Introduction

Despite the initial hope that embryonic stem cells (ESCs) could feature an immune privilege, it is now increasingly recognized that these cells trigger an immune reaction leading to their rejection, in both allogeneic and xenogeneic¹ settings. As the use of patient-specific immunologically matched cell lines derived by nuclear transfer has not yet been proven to be feasible in humans, a potential clinical application of ESCs would likely require an adjunctive immunosuppressive regimen. The well-documented adverse effects associated with immunosuppressive drugs strongly justify the search for alternate strategies and in this setting, the use of mesenchymal stem cells (MSCs) is appealing as these cells express low levels of major histocompatibility complex (MHC) class I, lack expression of MHC class II and of costimulatory molecules, and have been credited to be immune privileged through various mechanisms including inhibited proliferation of T and B lymphocytes, escape from natural killer cell–targeting mechanisms, and impairment of dendritic cell maturation.^2,3 Theoretically, these effects could underlie the host tolerance to MSCs that has been reported in animal models of myocardial infarction treated by allogeneic^4,5,6 and even xenogenic MSCs.⁷ This study was thus undertaken to assess whether the transplantation of human MSCs, in an immunocompetent rat model of myocardial infarction, could create a suppressive local microenvironment that would mitigate the expected rejection of coinjected ESCs and thus favorably affect cell engraftment and functional recovery.

Results

Assessment of cardiac function

Baseline left ventricle (LV) function was not different among the four groups. However, 2 months after transplantation, left ventricular ejection fraction (LVEF) was significantly higher in the ESC and combined (ESC + MSC) groups compared with controls. At this time point, all hearts had incurred some LV remodeling but the overall comparison of data failed to demonstrate a significant group effect (P = 0.33 and P = 0.13 for the comparisons LV end-diastolic and end-systolic volumes, respectively) (Table 1).

Table 1 - Evolution of LV function (LVEF), EDV, and ESV in control, hESC, MSC, MSC + hESC, and hESC + FK506 groups.

Full table (62K)

Immunohistochemical characterization of engrafted cells

Two months after transplantation, only few human cells could still be identified in the injection sites (Figure 1). Engraftment rates, expressed as the percentage of lamin-positive areas relative to LV infarcted areas, averaged 1.5, 2.9, and 3.8% in the ESC, MSC, and combined groups, respectively [combined group versus human ESCs (hESCs), P = 0.01]. These ratios were paralleled by the quantitative RT-PCR lamin A/C data, which yielded values (mean SD) of 0.0016 0.004, 0.0170 0.034, and 0.0192 0.0492 ng/l in the ESC-alone, MSC-alone, and ESC + MSC combined groups, respectively. Qualitatively, engrafted cells of all groups expressed markers of endothelial, smooth muscle, or myofibroblasts and ventricular cardiac cells to a roughly similar extent although the endothelial phenotype tended to be more abundant in the MSC group (Figure 2).

Figure 1.

Immunostaining of cryosections using an anti-human lamin antibody: (a) mesenchymal stem cell (MSC), (b) human embryonic stem cell (hESC), and (c) MSC + hESC cell-engrafted myocardium. Images were acquired in confocal microscopy (green channel 515–535 nm, 63). Bar = 10 m.

Full figure and legend (24K)

Figure 2.

Immunostaining of cryosections using markers of differentiation: an anti-smooth muscle cells antibody in (a) mesenchymal stem cell (MSC) group, (b) an anti-CD31 antibody in MSC group, (c) and anti-myosin heavy chain antibody in MSC group. Images were acquired in confocal microscopy (green channel 515–535 nm, 63). Bar = 10 m.

Full figure and legend (16K)

Hearts of all groups (including controls) demonstrated a similar infiltration of injected areas by CD4⁺ cells. CD3⁺ lymphocytes were also found in all hearts with a trend toward a smaller degree of infiltration in hearts injected with MSCs combined with ESCs. In line with this finding, regulatory CD4⁺CD25⁺ FoxP3-expressing lymphocytes tended to be present in greater amounts in MSC-treated hearts (regardless of whether ESCs were coinjected) compared with those of the control and ESC-alone groups (Figure 3).

Figure 3.

Immunostaining of cryosections using markers of rejection against (a) CD3, (b) CD4, and (c) regulatory CD4CD25 FoxP3-expressing lymphocytes in each group. Images were acquired in confocal microscopy (20). Bar = 50 m.

Full figure and legend (50K)

No cardiac teratoma was detected in any of the ESC-injected hearts; nor was there extracardiac tumors identified in these groups.

