Marlyne Goulet

Dystrophin expression in muscles of duchenne muscular dystrophy patients after high-density injections of normal myogenic cells.

A clinical trial was conducted to test a new protocol of normal muscle precursor cell (MPC) allotransplantation in skeletal muscles of patients with Duchenne muscular dystrophy (DMD). Cultured MPCs obtained from one of the patients parents were implanted in 0.25 or 1 cm3 of a Tibialis anterior in 9 patients with DMD. MPC injections were placed 1 to 2 mm from each other, and a similar pattern of saline injections was done in the contralateral muscle. The patients were immunosuppressed with tacrolimus. Muscle biopsies were performed at the injected sites 4 weeks later. In the biopsies of the cell-grafted sites, there were myofibers expressing donors dystrophin in 8 patients. The percentage of myofibers expressing donors dystrophin varied from 3.5% to 26%. Evidence of small myofiber neoformation was observed in some patients. Donor-derived dystrophin transcripts were detected by reverse transcriptase-polymerase chain reaction in the cell-grafted sites in all patients. The protocol of immunosuppression was sufficient to obtain these results, although it is not certain whether acute rejection was efficiently controlled in all the cases. In conclusion, intramuscular allotransplantation of normal MPCs can induce the expression of donor-derived dystrophin in skeletal muscles of patients with DMD, although this expression is restricted to the sites of MPC injection.

Efficacy of Myoblast Transplantation in Nonhuman Primates Following Simple Intramuscular Cell Injections: Toward Defining Strategies Applicable to Humans

Nonhuman primates were used to define myoblast transplantation strategies applicable to humans. Nevertheless, previous experiments were based on the use of myotoxins concomitant with the myoblast injections. Since myotoxins must be avoided for clinical applications, we analyzed the efficacy of simple myoblast injections (i.e., myoblasts resuspended only in saline) into monkey muscles. We also evaluated different FK506 dosages (in combination or not with mycophenolate mofetil) for immunosuppression. Allogeneic myoblasts transduced with the beta-galactosidase (beta-Gal) gene were implanted in the muscles of 19 monkeys by injections placed 1 to 2 mm from each other. A biopsy was performed at the implanted sites 1 month later, and histologically studied for demonstration of beta-Gal+ myofibers, lymphocyte infiltration, and CD8+ cells. The presence of antibodies against the donor myoblasts and the blood levels of FK506 were analyzed. Our results show that: (1) If myoblast injections are sufficiently close to each other, high percentages of hybrid myofibers can be obtained following myoblast transplantation in primates (25 to 67% with an interinjection distance of 1 mm). (2) Efficient immunosuppression can be reached by increasing FK506 dosages, but also by combining this drug with mycophenolate mofetil, a combination that reduces toxic effects. The present results represent a step towards a better designing of myoblast transplantation strategies in humans.

Successful myoblast transplantation in primates depends on appropriate cell delivery and induction of regeneration in the host muscle

Myoblast transplantation (MT) may be a potential treatment for severe recessive hereditary myopathies. The limited results of MT in clinical trials led us to improve this technique in monkeys, an animal model phylogenetically similar to humans. Three Macaca mulata monkeys were used as donors and six as receivers for MT. Myoblasts were grown in culture from muscle biopsies of adult monkeys and infected with a retroviral vector encoding the LacZ gene. Different numbers of cells (i.e., 4 x 10(6), 8 x 10(6), and 24 x 10(6) cells) were transplanted into different muscles and 8 x 10(6) cells (resuspended in a notexin solution) were injected in one muscle of four monkeys. For these transplantations, the cell suspension (in a volume of about 100 microl) was injected at 35 sites less than 1 mm apart. Two other monkeys received 100 x 10(6) myoblasts resuspended in 1 ml of HBSS or 1 ml of notexin. For these two monkeys, the myoblasts were injected at 200-250 sites within a small portion of the muscle. All monkeys were immunosuppressed with daily injections of FK506. Four weeks after MT, the transplanted muscle portions were biopsied and the presence of beta-galactosidase-positive (beta-Gal+) muscle fibers was investigated. The number of beta-Gal+ fibers was 822 +/- 150 (site grafted with 4 x 10(6) cells), 1253 +/- 515 (8 x 10(6) cells), 1084 +/- 278 (24 x 10(6)), and 2852 +/- 1211 (notexin). In the monkeys grafted with 100 x 10(6) myoblasts, the number of beta-Gal+ fibers was 4850 (site without notexin) and 9600 (site with notexin). We demonstrated that a precise mechanical distribution of myoblasts into the tissue improves substantially MT in primates. The presence of notexin with the transplanted cells further increased the success of their transplantation. These are the best results obtained either with MT or gene therapy in primates and they encourage the possibility to human MT trials.

