a more specific cell-ageing marker that accumulates as cell senescence progresses and eventually leads to cell degeneration [16]. Irrespective of cell-donor age, CD105+ MSCs showed similar lipofuscin increases in cells maintained in culture for long periods of time. These senescence data observed in human CD105+ MSCs should be taken into consideration when evaluating the potential clinical use of these cells, due to the inevitable in vitro expansion required prior to delivery into damaged tissue.

In summary, our data suggest that age does not influence the capacity of human CD105+ MSCs to differentiate into adipogenic and myogenic lineages. Indeed, if MSCs emerge as a candidate cell source for cardiac regeneration, these results support their use both in young and elderly patients with myocardial lesions. For this purpose, we speculate that cells could first be treated in vitro to trigger their myocyte fate, and then delivered into the damaged heart where the cardiac microenvironment may be conducive to the differentiation of these already committed cells to adult contractile cardiomyocytes. The presence of Cx-43 and its ability to form functional gap junctions in vitro is of paramount importance. This may allow early intercellular contacts after in vivo stem cell delivery and facilitate electromechanical coupling. Further research is required to demonstrate whether CD105+ MSCs efficiently commit to cardiomyocytes by chemical agents and/or other differentiation strategies.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
The authors thank Mercè Martí for electron microscopy technical assistance and Marco A. Fernández for flow cytometry assistance.

This work was supported by grants from Red de Trasplante Cardíaco (C03/03), Fundación Española del Corazón, Fundación Mapfre Medicina and Ministerio de Educación y Ciencia (SAF 2004-08044-C03-01). We also appreciate support from BMS.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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