Allogeneic mesoangioblasts give rise to alpha-sarcoglycan expressing fibers when transplanted into dystrophic mice

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Allogeneic mesoangioblasts give rise to alpha-sarcoglycan expressing fibers when transplanted into dystrophic mice Maria Guttinger a,⁎, Elisiana Tafi a , Manuela Battaglia b , Marcello Coletta a , Giulio Cossu c,d a

Institute for Cell Biology and Tissue Engineering, Fondazione Parco Biomedico San Raffaele, Via Castel Romano 100, 10028 Rome, Italy San Raffaele Scientific Institute, Telethon Institute for Gene Therapy (TIGET), Milan, Italy c Stem Cell Research Institute, San Raffaele Scientific Institute, Milan, Italy d Department of Biology, University of Milan, Italy b

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

Cell therapy for muscular dystrophy involves transplantation of either genetically modified

Received 1 March 2006

autologous cells or normal donor cells that will be rejected unless the host is adequately

Revised version received

immune suppressed. The extent of the immune response appears to be mitigated in this

9 August 2006

case of stem cells, by immune-suppressive and tolerogenic molecules that they release. We

Accepted 16 August 2006

previously reported significant morphological and functional amelioration of a mouse

Available online 18 August 2006

model of limb–girdle muscular dystrophy by transplantation of mesoangioblasts. These are vessel-associated stem cells that can be propagated in vitro and differentiate into several

Keywords:

types of mesoderm including skeletal muscle. In these experiments, both donor cells and

Muscle stem cells

host were syngeneic (C57Bl/6J) and thus possible immune reaction to the donor cells could

Muscular dystrophy

not be appreciated. To address this question, we transplanted H2-mismatched

Cell therapy

mesoangioblasts (BalbC) in the same dystrophic mice, and in addition, we treated the host with different pharmacological drugs (rapamycin, IL-10 or both). The results showed that donor cells give rise to fibers that express the mutated gene product (alphasarcoglycan) even in the absence of immune suppression; however, the combined action of rapamycin and IL-10 increases the number of alpha-sarcoglycan expressing fibers while reducing the levels of inflammatory cytokines. These results indicate that transplantation of mesoangioblasts into immunologically unrelated host leads to long-term survival of donor cells and this may be further enhanced by appropriate protocols of immune modulation, thus setting the stage for experimentation in large animals and in patients. © 2006 Elsevier Inc. All rights reserved.

Introduction Muscular dystrophies are primary defects of skeletal muscle, often depending upon a mutation in a structural gene of the muscle membranes, which progressively reduce motility of the patients, leading in the most severe cases

to paralysis and death [1]. Among novel therapeutic strategies, stem cell transplantation is becoming a real therapeutic opportunity [2], thanks to much recent work describing different types of stem/progenitor cells that show extended proliferation in vitro and the ability to generate normal muscle fibers when transplanted into a dystrophic

⁎ Corresponding author. Fax: +3906 80319054. E-mail address: [email protected] (M. Guttinger). 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.08.012

