Central tolerance to myogenic cell transplants does not include muscle neoantigens.

BACKGROUND: Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance. However, given its immunogenicity, we asked whether central tolerance to donor major histocompatibility complex would allow long-term expression of dystrophin, a tissue-specific neoantigen in dystrophic recipients. METHODS: Central tolerance was induced in C57BL/10J mdx (dystrophic) mice by allogeneic bone marrow transplantation (BMT) after conditioning with either lethal total body irradiation (TBI) or an established nonmyeloablative protocol (anti-CD154, anti-CD8 mAbs, and low-dose TBI). Recipients subsequently received donor-strain MT or skin grafts. RESULTS: Long-term hematopoietic chimeras generated using either lethal TBI or the nonmyeloablative regimen were tolerant to donor skin grafts and both primary and secondary donor MT (>90 days). Myogenic cell transplantation survival was decreased when chimerism was transient, which was most common with nonmyeloablative conditioning and fully rather than haplo-mismatched donors. Interestingly, regardless of conditioning, MT was associated with localized muscle infiltration with Foxp3CD4, CD25CD4, and PerforinCD8 cells, whereas skin grafts lacked infiltration. CONCLUSIONS: Central tolerance achieved using regimens that eliminate nearly all endogenous peripheral lymphocytes (i.e., lethal irradiation) or a nonmyeloablative protocol that depleted peripheral CD8 cells, results in lymphocytic infiltration in muscles that received MT but not in skin allografts. This suggests that muscle-specific infiltration may result from lack of negative selection for peripheral neoantigens in the thymus after BMT and that tolerance after MT may rely on peripheral regulatory mechanisms.

1,25-dihydroxyvitamin D3 increases the transplantation success of human muscle precursor cells in SCID mice.

Human muscle precursor cell (hMPC) transplantation is a potential therapy for severe muscle trauma or myopathies. Some previous studies demonstrated that 1,25-dihydroxyvitamin-D3 (1,25-D3) acted directly on myoblasts, regulating their proliferation and fusion. 1,25-D3 is also involved in apoptosis modulation of other cell types and may thus contribute to protect the transplanted hMPCs. We have therefore investigated whether 1,25-D3 could improve the hMPC graft success. The 1,25-D3 effects on hMPC proliferation, fusion, and survival were initially monitored in vitro. hMPCs were also grafted in the tibialis anterior of SCID mice treated or not with 1,25-D3 to determine its in vivo effect. Graft success, proliferation, and viability of transplanted hMPCs were evaluated. 1,25-D3 enhanced proliferation and fusion of hMPCs in vitro and in vivo. However, 1,25-D3 did not protect hMPCs from various proapoptotic factors (in vitro) or during the early posttransplantation period. 1,25-D3 enhanced hMPC graft success because the number of muscle fibers expressing human dystrophin was significantly increased in the TA sections of 1,25-D3-treated mice (166.75 +/- 20.64) compared to the control mice (97.5 +/- 16.58). This result could be partly attributed to the improvement of the proliferation and differentiation of hMPCs in the presence of 1,25-D3. Thus, 1,25-D3 administration could improve the clinical potential of hMPC transplantation currently developed for muscle trauma or myopathies.

A new pro-migratory activity on human myogenic precursor cells for a synthetic peptide within the E domain of the mechano growth factor.

Duchenne muscular dystrophy (DMD) is an inherited disease that leads to progressive muscle wasting. Myogenic precursor cell transplantation is an approach that can introduce the normal dystrophin gene in the muscle fibers of the patients. Unfortunately, these myogenic precursor cells do not migrate well in the muscle and thus many injections have to be done to enable a good graft success. Recent reports have shown that there is extensive splicing of the IGF-1 gene in muscles. The MGF isoform contains a C-terminal 24 amino acids peptide in the E domain (MGF-Ct24E) that has intrinsic properties. It can promote the proliferation while delaying the differentiation of C(2)C(12) cells. Here, we demonstrated that this synthetic peptide is a motogenic factor for human precursor myogenic cells in vitro and in vivo. Indeed, MGF-Ct24E peptide can modulate members of the fibrinolytic and metalloproteinase systems, which are implicated in the migration of myogenic cells. MGF-Ct24E peptide enhances the expression of u-PA, u-PAR and MMP-7 while reducing PAI-1 activity. Moreover, it has no effect on the gelatinases MMP-2 and -9. Those combined effects can favour cell migration. Finally, we present some results suggesting that the MGF-Ct24E peptide induces these cell responses through a mechanism that does not involve the IGF-1 receptor. Thus, this MGF-Ct24E peptide has a new pro-migratory activity on human myogenic precursor cells that may be helpful in the treatment of DMD. Those results reinforce the possibility that the IGF-1Ec isoform may produce an E domain peptide that can act as a cytokine.

Exercise improves the success of myoblast transplantation in mdx mice.