Fibrosis

All cell types reduced fibrosis as a significantly larger part of the LV was occupied by scar tissue in hearts injected with the control medium (41%) compared with those transplanted with ESCs (34.2%; P = 0.03 versus controls), MSCs (28.2%; P < 0.0001 versus controls), and the combination of ESCs and MSCs (30.9%; P < 0.01 versus controls). There was no difference in the extent of fibrosis among the three cell-treated groups (Figure 4).

Figure 4.

Immunostaining of cryosections using markers of fibrosis (Sirius red): from control (a), human embryonic stem cell (hESC) (b), mesenchymal stem cell (MSC) (c), and MSC + hESC groups (d). Pictures were taken in the core of the infarct area. Images were acquired in microscopy (5). Bar = 1 mm.

Full figure and legend (102K)

Angiogenesis

Vascular density, expressed as the number of rat endothelial cell antigen–positive cells per mm², was consistently greater in the three cell-transplanted groups than in the controls and averaged 470 120, 528 107, 575 117, and 538 106, in the control, ESC, MSC, and ESC + MSC groups, respectively (ESC versus controls: P = 0.058; MSC versus controls, P = 0.003; MSC + ESC versus controls: P = 0.057) (Figure 5).

Figure 5.

Immunostaining of cryosections using markers of angiogenesis (RECA): from control (a), human embryonic stem cell (hESC) (b), mesenchymal stem cell (MSC) (c), and MSC + hESC groups (d). Pictures were taken in the core of the infarct area. Images were acquired in microscopy (10). Bar = 0.5 mm.

Full figure and legend (108K)

Apoptosis

Apoptosis, as assessed by caspase 3 activation, did not significantly differ among groups, the intensity of emitted fluorescence being 335.3 4.6, 347.2 69.5, 298.6 24.8, and 297.8 22.3 in the control, MSC, ESC, and MSC + ESC groups, respectively (overall P = 0.13).

Discussion

The major finding of this study is that the combined transplantation of ESCs and MSCs provided a greater functional benefit, compared with controls, than either treatment alone. However, this benefit does not seem to be predominantly mediated by a limitation of graft rejection and might rather involve cell-derived trophic effects on the host tissue.

Data analysis

In hearts receiving combined cell transplantation, as well as in those treated by a single-cell type, MSCs and ESCs both expressed markers of the cardiac, smooth muscle, myofibroblast, and endothelial lineages. In the case of MSCs, this observation is consistent with the well-documented multidifferentiation potential of these cells.⁸ Our finding that they failed to elicit a significant improvement of function compared with controls is at variance with some previous reports,^4,5,6 a discrepancy that could be explained by differences in experimental protocols including cell origin, dosing, and longer duration of follow-up in this study. In the case of ESCs, the expression of cardiac markers supports our previous findings that preimplantation specification of ESCs by appropriate cytokines is effective in driving the treated cells toward a cardiomyogenic lineage⁹ and that ES-derived cardiac progenitors are functionally effective.^10,11 Although no teratoma was observed in any ESC-treated heart, this reassuring safety finding needs to be interpreted cautiously because xenotransplantation may be less likely to cause these tumors than allotransplantation.¹² It is clear that any potential clinical use of ESCs would require a stringent selection of cardiac-specified cells so as to only deliver a "pure" population of cardiac progenitors and, consequently, to maximize the lifeguards against a graft-derived uncontrolled cell proliferation.

Regardless of the phenotype adopted by the injected cells, a consistent finding was a low rate of sustained engraftment in all treated groups, as demonstrated by both immunostaining and quantitative RT-PCR. Several factors are known to contribute to graft loss, including ischemia, anoikis, and inflammation. In addition, exposure of human cells to the fast heart rates of rats may have further exacerbated their damage if one keeps in mind that rapid pacing is an effective means of inducing heart failure in large mammals.¹³ However, as all these events are assumed to have been distributed evenly across the different groups, the finding that the smallest number of surviving cells was found in the ESC group suggests that these hearts may have incurred an additional component of cell death as a result of rejection. This assumption is consistent with the increased recognition that ESCs are immunogenic^14,15 and thus rapidly rejected as their differentiation increases expression of MHC I that can then be recognized by cytotoxic T lymphocytes.¹⁶ Even though more human cells could be detected in the MSC and combined groups, the residual number of surviving cells was still too small to explain the augmentation of pump function in these two groups by a direct contribution of the cellular transplants to contractile activity. This raises the issue of the mechanism(s) by which implanted cells improved recovery of LV function.