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muscle. Currently, two alternative strategies are available for experimental and pre-clinical testing: transplantation of autologous, genetically corrected cells or transplantation of normal donor cells. The first strategy requires efficient gene correction that in cases such as Duchenne muscular dystrophy is problematic due to the gigantic dimension of the mutated dystrophin gene, whose cDNA is too large to be accommodated inside a viral vector [3]. Gene correction may be efficiently achieved by either skipping one of the mutated exon [4], or replacement with a truncated version of the cDNA [5] (micro- or minidystrophin). Anyway, the efficacy of these methods, especially in large animals or patients, remains to be analyzed in detail. Moreover, regulatory issues with viral vectors, especially lentiviral, remain to be addressed. Transplantation of mesoangioblasts from normal donors does not pose any of the above problems, but would require immune suppression to avoid allogeneic responses and cell grafts rejection by T lymphocytes. In the case of Duchenne dystrophy, patients should be treated during childhood, with a lifetime immune suppression and all its undesirable consequences. For this reason, the possibility of inducing immune tolerance against donor cells and their derived muscle fibers would be of great importance. Regulatory T cells (Treg) are CD4+ T cell subsets responsible for developing and maintaining immunologic tolerance [6]. Among Treg cells, the naturally occurring Treg type is the most defined subset. These cells are generated in the thymus, co-express CD4 and interleukin-2 receptor alpha (IL-2Rα) chain (CD4+CD25+), represent 5–10% of the CD4+ T lymphocytes in adult mice and humans and play a key role in controlling both the innate and the adaptive immunity [7,8]. CD4+CD25+ cells require cell–cell contact to suppress proliferation and cytokine production by both CD4+ and CD8+ T cell [9] and have been shown to protect mice from autoimmune disorders [10–12] and transplant rejection [13,14]. Full suppressive activity needs the presence of interleukin-10 (IL-10) and TGF-β; interestingly, high levels of IL-10 production in vivo correlate with tolerance in SCID patients transplanted with HLA-mismatched hematopoietic stem cells [15]. Thus, naturally occurring CD4+CD25+ cells represent a valid alternative tool in regenerative medicine, aiming to promote cell engraftment through the tolerization toward alloantigens. Rapamycin is a T-cell-targeted immunosuppressive drug, currently used in the clinic of transplantation medicine to reduce allograft rejection [16]. Recently, it has been demonstrated that rapamycin is able to selectively expand the naturally occurring CD4+CD25+ cells, blocking in the mean time the expansion of effector T cells [17,18]. In addition, rapamycin combined with IL-10 efficiently blocks type 1 diabetes development and induces long-term immunotolerance in the absence of chronic immunosuppression in nonobese diabetic (NOD) mice [18]. The aim of this work was to develop a novel pre-clinical protocol of cell therapy for muscular dystrophy, based on local delivery of non-autologous, vessel-derived stem cells (mesoangioblasts) to dystrophic mice, in order to verify whether long-term correction of the dystrophic phenotype may be achieved by the use of allogeneic normal donor cells. Mesoangioblasts have been shown to efficiently restore

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muscle structure and function in a mouse model of limb– girdle muscular dystrophy [19]. In that case, however, donor wild-type or dystrophic cells were derived from the same strain of the dystrophic animals (C57BL/6) and thus the problem of allogeneic rejection was not faced. Indeed much current experimental evidence suggests that stem cells have developed strategies to escape immune surveillance by secreting and/or inducing secretion of inhibitory cytokines in neighbor cells [20–24]. It remains to be seen whether the differentiated cells that they must generate to correct the function of a given tissue, may also enjoy the same immune privilege. Here we have transplanted wild-type Balb/c mesoangioblasts, following the same protocol and treating different groups of animals with different pharmacological regimens. The results indicate that allogeneic donor cells give rise to muscle fibers that express the mutated gene product for at least three months even in the absence of immune suppression, but this phenomenon is amplified by a combination of rapamycin and IL-10.

Materials and methods Mice Balb/c mice, purchased from Charles River Laboratories (Calco, Italy), and α-SG null mice on C57BL/6 genetic background previously described [19] were used in this study. All mice were kept under specific pathogen-free conditions either at the S. Raffaele Scientific Institute, Milan, or at Biomedical S. Raffaele Science Park of Rome.

Mesoangioblasts transplantation and mice treatment Balb/c mesoangioblasts were isolated from the embryonic aorta as previously described [25]. 5 × 105 cells in 20 μl of sterile PBS were delivered i.m. in the left tibialis anterior muscle. As a positive control, D16 normal mesoangioblasts, from C57BL/6 mice, [19] were also delivered with the same protocol and served as positive, syngeneic control; these mice were analyzed at day 90. SG−/− mice received three mesangioblast injections: at day 0, day 30 and day 60. According to the treatments, mice were grouped as follows: Group 1: untreated mice, did not receive any pharmacological treatment. Group 2: rapamycin (Rapamune, A.G. Scientific, Inc., San Diego, CA), diluted in PBS saline solution (EuroClone), 50% EtOH (Sigma) and administered at 1 mg/kg i.p. once a day for the first 5 days post-transplant, followed by equal doses every other day for 45 days. The treatment was then suspended and started again at day 60 for one week. Group 3: human IL-10 cross-reacting with mouse (BD Biosciences, Mountain View, CA), diluted in PBS and administered i.p. twice a day at a dose of 0.05 μg/ kg for 45 days. The treatment was then suspended and started again at day 60 for one week. Group 4: a combination of rapamycin and IL-10, both delivered as described above. Groups included ten animals; for each group, five mice were sacrificed and analyzed at day 30 after only one injection, and the other five at day 90 (i.e., 25 days after the end of any type of immune suppression).

