Daniel Skuk

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.

Myoblast transplantation: the current status of a potential therapeutic tool for myopathies

The implantation of cultured myogenic cells into the body (myoblast transplantation) is an experimental strategy that is being explored for the potential treatment of myopathies. Its potential benefits should be: (1) to slow down or to stop muscle degeneration, and/or (2) to increase force in wasted muscles. For these objectives, myoblast transplantation may act by two actions: (1) genetic complementation (as a vehicle of normal genes in the case of genetic myopathies), and (2) increasing the myogenic pool of the muscle. During the last decade, myoblast transplantation seemed stagnant in a contradiction of experiments producing good results in mice, against the poor results of human trials. This contradiction was apparent, since the conditions used in mouse models were largely different from those used in dystrophic patients. Our monkey experiments demonstrated that promising results can be observed in large muscles of primates, but under conditions that differ from those previously used in patients. These conditions are: (1) an appropriate immunosuppression, and (2) a careful distribution of sufficient quantities of myoblasts into the recipient muscles. Most of the work on myoblast transplantation is addressed to improve this method by: (1) reducing or avoiding the toxicity of sustained immunosuppression, (2) favoring donor-myoblast migration into the recipient muscle, and (3) defining the factors implicated in the early donor-cell survival following intramuscular implantation. Other research subjects in this field are the potential use of pluripotent stem cells instead of satellite cells, and the potential delivery of the exogenous myogenic cells by the blood stream.

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.

Progress in myoblast transplantation: a potential treatment of dystrophies

Myoblast transplantation (MT) consists of injecting normal or genetically modified myogenic cells into muscles, where they are expected to fuse and form mature fibers. As an experimental approach to treat severe genetic muscle diseases, MT was tested in dystrophic patients at the beginning of the 1990s. Although these early clinical trials were unsuccessful, MT has progressed through the research on animal models. Many factors that may condition the success of MT were identified in the last years. The present review updates our knowledge on MT and describes the different problems that have limited its success. Factors that were first underestimated, like the specific immune response after MT, are presently well characterized. Destruction of the hybrid fibers by activated T‐lymphocytes and production of antibodies against the transplanted myoblasts take place after MT and are responsible for the graft rejection. The choice of the immunosuppression seems to be very important, and FK506 is the best agent known to allow the best results after MT. Under FK506 immunosuppression, very efficient MT were obtained both in mice and monkeys. Moreover, in dystrophic mice it was demonstrated that MT ameliorates some phenotypical characteristics of the disease. The improvement of the survival of the transplanted cells and the increase of their migration into the injected tissue are presently under investigation. Some of the present research is directed also to bypass the immunosuppression by using the patients own cells for MT. In this sense, efforts are conducted to introduce the normal gene into the patients myoblasts before MT and to improve the ability of these cells to proliferate in vitro. Micros. Res. Tech. 48:213–222, 2000.

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 cell transplantation as a potential treatment of myopathies: clinical and preclinical relevant data

Introduction: Myopathies produce deficits in skeletal muscle function and, in some cases, literally progressive loss of skeletal muscles. The transplantation of cells able to differentiate into myofibers is an experimental strategy for the potential treatment of some of these diseases. Areas covered: Among the two routes used to deliver cells to skeletal muscles, that is intramuscular and intravascular, this paper focuses on the intramuscular route due to our expertise and because it is the most used in animal experiments and the only tested so far in humans. Given the absence of recent reviews about clinical observations and the profusion based on mouse results, this review prioritizes observations made in humans and non-human primates. The review provides a vision of cell transplantation in myology centered on what can be learned from clinical trials and from preclinical studies in non-human primates and leading mouse studies. Expert opinion: Experiments on myogenic cell transplantation in mice are essential to quickly identify potential treatments, but studies showing the possibility to scale up the methods in large mammals are indispensable to determine their applicability in humans and to design clinical protocols.

Myoblast transplantation for inherited myopathies: a clinical approach

Myoblast transplantation (MT) is an experimental strategy for the potential treatment of myopathies. MT has two properties that make it potentially beneficial: genetic complementation and myogenic potential. Preclinical experiments on monkeys have shown that promising results can be obtained with MT in large muscles of primates depending on two conditions: appropriate immunosuppression and cell delivery by a method of high-density injections. Preclinical work on MT is being, or may be, addressed to: develop efficient methods of donor cell delivery applicable to clinics; control or avoid acute rejection by methods with the fewest secondary effects; understand the factors that condition the early survival of donor cells following transplantation; increase the success of each individual injection; re-engineer a functional structure in muscles that degenerates to fibrosis and fat substitution; and search for precursor cells with potential advantages over myoblasts.

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.