Mechanistic hypotheses

MSCs have been reported to be immune privileged and tolerogenic by mechanisms that involve both cell-to-cell contact and release of soluble anti-inflammatory mediators.^2,17 In this study, although the trend for MSCs to be associated with smaller numbers of infiltrating CD3⁺ cells and higher numbers of regulatory FoxP3⁺ lymphocytes could be consistent with some immunomodulation, these effects should be interpreted cautiously in the absence of a strict quantification; in any case, they were seemingly unable to significantly enhance survival of the coinjected ESCs as engraftment rates were not significantly different between the MSC-alone and MSC + ESC groups (P = 0.23), although the latter was injected with a twofold greater number of cells. The inability of MSCs, at least when given as in this study, to act as effective immunosuppressants compared with standard antirejection therapies is further demonstrated by our RT-PCR-based finding of a 20-fold higher number of engrafted ESCs (0.2295 0.3444 ng/l) in a parallel series of experiments where ESC-transplanted hearts were treated with FK-506. These results are actually in line with recent studies that have challenged the view that MSCs were immune privileged by showing that these cells could indeed function as antigen-presenting cells,^18,19 were susceptible to activated natural killer–mediated lysis,^20,21 and lost their in vitro immunoprivileged properties following in vivo implantation because of a gradual expression of MHC II parallel to differentiation.²² Altogether, these data could explain why MSCs have been shown to undergo rejection following either allogeneic MHC-mismatched injection^23,24,25 or xenotransplantation in an infarction model similar to the one used in this study.²⁶

Paracrine effects then represent a sound alternate mechanism by which MSCs may have optimized the benefits of ESCs. This paracrine hypothesis has been raised by the consistent observation that, like in this study, the functional benefits of cell transplantation sharply contrast with the negligible amount of cells remaining engrafted after a few weeks.^27,28 Then, the current prevailing view is thus that engrafted cells release survival-promoting cytokines and growth factors which activate endogenous protective pathways whose effects extend beyond the physical disappearance of the trigger cells. Indeed, MSCs have been shown to reduce native cardiomyocyte apoptosis,^6,29,30 increase angiogenesis,^6,31 and induce changes in the extracellular matrix composition leading to limitation of fibrosis.^5,32,33 Likewise, factors released from ESCs were reported to inhibit H₂O₂-induced apoptosis of a cardiomyocyte cell line.³⁴ The present results are consistent with this hypothesis in that the two cell types reduced fibrosis and increased angiogenesis compared with controls despite the scarcity of their engraftment whereas the lack of between-group differences in the extent of apoptosis could have been due to the relatively late sampling time (2 months) relative to the index ischemic injury. Overall, however, there was a definite trend for the combined group to yield the best outcomes. Because MSCs and ESCs have different transcriptional profiles,³⁵ it is thus tempting to speculate that the two populations of cells expressed a different (although possibly in part overlapping) blend of trophic factors that acted synergistically to improve tissue protection and the related recovery of LV function. This hypothesis is well consistent with the recent observation made in a mouse model of myocardial infarction that the improvement of LV function after transplantation of skeletal myoblasts or ESCs correlates with the release of cardioactive cytokines which trigger endogenous cytoprotective pathways.²⁷

Study limitations

A first limitation of this study is the lack of specific cell labeling before transplantation which then made impossible to distinguish between the mesenchymatous or embryonic origin of the engrafted human cells. Clearly, future studies should include tracking methods to accurately delineate the fate of the individual cell transplants. A second issue is the twofold greater number of injected cells in the combined group compared with the single-cell transplantation groups, which could arguably have biased the results in favor of the former one. This dosing regimen was based on the observation that dose–response studies have shown 5 million to be the optimal ESC number in this model.¹⁰ This value, in turn, dictated the number of MSCs because the study of dose–effect relationships in mixed lymphocyte reaction experiments has shown a 1:1 ratio very effective for inhibiting T-lymphocyte proliferation.^36,37 To match the cell number of the combined group, we should thus have increased the ESC number to 10 million in the ESC-alone group, which we were reluctant to do because no added benefit has been reported beyond 5 million ESCs¹⁰ whereas the risk of tumor is markedly increased.³⁸ Indeed, although a twofold greater cell number was injected in the MSC + ESC group compared with the MSC-alone group, differences in engraftment rates were not significant, which supports the assumption that, beyond a certain threshold, outcomes may be less affected by graft sizes than by the cell-specific paracrine effects on the host myocardium.³⁹ Third, the timing of therapy may have played a confounding role. Namely, it has recently been shown⁴⁰ that proinflammatory cytokines were required for MSCs to exert immunosuppressive effects because these cytokines upregulate inducible nitric oxide synthase and several leukocyte chemokines which may bring immune cells close to MSCs and thus allow MSC-derived nitric oxide to suppress T-cell function. It is thus possible that despite the needle-induced tissue injury, our chronic infarct model did not result in local proinflammatory cytokine levels high enough to trigger this chain of events. In addition, whereas ESCs tend to remain clustered around the needle tracks, MSCs can migrate away from the injection sites, which could have further prevented protection of the graft from rejection. Finally, in this study, MSCs were xenotransplanted in postinfarction scars. We have already mentioned one important limitation of the model due to the discrepancy in heart rates between human and rat cardiomyocytes and the damage which can thus be inflicted to the former when they have to withstand sustained tachycardic rates. Furthermore, given the likely influence of local signals on MSC functional responses,² our conclusions should also be cautiously considered as model specific and may not apply to other conditions of MSC use such as systemic infusions to induce tolerance or MSC sources different from the bone marrow.⁴¹