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Immunohistochemistry For frozen sectioning, tissues were washed in PBS and embedded in OCT compound (Bayer Diagnostic). 5-μm frozen sections were cut onto SuperFrost Plus (Menzel-Glaser)-coated slides. Section were fixed briefly in cold 4% paraformaldehyde solution at 4°C and washed with PBS–1% BSA–0.2% Triton X-100 (Sigma). Sections were then blocked in 20% normal goat serum and incubated for 1 h with primary antibody. The following primary antibodies were used: rabbit anti-laminin (Sigma), mouse anti-alpha-sarcoglycan (Novocastra Laboratories Ltd.), rat anti-CD4 (L3T4) (BD Biosciences), rat anti-CD8 (3.955), biotin-conjugated rat-anti-CD25 (7D4) and biotin-conjugated rat-anti-CD11b (M1/70) (BD Biosciences). After extensive washing, incubation with the following appropriate secondary reagent was done: rhodamine-conjugated goat anti-mouse F (ab′)2, fluorescein isothiocyanate-conjugated goat anti-rabbit F (ab′)2, rhodamine-conjugated goat anti-rat F(ab′)2, fluorescein (DTAF)-conjugated streptavidin (Jackson ImmunoResearch). To avoid unspecific reactions due to secondary antibody cross-

staining, in the case of CD4–CD25 and CD8–CD11b double immunofluoresce stainings, we first used unlabelled antibody (CD4 or CD8) immediately followed by secondary reagent, and then we used biotin-conjugated antibody (CD25 or CD11b) followed by fluorescein-conjugated streptavidin. Isotype controls were included in all experiments. Staining was analyzed using a Nikon TE2000-E confocal microscope and Nikon Metamorph software. Percentage of α-sarcoglycan positive fibers was calculated by counting the number of positive fibers and the total number of fibers (stained for laminin) in at least ten randomly selected microscopic fields of non-adjacent transverse cryostat sections taken at different cranio-caudal levels of the muscle.

Real-time RT-PCR Total RNA was isolated from whole transplanted and untransplanted tibialis anterior muscles using Trizol reagent (Life Sciences, Paisley, UK). Single-stranded cDNA was synthesized with 1 μg of total RNA using Superscript II reverse

Fig. 1 – Immunofluorescence analysis of α-SG and laminin expression in mice transplanted with syngeneic or allogeneic mesoangioblasts under different pharmacological treatment. Laminin (green) and α-SG (red) immunofluorescence on tibialis anterior muscle sections from wild-type (wt) control mice (A), α-SG null mice transplanted with syngeneic D16 mesoangioblasts (B) or with Balb/c mesoangioblasts under either no pharmacological treatment (C), or under rapamycin (D), IL-10 (E) or rapamycin/IL-10 (F) treatment, respectively. Sections were also stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 100 μm.

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transcriptase (Invitrogen) and random hexamers or oligo-dT primers in a total volume of 20 μl. A Taqman-based system and an ABI Prism 7000 sequence detector were used for realtime RT-PCR. All reagents were obtained from Applied Biosystems as Assays-on-Demand Gene Expression products (Warrington, UK). The reactions were run in 96-well plates for 40 cycles of 15 s at 95°C and 60 s at 60°C, after an initial 8.5 min at 95°C to activate the iTaq DNA polymerase. A dissociation thermal protocol was added to analyze the melting peaks of the generated PCR products. Relative expression level of the target gene was normalized to the geometric mean of the Gapdh control gene. α-SG−/− cDNA was used as control for all runs; all mRNAs levels were determined in relation to this [26].

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were cultured in 96-well plates and stimulated with immobilized anti-CD3 mAb (10 μg/ml) (BD Biosciences clone 17A2) and soluble anti-CD28 mAb (1 μg/ml) (BD Biosciences clone 37.51). Alternatively, a mixed lymphocyte culture was performed: total splenocytes (4 × 105/well) were cultured in the presence of either 5 × 105/well irradiated (10.000 rad) mesoangioblasts or 2 × 105/well irradiated (3000 rad) Balb/c splenocytes. Released cytokines were measured in the culture supernatants either after 48 h (for IL-2, IL-4, IL-5, IFN-γ, TNF-α) or after 72 h (for IL-10, IFN-γ, TNF) by flow cytometry-based assay using standard commercially available kits (CBA, BD Biosciences).