Transplantation of normal muscle precursor cells is a potential approach to restore dystrophin expression within dystrophin [deficient] mdx mice, a model of Duchenne Muscular Dystrophy. This study aims to evaluate whether exercise could improve graft success and hybrid fiber distribution within mdx muscle. eGFP(+) Muscle precursor cells were transplanted into tibialis anterior muscles of mdx mice using a single injection trajectory. During the following weeks, muscle fiber breaks were induced by making mdx mice swim. To evaluate fiber damage, Evans blue solution was injected intraperitoneally to mice 16h before their sacrifice. Tibialis anterior muscles were then harvested and eGFP, dystrophin and Evans blue labeling were analyzed by fluorescent microscopy. Twenty minutes of exercise (i.e., swimming) were used to induce damage in about 30% of TA muscle fibers. Graft success, evaluated as the percentage of hybrid fibers which are eGFP(+), was improved by 1.9-fold after swimming 3 times per week during 4 weeks and by 1.8-fold after daily swimming. Hybrid muscle fiber transversal and longitudinal distribution were also increased after repeated physical efforts. Exercise induced fiber breaks, which improved MPC recruitment and fusion and increased long-term graft success and also transverse and longitudinal distribution of hybrid fibers.

Induction of tolerance across fully mismatched barriers by a nonmyeloablative treatment excluding antibodies or irradiation use.

A mixed-chimerism approach is a major goal to circumvent sustained immunosuppression, but most of the proposed protocols need antibody treatment or host irradiation. Another promising experience involves busulfan combined with cyclophosphamide treatment. Additionally, recent publications demonstrated that, differing from busulfan, treosulfan administration does not present severe organ or hemato toxicities. Currently, Duchenne muscular dystrophy (DMD) patients are treated with chronic immunosuppression for muscle precursor cell transplantation (MT). We have developed a safe tolerance approach within this cellular allotransplantation therapy background. Thus, we have conditioned, prior to a donor BALB/c MT, the dystrophic mouse model C57Bl10J mdx/mdx, with our treatment based on a donor-specific transfusion, then a treosulfan treatment combined with single cyclophosphamide dose, and finally a donor bone marrow transplantation (TTCB). A first MT was performed in all mixed chimeric mice resulting from the TTCB treatment in the left tibialis anterior (TA) muscles. A second MT from the same donor strain was performed 100 days later in the right TA without any additional therapy. Results show that all treated mice developed permanent mixed chimerism. Long-lasting donor-positive fibers were present in both TAs of the mice, which received MT after the TTCB treatment. Only a basal level of infiltration was observed around donor fibers and mixed chimeric mice rejected third-party haplotype skin grafts. Thus, mixed chimerism development with this TTCB conditioning regimen promotes donor-specific stable tolerance, avoiding costimulatory blockade antibodies or irradiation use and side effects of sustained immunosuppressive treatments. This protocol could be eventually applied for MT to DMD patients or others tissue transplantations.

Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase: stable expression of a full-length-dystrophin fusion gene by human myoblasts.

Ex vivo gene therapy offers a potential treatment for Duchenne muscular dystrophy by transfection of the dystrophin gene into the patient's own myogenic precursor cells, followed by transplantation. We used nucleofection to introduce DNA plasmids coding for enhanced green fluorescent protein (eGFP) or eGFP-dystrophin fusion protein and the phage phiC31 integrase into myogenic cells and to integrate these genes into a limited number of sites in the genome. Using a plasmid expressing eGFP, we transfected 50% of a mouse muscle-derived stem cell line and 60% of normal human myoblasts. Co-nucleofection of a plasmid expressing the phiC31 integrase and an eGFP expression plasmid containing an attB sequence produced 15 times more frequent stable expression, because of site-specific integration of the transgene. Co-nucleofection of the phiC31 integrase plasmid and a large plasmid containing the attB sequence and the gene for an eGFP-full-length dystrophin fusion protein produced fluorescent human myoblasts that were able to form more intensely fluorescent myotubes after 1 month of culture. A nonviral approach combining nucleofection and the phiC31 integrase may eventually permit safe autotransplantation of genetically modified cells to patients.

Growth factors improve the in vivo migration of human skeletal myoblasts by modulating their endogenous proteolytic activity.

BACKGROUND: A main technological problem related to the clinical application of myoblast transplantation is the poor migration of transplanted cells. In this study, we investigated a new physiologic approach that consists of coinjecting motogenic factors insulin growth factor (IGF)-1 or basic fibroblast growth factor (bFGF) to enhance the migration of human skeletal myoblasts. Among the different ways by which those factors can induce the cell migration processes, we investigated their capacity to enhance cell endogenous proteolytic activity that will help transplanted cells to migrate through the extracellular matrix. METHODS: In vitro, myoblasts were coincubated with bFGF or IGF-1. Growth factors effects on cell migration were evaluated using invasion chambers, and their effects on proteolytic systems were evaluated by zymography, Western blot, and reverse transcription polymerase chain reaction. In vivo, myoblasts were coinjected with growth factors and the intramuscular migration capacity was assessed using the microtube technique. RESULTS: In vitro, the presence of IGF-1 or bFGF significantly enhanced the expression of the gelatinase matrix metalloproteinase-9 and focalized the fibrinolytic system activity at the cell membrane. In vitro and in vivo, both bFGF and IGF-1 showed strong chemokinetic potentials and improved the migration of human myoblasts. Moreover, the implication some proteinases in the in vivo enhanced migration was confirmed using specific inhibitors (BB94 or amiloride). CONCLUSIONS: These results suggest that IGF-1 or bFGF coinjection with human myoblasts increased their proteolytic activities and consequently their migratory capacity. This study may help to develop approaches that will reduce the number of injection sites for the treatment of Duchenne muscular dystrophy patients.