In conclusion, the present data show that cotransplantation of ESCs and MSCs provided better preservation of LV function compared with single-cell treatment alone. The lack of clear evidence for an immunosuppressive or tolerogenic action of MSCs rather suggests that the benefits of this combined regimen were mediated by synergistic trophic effects that enhanced repair of injured host tissue. As such, these data fit the paradigm that outcomes of cell transplantation might be optimized by the combination of different cell types. From this standpoint, it is noteworthy that a clinical trial is currently investigating the effects of a cotransplantation of islet and MSCs in type I diabetic patients (NCT00646724).

Materials and Methods

Animals. Immunocompetent female Wistar rats (mean weight 250 g; Charles River Laboratories, Arbresle, France) were used in this study. All procedures were in compliance with the Guide for Care and Use of Laboratory animals (NIH publication No 85-23, revised 1996) and Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care.

hESC cultures. HUES-1 and I6 cell lines were cultured on Mouse Embryonic Fibroblasts prepared from E14 mouse embryos using knockout–Dulbecco's minimum essential medium supplemented with mercaptoethanol, glutamine, nonessential amino acids, 15% knockout–serum replacement, and 10 or 5 ng/ml fibroblast growth factor, respectively. The medium was changed every day. Cell colonies were dissociated into single cells or cell clusters every 4–5 days using trypsin (HUES-1) or collagenase (I6), respectively. A similar enzymatic digestion was used before cell transplantation in infarcted rats. ESCs were treated for 48 hours with 10 ng/ml BMP2 in the presence of 1 mol/l SU5402, a fibroblast growth factor receptor inhibitor, in low knockout–serum replacement (5%) containing knockout–Dulbecco's minimum essential medium, a process which has been previously been shown to upregulate genes specific for the cardiac lineage.⁹

Human MSC cultures. Bone marrow samples from healthy donors were obtained from filters used during bone marrow processing for allogeneic transplantation. Bone marrow mononuclear cells were isolated by Ficoll (Invitrogen, Cergy-Pontoise, France) or Ficoll-Hystopaque density gradient (1.077 gr/l, Sigma) and cultured at an initial density of 5 10⁴ cells/cm² in -minimum essential medium (Invitrogen), supplemented with 10% defined fetal calf serum (HyClone, Logan, UT), L-glutamine (2 mmol/l; Invitrogen), antibiotic/antimycotic (Invitrogen), and basic fibroblast growth factor (1 ng/ml; R&D Systems, Lille, France). After 24–48 hours, nonadherent cells were removed and the medium was changed. Cultures were fed every 2 or 3 days until confluence. Adherent cells were then trypsinized, harvested, and subcultured by seeding 1 10³ cells per cm². MSCs were expanded until passage 2, and then collected and injected into animals.

For surface phenotype analysis, MSCs were stained with a core set of antibodies directed to CD34, CD45, CD73, CD90, CD13, CD49a CD44, CD105, HLA-ABC, and HLA-DR or matched isotype controls (all from Becton Dickinson, France). Immunofluorescence analysis was performed using a five-parameter flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA).

Myocardial infarction model. Rats were anaesthetized with isoflurane (Baxter, Maurepas, France), 3% at induction and 2% for maintenance and tracheally ventilated at a rate of 70/minute and with an 0.2-ml average insufflate volume (Alphalab, Minerve, Esternay, France). Analgesia was performed with a 10 mg/kg subcutaneous injection of ketoprofen (Merial, Lyon, France). The heart was exposed through a left thoracotomy and the left coronary artery was permanently ligated between the pulmonary artery trunk and the left atrial appendage.

Experimental design. On the 21th day after infarction, a baseline echocardiography was performed and only rats with a LVEF between 20 and 45% were selected for the study. These animals were then reoperated on by median sternotomy and randomized to receive injections of human MSCs (5 10⁶, n = 10), hESC (5 10⁶ HUES-1, n = 5; 5 10⁶ HUES-I6, n = 5), a combination of MSCs and hESCs [5 10⁶ MSC + 5 10⁶ HUES-1 (n = 5) or 5 10⁶ HUES-I6 (n = 5)] or culture medium (controls, n = 10). As an additional control, six rats were injected with hESCs (5 10⁶ HUES-1) and immunosuppressed by FK506 (Astellas, Tokyo) at the dose of 1 mg/kg/day during 2 weeks, beyond which the dosage was reduced to three times a week. All injections consisted of a 150 l volume that was delivered in three sites (one in the core of the infarct and two at its borders). In the coinjection group, MSCs and hESCs were mixed a few minutes before the injection.