Antibodies and flow cytometry T lymphocyte proliferation assay Total splenic cells were isolated by mechanical disruption of the spleen and red blood cells were lysed by quick sterilewater treatment; then CD4+ T lymphocytes were isolated using anti-CD4 mAb-coated microbeads and MiniMacs columns (Miltenyi Biotec). CD4+ cells were then cultured in triplicate in 96 wells at a concentration of 2 × 105 cells/well; as antigen source either irradiated (10.000 rad) Balb/c mesangioblasts 105 cells/well, or lysates in 6 M urea 10 μg/ml from autologous transplanted or untransplanted contralateral muscles. The polyclonal mitogen concanavalin A (ConA, Sigma), 5 μg/ml, was used for proliferation control. After culture at 37°C for three days, 3H-thymidine was added, cells were harvested 18 h later and thymidine incorporation was measured.

The following antibodies (BD PharMingen) were used in this study: for flow cytometry anti-CD4 (RM4-5) and anti-CD25 (7D4). Antibodies were labeled with phycoerythrin (PE) and APC, respectively. To minimize unspecific staining, total splenocytes were first pre-incubated with unlabeled mAb to FcγRIII/II (CD16/32) on ice for 15 min. Then, after washing with cold PBS/5%FCS, cells were incubated with the indicated antibodies on ice for 15 min, washed and analyzed with a FACSCalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences).

Statistical analysis Data from multiple experiments were expressed as mean ± SD. Statistical analysis was performed using ANOVA test.

Cytokines profiles

Results and discussion CD4+ T lymphocytes were isolated from total splenic cells using anti-CD4 mAb-coated microbeads and MiniMacs columns (Miltenyi Biotec). Purified CD4+ T cells (2 × 105/well)

To analyze survival and muscle differentiation of donor allogeneic mesoangioblasts after transplantation in a

Fig. 2 – Quantitative RT-PCR analysis of α-SG expression in the tibialis anterior muscle from α-SG null mice transplanted with syngeneic or allogeneic mesoangioblasts under different pharmacological treatment. mRNA from whole tibialis anterior muscle was reverse-transcribed and used as the template for PCR with specific primers and probes for α-SG. Relative expression level of the target gene was normalized to the geometric mean of the GAPDH control gene. α-SG−/− cDNA, which is given a value of 1, was used as control for all runs; all mRNAs levels were determined in relation to this. W.T.: wild type; Syn.: syngeneic transplantation; Untr.: allogeneic transplantation, without any pharmacological treatment; Rapa: allogeneic transplantation with rapamycin treatment; IL-10: allogeneic transplantation with interleukin 10 treatment; Rapa + IL-10: allogeneic transplantation with Rapa + IL-10 treatment.