Myoblast transplantation in monkeys: control of immune response by FK506.

Myoblasts were grown from monkey muscle biopsies and infected in vitro with a defective retroviral vector containing a cytoplasmic β-galactosidase (β-gal) gene. These myoblasts were then transplanted to 14 different monkeys, 6 of which were immunosuppressed with FK506. Without immunosuppression, only a few myoblasts and myotubes expressing β-gal were observed 1 week after the transplantation, but no cells expressing β-gal were observed after 4 weeks. This result was attributed to immune responses since infiltration by CD4+ or CD8+ lymphocytes was abundant 1 week after transplantation but not after 4 weeks. The expression of interleukin 6 (IL-6), interleukin 2 (IL-2), granulocyte/macrophage colony stimulating factor (GM-CSF), transforming growth factor-beta (TGF-β) and granzyme B mRNAs was increased in the myoblast-injected muscle indicating that the infiltrating lymphocytes were activated. Moreover, antibodies against the donor myoblasts were detected in 3 out of 6 cases. When the monkeys were immunosuppressed with FK506, muscle fibers expressing beta-galactosidase (β-gal) were present 1, 4 and 12 weeks after the transplantation. There was neither significant infiltration by CD4 or CD8 lymphocytes, nor antibodies detected. The mRNA expression of most cytokines was significantly reduced as compared to the nonimmunosuppressed monkeys. These results indicate that FK506 is effective in controlling short-term immune reactions following myoblast transplantation in monkeys and suggest that it may prove useful for myoblast transplantation in Duchenne Muscular Dystrophy patients.

Dynamics of the early immune cellular reactions after myogenic cell transplantation.

The role of immune cells in the early donor cell death/survival following myoblast transplantation is confusing, one of the reasons being the lack of data about the immune reactions following cell transplantation. We used outbred mice as hosts for transplantation of primary cultured muscle cells and T-antigen-immortalized myoblasts. The host muscles were analyzed 1 h to 7 days after cell injection. No net loss of the donor primary cultured cell population was observed in this period. The immune cellular reaction in this case was: 1) a brief (<48 h) neutrophil invasion; 2) macrophage infiltration from days 1 to 7; 3) a specific response involving CTL and few NK cells (days 6 and 7), preceded by a low CD4+ cell infiltration starting at day 3. In contrast, donor-immortalized myoblasts completely disappeared during the 7-day follow-up. In this case, an intense infiltration of CTL and macrophages, with moderate CD4+ infiltration and lower amounts of NK cells, was observed starting at day 2. The nonspecific immune response at days 0 and 1 was similar for both types of donor cells. The present observations set a basis to interpret the role of immune cells on the early death/survival of donor cells following myoblast transplantation.

Intramuscular Transplantation of Human Postnatal Myoblasts Generates Functional Donor-Derived Satellite Cells

Myogenic cell transplantation is an experimental approach for the treatment of myopathies. In this approach, transplanted cells need to fuse with pre-existing myofibers, form new myofibers, and generate new muscle precursor cells (MPCs). The last property was fully reported following myoblast transplantation in mice but remains poorly studied with human myoblasts. In this study, we provide evidence that the intramuscular transplantation of postnatal human myoblasts in immunodeficient mice generates donor-derived MPCs and specifically donor-derived satellite cells. In a first experiment, cells isolated from mouse muscles 1 month after the transplantation of human myoblasts proliferated in vitro as human myoblasts. These cells were retransplanted in mice and formed myofibers expressing human dystrophin. In a second experiment, we observed that inducing muscle regeneration 2 months following transplantation of human myoblasts led to myofiber regeneration by human-derived MPCs. In a third experiment, we detected by immunohistochemistry abundant human-derived satellite cells in mouse muscles 1 month after transplantation of postnatal human myoblasts. These human-derived satellite cells may correspond totally or partially to the human-derived MPCs evidenced in the first two experiments. Finally, we present evidence that donor-derived satellite cells may be produced in patients that received myoblast transplantation.

Ischemic Central Necrosis in Pockets of Transplanted Myoblasts in Nonhuman Primates : Implications for Cell-Transplantation Strategies