Two months after the procedure, a new echocardiographic assessment was performed, after which the rats were killed.

Functional assessment. Pre- and post-transplantation cardiac function was evaluated by transthoracic echocardiography (Sequoia 516; Siemens, Seine-St Denis, France; equipped with a 15 MHz transducer) in animals sedated with 2% isoflurane (Baxter). Parasternal long- and short-axis views were obtained with both M-mode and two-dimensional images. Left ventricular end-diastolic surface, LV end-systolic surface, LV end-diastolic length, and LV end-systolic length were measured on parasternal long axis views with two-dimensional images. Volumes were calculated as (8/3) (surface²/length). Ejection fraction was calculated as left ventricular end-diastolic surface – left ventricular end-systolic volume/left ventricular end-systolic volume 100. All measurements were made in triplicate and averaged by an investigator blinded to the treatment group.

Histological and immunohistochemical analyses. Two months after cell injections, rats were euthanized under general anaesthesia. Explanted hearts were cut into two halves that were immediately fixed in Optimal Temperature Cutting medium (Tissutec) and frozen at -180 °C in liquid nitrogen. Blocks were then sliced in 7-m-thick cryosections using an ultramicrotome (LM 1850; Leica). Examinations were performed with a microscope (Leica DMIL; Leica, Wetzlar, Germany) equipped with a digital camera (Qicam, QImaging, Burnaby, BC, Canada). For each heart, 10 high-power fields were randomly assessed at different section levels. The delineation of the infarcted area and the search for teratoma were based on standard hematoxylin and eosin staining. The presence of human cells was detected by immunofluorescence using an antibody directed against a specific nuclear human protein (lamin A/C; Novocastra, Newcastle upon Tyne, UK) and the percentage of engraftment was then estimated by computerized planimetry using the Metamorph software (Universal Imaging Corporation, Downingtown, PA). The phenotype of the engrafted human lamin A/C-positive cells was characterized by incubating slides with primary antibodies directed against markers of ventricular cardiomyocytes (mouse monoclonal anti-myosin heavy chain, 1:10; Chemicon, Saint-Quentin en Yvelines, France), endothelial cells (mouse monoclonal anti-CD31, 1:150; BioLegend, San Diego, CA), mesenchymal cells (polyclonal rabbit anti-CD105, 1:150; AnaSpec, Anaheim, CA), and smooth muscle cells (mouse monoclonal anti-SMA, 1:150; Zymed, San Francisco, CA). The proteins were revealed using fluorescein isothiocyanate- or Texas red–conjugated secondary antibodies and dual labeling was confirmed by confocal microscopy (Zeiss LSM-510 meta). Patterns of rejection were assessed using polyclonal rabbit antibodies against CD3⁺ (1:75ème; Dakocytomation, Carpinteria, CA) and CD4⁺ (1:200; Santa Cruz, CA) and mouse monoclonal antibodies against regulatory CD4⁺CD25⁺ FoxP3-expressing lymphocytes (1:100; BioLegend). To determine whether transplanted cells had triggered changes in angiogenesis in the host tissue, sections were stained with mouse monoclonal against rat endothelial cell antigen (clone HIS52, 1:30ème; Serotec, Oxford, UK) conjugated with a biotinylated anti-mouse IgG secondary antibody (Vector Laboratories, Burlingame, CA), and the number of rat endothelial cell antigen–positive vessels was manually counted in 40 high-power fields and expressed as the number of vessels per high-power field (0.2 mm²). The extent of fibrosis was assessed by Sirius red staining and expressed as the ratio between the area of scar tissue (in m²) to the LV area. Apoptosis was assessed by measuring the activation of caspase 3 (in a cell lysate) using The ApoAlert Caspase-3 Assay Plate (Clontech, Mountain View, CA) which contains a fluorogenic substrate specific for caspase 3 immobilized in the wells of a 96-well plate. When cell lysate containing the active caspase is applied to the wells, the caspase cleave its substrate and a fluorescent product is released that can be detected with a standard fluorescence plate reader.

Nuclei were counterstained using 4',6-diamidino-2-phenylindole.

In addition, a whole-body autopsy of each transplanted rat, including brain, lungs, liver, spleen, pancreas, kidneys, periaortic lymph nodes, thymus, spine, and ovaries, was systematically performed for the detection of an extracardiac tumor.

PCR analysis and real-time RT-PCR. RNA was extracted from HES and MSC cells or slices of rat myocardium using the RNA Mini Kit (Qiagen, Courtaboeuf, France). One microgram of RNA was reverse transcribed using the Mu-MLV reverse transcriptase (Invitrogen, Cergy, France) and oligo(16)dT.