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dystrophic muscle, we used the alpha-sarcoglycan null mouse (α-SG−/−), a mouse model of limb–girdle myopathy, on C57BL/6 genetic background (H2b) [27]. We first isolated mesoangioblast cell lines from the dorsal aorta of E10 embryos from H2-mismatched mouse strains; i.e., Balb/c (H2d), C3H (H2k) and CD1 (outbred) [28]. Unexpectedly, we observed a different mesoangioblast cloning efficiency among different strains tested; for example, Balb/c and C3H generated five-fold-less clones compared to C57BL/6 and CD1. However, once established in culture, all clones from the different mouse strains appeared almost identical with respect to marker expression (CD34, Sca-1, Pax3, MEF2C, etc.), proliferation rate or differentiation potency towards smooth, skeletal or cardiac muscle (data not shown). Balb/c mesoangioblasts were chosen for in vivo experiments and subjected to an experimental protocol similar to that previously utilized for H2-matched (C57BL/6) mesoangioblasts [19]. Donor cells were transplanted three times (days 0, 30 and 60) into ten α-SG−/−-untreated mice (group 1). Group 2 (ten mice) received rapamycin [29]; group 3 (ten mice) was treated with IL-10 [30]; group 4 (ten mice) received both rapamycin and IL-10, which promote tolerance to allogeneic islet transplantation [18]. In addition, four SG−/− mice were transplanted with C57BL/6 syngeneic mesoangioblasts, as a control group. We performed three serial injections to increase the number of positive fibers and were forced to use intra-muscular (i.m.) rather than intraarterial injections because preliminary experiments had shown that the surgery associated with the latter procedure almost invariably results in severe infections in immune suppressed animals. One half of the mice (n = 5) was analyzed at day 30 after only one injection, the other half at day 90 (i.e., 25 days after the end of any type of immune suppression). Analysis performed at day 30 did not show any appreciable difference between the four groups, both in terms of the limited distribution of α-SG, and the absence of any inflammatory infiltrate. This poor immune response was expected for the groups under continuous pharmacological treatment, which appeared to be all of equivalent in efficacy, but was also observed for the untreated group. This result could be explained by the limited exposure of newly generated fibers to the immune system. On the contrary, as shown in Fig. 1, immunofluorescence analysis performed on transplanted tibialis anterior muscle sections at day 90 showed a different level α-SG expression after different treatments: donor mesoangioblasts gave rise to few small clusters of α-SG positive fibers even in the absence of any treatment, a significant result because in this model of muscular dystrophy there are no revertant fibers [27]. However, the number of α-SG positive fibers was significantly (p < 0.001 by ANOVA test) higher (about 10% in comparison with 5% of untreated group) in mice similarly transplanted, but treated with rapamycin. IL-10 alone resulted in no significant increase over untreated, whereas the combined treatment of rapamycin and IL-10 produced the most extensive expression of α-SG (15%) a value significantly (p < 0.011) higher than what observed with rapamycin alone and comparable (though somehow lower) with the expression observed after injection of D16 syngeneic mesoangioblasts.

Analysis of the SG transcripts obtained by real-time RTPCR on cDNAs from transplanted muscles at day 90 confirmed the results described above. As shown in Fig. 2, production of SG could be detected in all transplanted groups when compared with untransplanted α-SG−/− dystrophic mice; after transplantation of allogeneic mesoangioblasts, a low level of expression was detected in the absence of any treatment and in mice treated with IL-10 alone; a significantly (p < 0.001 by ANOVA test) higher level was detected in mice treated with rapamycin and still slightly higher in mice treated with rapamycin/IL-10, although this value was still 40% lower than the level observed in mice transplanted with D16 syngeneic mesoangioblasts. No appreciable differences in SG transcript levels among the different groups were observed at day 30 (data not shown). To identify and characterize lymphatic infiltrates in the transplanted regions, CD4/CD25 and CD8/CD11b double immunofluorescence staining was used. Practically no infiltrates were detected at day 30. As summarized in Fig. 3A, at day 90 CD4+, as well as CD8+, CD4+CD25+ T cells and monocytes, as defined by CD11b marker, were detectable in all transplanted

Fig. 3 – (A) Analysis of cellular infiltrates. Cellular infiltration in transplanted muscle was investigated by immunofluorescence using anti-CD4, CD8, CD25 and CD11b. For each marker or combination (CD4/CD25), positive cells were counted: average numbers of twenty fields/animal are reported. (B) Presence of CD4+CD25+ Treg cells in transplanted muscles. CD4 (red) and CD25 (green) immunofluorescence on the tibialis anterior muscle from α-SG transplanted mice under rapamycin/IL-10 treatment. Sections were also stained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 20 μm.

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Fig. 4 – (A) Frequency of CD4+CD25+ T cells in spleen. Percentages of Treg cells was evaluated on gated CD4+ cells by antibody staining and FACS analysis in splenocytes of transplanted mice, either untreated or treated with rapamycin (Rapa), interleukin 10 (IL-10) or rapamycin + IL-10 (rapa + IL-10). (B) IL-5 and IL-10 production by CD4+ T cells of transplanted mice. Purified splenic CD4+ T cells from transplanted mice either untreated or treated with rapamycin (Rapa), interleukin 10 (IL-10) or rapamycin + IL-10 (rapa + IL-10) were stimulated in vitro with allogeneic mesoangioblasts. Cytokines present in the supernatants were assessed by flow cytometry-based assay using standard commercially available kits (CBA, BD Biosciences).