Background. Several cell-transplantation strategies implicate the injection of cells into tissues. Avascular accumulations of implanted cells are then formed. Because the diffusion of oxygen and nutrients from the surrounding tissue throughout the implanted cell accumulations may be limited, central ischemic necrosis could develop. We analyzed this possibility after myoblast transplantation in nonhuman primates. Methods. Macaca monkeys were injected intramuscularly with different amounts of myoblasts per single site. These sites were sampled 1 hr later and at posttransplantation days 1, 3, 5, and 7 and analyzed by histological techniques. Results. One day posttransplantation, the largest pockets of implanted cells showed cores of massive necrosis. The width of the peripheral layer of living cells was ∼100–200 &mgr;m. We thus analyzed the relationship between the amount of myoblasts injected per site and the volume of ischemic necrosis. Delivering 0.1×106 and 0.3×106 myoblasts did not produce ischemic necrosis; pockets of 1×106, 3×106, 10×106, and 20×106 myoblasts exhibited, respectively, a mean of 2%, 9%, 41%, and 59% of central necrosis. Intense macrophage infiltration took place in the muscle, invading the accumulations of necrotic cells and eliminating them by posttransplantation days 5 to 7. Conclusions. The desire to create more neoformed tissue by delivering more cells per injection site is confronted with the fact that the acute survival of the implanted cells is restricted to the peripheral layer that can profit of the diffusion of oxygen and nutriments from the surrounding recipient’s tissue.

Use of repeating dispensers to increase the efficiency of the intramuscular myogenic cell injection procedure.

Intramuscular myoblast transplantation in humans and nonhuman primates requires precise repetitive cell injections very close to each other. Performed with syringes operated manually throughout large regions, this procedure takes a lot of time, becoming tiring and thus imprecise. We tested two repetitive dispensers with Hamilton syringes as cell injection devices to facilitate this procedure. Monkeys received intramuscular allotransplantations of β-galactosidase-labeled myoblasts, using either a monosyringe or a multisyringe repeating dispenser. The monosyringe repeating dispenser allowed performing cell injections faster and easier than with a manually operated syringe. The multisyringe dispenser accelerated the procedure still more, but it was not ergonomic. Biopsies of the myoblast-injected sites 1 month later showed abundant β-galactosidase-positive myofibers, with the same density and morphological pattern observed following myoblast transplantation with a syringe operated manually. We recommend the monosyringe repeating dispenser for myoblast transplantation in skeletal muscles and maybe in the heart.

Growth factor coinjection improves the migration potential of monkey myogenic precursors without affecting cell transplantation success.

Duchenne muscular dystrophy (DMD) is an inherited disease and a main target of myogenic cell transplantation (MT). After the failure of the first clinical trials with DMD patients, the poor migration of transplanted cells has been suspected to be a major problem for a more effective clinical application of MT. Previous investigations suggested that the quantity and dispersion of myofibers containing donor cell nuclei might be improved by increasing the migration of the transplanted cells outside the injection sites. Because the coinjection of motogenic factors with human myoblasts enhanced their intramuscular migration following MT in SCID mice, the present study aimed to investigate whether this approach was appropriate to increase MT success in muscles of nonhuman primates. In vitro studies indicated that IGF-1 or bFGF increased components of proteolytic systems involved in myoblast migration. In vitro and in vivo experiments also demonstrated that coinjection of bFGF or IGF-1 was able to improve monkey myogenic cell migration and invasion. Sixty hours after MT in skeletal muscle tissue, the migration distances reached by monkey myoblasts increased by nearly twofold when one of the growth factors was coinjected with the cells. However, long-term observations in adult monkeys suggest that promigratory treatments are not intrinsically sufficient to improve the success of MT. Even if short-term observations reveal that grafted cells are not always trapped inside the injection site and in spite of the fact that both factors enhanced transplanted cell migration, myofibers including grafted cell nuclei were still restrained to the injection trajectory without notable difference in their amount or their dispersion. The incapacity of transplanted cells to fuse with undamaged myofibers, which are located outside the injection sites, is a priority problem to solve in order to improve transplantation success and reduce the number of injections required for the treatment of DMD patients.

Dysferlin expression after normal myoblast transplantation in SCID and in SJL mice

Limb girdle muscular dystrophy type 2B form and Miyoshi myopathy are both caused by mutations in the recently cloned gene dysferlin. In the present study, we have investigated whether cell transplantation could permit dysferlin expression in vivo. Two transplantation models were used: SCID mice transplanted with normal human myoblasts, and SJL mice, the mouse model for limb girdle muscular dystrophy type 2B and Miyoshi myopathy, transplanted with allogeneic primary mouse muscle cell cultures expressing the beta-galactosidase gene under control of a muscle promoter of Troponin I. FK506 immunosuppression was used in the non-compatible allogeneic model. One month after transplantation, human and mouse dysferlin proteins were detected in all transplanted SCID and SJL muscles, respectively. Co-localization of dysferlin and human dystrophin or beta-galactosidase-positive fibers was observed following the transplantation of myoblasts. Dysferlin proteins were monitored by immunocytochemistry and Western blot. The number of dysferlin-positive fibers was 40-50% and 20-30% in SCID and SJL muscle sections, respectively. Detection of dysferlin in both SCID mice and dysferlin-deficient SJL mouse shows that myoblast transplantation permits the expression of the donor dysferlin protein.