Real-time quantitative PCR was performed using a LightCycler (Roche Diagnostics, Mannheim, Germany) or a Chromo4 thermal cycler (Bio-Rad Laboratories, Marne-la-Coquette, France). Amplification was carried out according to manufacturers' instructions. Twelve-microliter reaction mixture contained 12 l of Roche SYBR Green I mix (including Taq DNA polymerase, reaction buffer, deoxynucleoside trisphosphate mix, SYBER Green I dye, and 3 mmol/l MgCl₂), 0.25 mol/l concentration of the appropriate F- and R-primer, and 2 l of cDNA. The amplification programme included the initial denaturation step at 95 °C for 15 or 8 minutes, and 40 cycles of denaturation at 95 °C for 10 seconds, annealing at 65 °C for 8 seconds (LightCycler), and extension at 72 °C for 8 or 30 seconds. The temperature transition rate was 20 °C/second (LightCycler). Fluorescence was measured at the end of each extension step. After amplification, a melting curve was acquired by heating the product at 20 °C/second to 95 °C, cooling it at 20 °C/second to 70 °C, keeping it at 70 °C for 20 second, and then slowly heating it at 20 °C/second to 95 °C. Fluorescence was measured through the slow heating phase. Melting curves were used to determine the specificity of PCR products, which were confirmed using conventional gel electrophoresis. Data were analyzed according to Pfaffl.⁴² Primers specific for human genes are described in Table 2. Gene expression was determined by comparing the intensity of emitted fluorescence in experimental samples with that of a control series containing different known concentrations of total human cDNA [ng/l].

Table 2 - PCR primer sequences.

Full table (51K)

Statistical analysis. Data analysis was performed in a blind fashion by an independent statistician (L.T.). Data are summarized using median (25% percentile; 75% percentile). The changes in LV function parameters (LVEF, EDV, ESV) were compared between treatment groups using nonparametric analysis of covariance with the presacrifice measurement as outcome and the pretransplantation measurement and treatment groups as covariates. Nonparametric tests were used because of the small sample size and because the data did not meet distributional assumptions of parametric tests.

The percentage of lamin-positive areas relative to LV infarcted areas, the mean numbers of vessels per mm², the mean percentages of fibrosis, and the intensity of emitted fluorescence reflecting apoptosis were compared between groups using a two-sample t-test for clustered data, taking into account the intraheart correlation (as multiple slices were used for a single heart). Finally, PCR results were compared between groups using the Wilcoxon nonparametric test.

Statistical significance was set at the 0.05 threshold.