muscles. The magnitude of cellular infiltration was higher in untreated mice, and ratios between effector and regulatory cells were different in treated mice. In particular, the group treated with rapamycin had the highest numbers of CD4+CD25+ T cells (p < 0.001 in relation to all other groups), but also of CD8 T cells, although this difference was statistically significant (p < 0.01) only against the group treated with rapamycin + IL-10. Indeed, rapamycin/IL-10-treated mice,

but not the IL-10-treated ones, showed a reduction in the number of total infiltrates and CD4 and CD8 cells (p < 0.001 for CD4 cells and p < 0.01 for CD8 cells) compared to untreated mice, indicating that rapamycin and IL-10 exert their action in a synergistic way. The increment in CD4+CD25+ T cells observed in situ was partially reflected in spleens. As revealed by antibody staining and FACS analysis (Fig. 4), the frequency of CD4+CD25+ T cells

Fig. 5 – T lymphocyte proliferation assay. Purified splenic CD4+ T cells from transplanted mice either untreated or treated with rapamycin (Rapa), interleukin 10 (IL-10) or rapamycin + IL-10 (rapa + IL-10) were stimulated in vitro with allogeneic mesoangioblasts (mesoang.) or total protein lysate from untransplanted (untr.lys.) or transplanted (transpl.lys.) muscles. Specific proliferation was measured by thymidine (3H-TdR) incorporation.

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was only slightly increased in rapamycin and rapamycin + IL10 groups. These data suggest that CD4+CD25+ Treg cells might accumulate in the transplanted regions. However, the mere expression of CD25 does not account for regulatory properties. Interestingly, cytokine production profiles of purified CD4+ T lymphocytes and irradiated mesoangioblasts revealed that in spleens of treated mice there was a high proportion of Tr1 cells producing high levels of IL-10 and IL-5. IL-10 was produced to a much lesser extent when CD4 cells were polyclonally stimulated with anti-CD3/CD28 antibodies (data not shown), suggesting a certain degree of antigen specificity in the production of this cytokine. Levels of effector Th1 (IL-2, IFN-γ) and Th2 (IL-4) cytokines were very similar in all groups (data not shown). To verify the establishment of an effective tolerization, proliferation assays were used. CD4 cells were stimulated with irradiated Balb/c allogeneic mesoangioblasts and protein extracts from either transplanted or contralateral tibialis anterior muscles. As shown in Fig. 5, all groups, including the untreated one, poorly reacted to donor mesoangioblasts, confirming that stem cells enjoy some kind of immune privilege [22]; on the other hand, responses to transplanted muscles were high in all mice. However, a significant (p < 0.001 by ANOVA test) reduction in the proliferation extent was observed in rapamycin group and rapamycin + IL-10 group. Protein extracts from contralateral untransplanted muscle did not elicit a proliferative response. These results indicate that mesoangioblasts are immune privileged, but the newly differentiated muscle fibers that they form are much more immunogenic [31,32]. Nevertheless, the fibers that were formed in the absence of any immune suppression should have been completely destroyed by the immune reaction that we could measure by the lymphocyte proliferation assay. One possible explanation for this apparent contradiction may be found in the continuous presence of immune-suppressive molecules produced by those stem cells that had not differentiated. It is known that only a fraction of transplanted cells undergo terminal muscle differentiation in the host: the remaining population may be responsible for this protective effect that could not be detected in an in vitro assay. This possibility may also explain, at least in part, a recently reported similar result [33]. In this case, human stem cells derived from adipose tissue were transplanted into mdx dystrophic mice and, unexpectedly, fibers expressing human dystrophin survived in the mouse host even in the absence of immune suppression. Furthermore, pharmacological treatment with rapamycin and IL-10 seems to favor the development of antigen-specific Treg cells that might contribute in preserving the newly generated muscular fibers. Recently, the possibility of inducing and expanding antigen-specific Treg cells has been reported [34], and this represents an appealing perspective for stem cell transplantation and regenerative medicine [35]. Thus, it is possible that stem cell therapies may in the future be tested in patients with modest and decreasing levels of immune suppression, thus increasing the overall feasibility of this therapeutic strategy. Additional strategies such as induced micro-chimerism may later contribute to total elimination of immune suppression and real perspective of clinical efficacy for stem cell transplantation.

Acknowledgments We thank Maria Grazia Roncarolo for suggestions and help with the planning of the experiments. This work was supported by a grant from Istituto Superiore di Sanità (Progetto Cellule Staminali). Support was also obtained by Leducq Foundation, European Community, Telethon, MDA, AFM and Duchenne Parent Project.

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