References

REFERENCES

1. Grinnemo, KH, Kumagai-Braesch, M, Månsson-Broberg, A, Skottman, H, Hao, X, Siddiqui, A et al. (2006). Human embryonic stem cells are immunogenic in allogeneic and xenogeneic settings. Reprod Biomed Online 13: 712–724. | PubMed | ChemPort |
2. Uccelli, A, Pistoia, V and Moretta, L (2007). Mesenchymal stem cells: a new strategy for immunosuppression? Trends Immunol 28: 219–226. | Article | PubMed | ChemPort |
3. Ryan, JM, Barry, FP, Murphy, JM and Mahon, BP (2005). Mesenchymal stem cells avoid allogeneic rejection. J Inflamm (Lond) 26: 2–8.
4. Amado, LC, Saliaris, AP, Schuleri, KH, St John, M, Xie, JS, Cattaneo, S et al. (2005). Cardiac repair with intramyocardial injection of allogeneic mesenchymal stem cells after myocardial infarction. Proc Natl Acad Sci USA 102: 11474–11479. | Article | PubMed | ChemPort |
5. Nagaya, N, Kangawa, K, Itoh, T, Iwase, T, Murakami, S, Miyahara, Y et al. (2005). Transplantation of mesenchymal stem cells improves cardiac function in a rat model of dilated cardiomyopathy. Circulation 112: 1128–1135. | Article | PubMed | ISI |
6. Uemura, R, Xu, M, Ahmad, N and Ashraf, M (2006). Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling. Circ Res 98: 1414–1421. | Article | PubMed | ISI | ChemPort |
7. MacDonald, DJ, Luo, J, Saito, T, Duong, M, Bernier, PL, Chiu, RC et al. (2005). Persistence of marrow stromal cells implanted into acutely infarcted myocardium: observations in a xenotransplant model. J Thorac Cardiovasc Surg 130: 1114–1121. | Article | PubMed |
8. Zimmet, JM and Hare, JM (2005). Emerging role for bone marrow derived mesenchymal stem cells in myocardial regenerative therapy. Basic Res Cardiol 100: 471–481. | Article | PubMed | ChemPort |
9. Tomescot, A, Leschik, J, Bellamy, V, Dubois, G, Messas, E, Bruneval, P et al. (2007). Differentiation in vivo of cardiac committed human embryonic stem cells in post-myocardial infarcted rats. Stem Cells 25: 2200–2205. | Article | PubMed | ChemPort |
10. Laflamme, MA, Gold, J, Xu, C, Hassanipour, M, Rosler, E, Police, S et al. (2005). Formation of human myocardium in the rat heart from human embryonic stem cells. Am J Pathol 167: 663–671. | PubMed | ISI | ChemPort |
11. Van Laake, LW, Passier, R, Monshouwer-Kloots, J, Verkleij, AJ, Lips, DJ, Freund, C et al. (2007). Human embryonic stem cell-derived cardiomyocytes survive and mature in the mouse heart and transiently improve function after myocardial infarction. Stem Cell Res 1: 9–24. | Article |
12. Erdö, F, Bührle, C, Blunk, J, Hoehn, M, Xia, Y, Fleischmann, B et al. (2003). Host-dependent tumorigenesis of embryonic stem cell transplantation in experimental stroke. J Cereb Blood Flow Metab 23: 780–785. | Article | PubMed | ISI |
13. Kääb, S, Nuss, HB, Chiamvimonvat, N, O'Rourke, B, Pak, PH, Kass, DA et al. (1996). Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ Res 78: 262–273. | PubMed | ISI | ChemPort |
14. Drukker, M, Katz, G, Urbach, A, Schuldiner, M, Markel, G, Itskovitz-Eldor, J et al. (2002). Characterization of the expression of MHC proteins in human embryonic stem cells. Proc Natl Acad Sci USA 99: 9864–9869. | Article | PubMed | ChemPort |
15. Swijnenburg, RJ, Tanaka, M, Vogel, H, Baker, J, Kofidis, T, Gunawan, F et al. (2005). Embryonic stem cell immunogenicity increases upon differentiation after transplantation into ischemic myocardium. Circulation 112 (9 suppl.): I166–I172. | PubMed | ISI |
16. Drukker, M, Katchman, H, Katz, G, Even-Tov Friedman, S, Shezen, E, Hornstein, E et al. (2006). Human embryonic stem cells and their differentiated derivatives are less susceptible to immune rejection than adult cells. Stem Cells 24: 221–229. | Article | PubMed |
17. Aggarwal, S and Pittenger, MF (2005). Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 105: 1815–1822. | Article | PubMed | ISI | ChemPort |
18. Chan, JL, Tang, KC, Patel, AP, Bonilla, LM, Pierobon, N, Ponzio, NM et al. (2006). Antigen-presenting property of mesenchymal stem cells occurs during a narrow window at low levels of interferon-gamma. Blood 107: 4817–4824. | Article | PubMed | ISI | ChemPort |
19. Stagg, J, Pommey, S, Eliopoulos, N and Galipeau, J (2006). Interferon-gamma-stimulated marrow stromal cells: a new type of nonhematopoietic antigen-presenting cell. Blood 107: 2570–2577. | Article | PubMed | ChemPort |
20. Sotiropoulou, PA, Perez, SA, Gritzapis, AD, Baxevanis, CN and Papamichail, M (2006). Interactions between human mesenchymal stem cells and natural killer cells. Stem Cells 24: 74–85. | Article | PubMed |
21. Spaggiari, GM, Capobianco, A, Becchetti, S, Mingari, MC and Moretta, L (2006). Mesenchymal stem cell-natural killer cell interactions: evidence that activated NK cells are capable of killing MSCs, whereas MSCs can inhibit IL-2-induced NK-cell proliferation. Blood 107: 1484–1490. | Article | PubMed | ChemPort |
22. Liu, H, Kemeny, DM, Heng, BC, Ouyang, HW, Melendez, AJ and Cao, T (2006). The immunogenecity and immunomodulatory function of osteogenic cells differentiated from mesenchymal stem cells. J Immunol 176: 2864–2871. | PubMed | ChemPort |
23. Nauta, AJ and Fibbe, WE (2007). Immunomodulatory properties of mesenchymal stromal cells. Blood 110: 3499–3506. | Article | PubMed | ChemPort |
24. Nauta, AJ, Westerhuis, G, Kruisselbrink, AB, Lurvink, EG, Willemze, R and Fibbe, WE (2006). Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 108: 2114–2120. | Article | PubMed | ISI | ChemPort |
25. Eliopoulos, N, Stagg, J, Lejeune, L, Pommey, S and Galipeau, J (2005). Allogeneic marrow stromal cells are immune rejected by MHC class I- and class II-mismatched recipient mice. Blood 106: 4057–4065. | Article | PubMed | ISI | ChemPort |
26. Grinnemo, KH, Månsson, A, Dellgren, G, Klingberg, D, Wardell, E, Drvota, V et al. (2004). Xenoreactivity and engraftment of human mesenchymal stem cells transplanted into infarcted rat myocardium. J Thorac Cardiovasc Surg 127: 1293–1300. | Article | PubMed | ISI | ChemPort |
27. Ebelt, H, Jungblut, M, Zhang, Y, Kubin, T, Kostin, S, Technau, A et al. (2007). Cellular cardiomyoplasty: improvement of left ventricular function correlates with the release of cardioactive cytokines. Stem Cells 25: 236–244. | Article | PubMed | ISI | ChemPort |
28. Iso, Y, Spees, JL, Serrano, C, Bakondi, B, Pochampally, R, Song, YH et al. (2007). Multipotent human stromal cells improve cardiac function after myocardial infarction in mice without long-term engraftment. Biochem Biophys Res Commun 354: 700–706. | Article | PubMed | ISI | ChemPort |
29. Tang, YL, Zhao, Q, Qin, X, Shen, L, Cheng, L, Ge, J et al. (2005). Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. Ann Thorac Surg 80: 229–236. | Article | PubMed | ISI |
30. Gnecchi, M, He, H, Noiseux, N, Liang, OD, Zhang, L, Morello, F et al. (2006). Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J 20: 661–669. | Article | PubMed | ISI | ChemPort |
31. Tang, J, Xie, Q, Pan, G, Wang, J and Wang, M (2006). Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion. Eur J Cardiothorac Surg 30: 353–361. | Article | PubMed |
32. Kudo, M, Wang, Y, Wani, MA, Xu, M, Ayub, A and Ashraf, M (2003). Implantation of bone marrow stem cells reduces the infarction and fibrosis in ischemic mouse heart. J Mol Cell Cardiol 35: 1113–1119. | Article | PubMed | ISI | ChemPort |
33. Berry, MF, Engler, AJ, Woo, YJ, Pirolli, TJ, Bish, LT, Jayasankar, V et al. (2006). Mesenchymal stem cell injection after myocardial infarction improves myocardial compliance. Am J Physiol Heart Circ Physiol 290: H2196–H2203. | Article | PubMed | ChemPort |
34. Singla, DK and McDonald, DE (2007). Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells. Am J Physiol Heart Circ Physiol 293: H1590–H1595. | Article | PubMed | ChemPort |
35. Ulloa-Montoya, F, Kidder, BL, Pauwelyn, KA, Chase, LG, Luttun, A, Crabbe, A et al. (2007). Comparative transcriptome analysis of embryonic and adult stem cells with extended and limited differentiation capacity. Genome Biol 8: R163. | Article | PubMed | ChemPort |
36. Di Nicola, M, Carlo-Stella, C, Magni, M, Milanesi, M, Longoni, PD, Matteucci, P et al. (2002). Human bone marrow stromal cells suppress T-lymphocyte proliferation induced by cellular or nonspecific mitogenic stimuli. Blood 99: 3838–3843. | Article | PubMed | ISI | ChemPort |
37. Arnulf, B, Lecourt, S, Soulier, J, Ternaux, B, Lacassagne, MN, Crinquette, A et al. (2007). Phenotypic and functional characterization of bone marrow mesenchymal stem cells derived from patients with multiple myeloma. Leukemia 21: 158–163. | Article | PubMed | ChemPort |
38. Behfar, A, Zingman, LV, Hodgson, DM, Rauzier, JM, Kane, GC, Terzic, A et al. (2002). Stem cell differentiation requires a paracrine pathway in the heart. FASEB J 16: 1558–1566. | Article | PubMed | ISI |
39. van Laake, LW, Passier, R, Doevendans, PA and Mummery, CL (2008). Human embryonic stem cell-derived cardiomyocytes and cardiac repair in rodents. Circ Res 102: 1008–1010. | Article | PubMed | ChemPort |
40. Ren, G, Zhang, L, Zhao, X, Xu, G, Zhang, Y, Roberts, AI et al. (2008). Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell 2: 141–150. | Article | PubMed | ChemPort |
41. Keyser, KA, Beagles, KE and Kiem, HP (2007). Comparison of mesenchymal stem cells from different tissues to suppress T-cell activation. Cell Transplant 16: 555–562. | PubMed |
42. Pfaffl, MW (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45. | Article | PubMed | ChemPort |

Acknowledgments

This work was partly funded by the LeDucq Foundation (Cardiac Progenitors Transatlantic Alliance network) and the French Fédération de Cardiologie. E.P. was supported by a grant from the Fondation pour la Recherche Médicale. The authors have no financial conflicts of interest related to the submitted manuscript.