Intramuscular transplantation of myogenic cells in primates: importance of needle size, cell number, and injection volume.

The aim of this study was to quantitatively define the main measurable technical parameters for the intramuscular transplantation of myogenic cells in primates. Myoblasts transduced with the gene coding for ß-galactosidase were injected into the skeletal muscles of 15 monkeys. The following parameters were studied: needle size, number of cells per injection, and volume of cell suspension per injection. Monkeys were immunosuppressed with tacrolimus. The cell-injected sites were biopsied 1 or 2 months later. Biopsies were examined histologically to assess the myoblast engraftment and the muscle structure. The conclusions were as follows: (1) Needles should be thin enough to avoid important tissue damage and allow muscle regeneration as satisfactory as possible. Among those tested, 27G should be the choice if the length is consistent with depth of injection. (2) At least 100,000 cells should be delivered per centimeter of needle trajectory. (3) The smallest volumes of cell suspension per injection should be used. In this study, 1 µl/cm of injection trajectory was sufficient. In principle, these parameters apply to muscles in which no damage occurred other than the injections.

Electroporation as a method to induce myofiber regeneration and increase the engraftment of myogenic cells in skeletal muscles of primates.

Engraftment of intramuscularly transplanted myogenic cells in mice can be optimized after induction of massive myofiber damage that triggers myofiber regeneration and recruitment of grafted cells; this generally involves either myotoxin injection or cryodamage. There are no effective methods to produce a similar process in the muscles of large mammals such as primates. In this study, we tested the use of intramuscular electroporation for this purpose in 11 macaques. The test sites were 1 cm of skeletal muscle. Each site was treated with 3 penetrations of a 2-needle electrode with 1 cm spacing, applying 3 pulses of 400 V/cm, for a duration of 5 milliseconds and a delay of 200 milliseconds during each penetration. Transplantation of ß-galactosidase-labeled myoblasts was done in electroporated and nonelectroporated sites. Electroporation induced massive myofiber necrosis that was followed by efficient muscle regeneration. Myoblast engraftment was substantially increased in electroporated compared with nonelectroporated sites. This suggests that electroporation may be a useful tool to study muscle regeneration in primates and other large mammals and as a method for increasing the engraftment of myoblasts and other myogenic cells in intramuscular transplantation.

Myopathy in a rhesus monkey with biopsy findings similar to human sporadic inclusion body myositis.

A rhesus macaque with generalized muscle atrophy and musculotendinous contractures was detected in our research center. Muscle biopsies showed myofibers with rimmed vacuoles and eosinophilic hyaline inclusions, accumulations of CD8+ and CD4+ lymphocytes and expression of major histocompatibility complex class I in myofibers. Intracellular inclusions were positive to Congo red. Semithin sections and transmission electron microscopy showed autophagic vacuoles within myofibers and myonuclei with inclusions of filaments. These morphological observations conform with the diagnostic criteria of human sporadic inclusion body myositis. This is the first report of this myopathy in nonhuman primates.

Transplanted myoblasts can migrate several millimeters to fuse with damaged myofibers in nonhuman primate skeletal muscle.

A major restriction of the intramuscular transplantation of myoblasts is that the grafted cells fuse mostly with myofibers along the injection trajectories. This has been attributed to a "lack of migration ability" of the grafted myoblasts. It has been assumed that grafted myoblasts remain motionless in the sites of delivery and fuse only with myofibers with which they come into contact. In the present study, we analyzed this phenomenon in 17 cynomolgus monkeys. We found that intramuscularly injected myoblasts within 1 hour after their injection are mainly located in the perimysium and not distributed along the injection trajectories. This suggested that the grafted myoblasts later migrate from the perimysium to fuse with myofibers that are damaged by the injections. Therefore, we analyzed whether ß-galactosidase-labeled myoblasts injected subcutaneously over skeletal muscles migrate in needle-damaged and nondamaged muscle regions. We observed that grafted myoblasts migrated up to 1cm in depth from the muscle surface into the muscles, although they seemingly fused mainly with damaged myofibers. Our findings suggest that myoblast transplantation is not necessarily restricted bya "lack of migration ability" of the grafted cells but by the fact that myoblasts fuse with regenerating myofibers and not with undamaged myofibers.

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.

A first semimanual device for clinical intramuscular repetitive cell injections.

Intramuscular cell transplantation in humans requires so far meticulous repetitive cell injections. Performed percutaneously with syringes operated manually, the procedure is very time consuming and requires a lot of concentration to deliver the cells exactly in the required region. This becomes impractical and inaccurate for large volumes of muscle. In order to accelerate this task, to render it more precise, and to perform injections more reproducible in large volumes of muscle, we developed a specific semimanual device for intramuscular repetitive cell injections. Our prototype delivers very small quantities of cell suspension, homogeneously throughout several needles, from a container in the device. It was designed in order to deliver the cells as best as possible only in a given subcutaneous region (in our case, skeletal muscles accessible from the surface), avoiding wasting in skin and hypodermis. The device was tested in monkeys by performing intramuscular allotransplantations of beta-galactosidase-labeled myoblasts. During transplantations, it was more ergonomic and considerably faster than manually operated syringes, facilitating the cell graft in whole limb muscles. Biopsies of the myoblast-injected muscles 1 month later showed abundant beta-galactosidase-positive myofibers with homogeneous distribution through the biopsy sections. This is the first device specifically designed for the needs of intramuscular cell transplantation in a clinical context.

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.

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 approximately 100-200 microm. We thus analyzed the relationship between the amount of myoblasts injected per site and the volume of ischemic necrosis. Delivering 0.1 x 10(6) and 0.3 x 10(6) myoblasts did not produce ischemic necrosis; pockets of 1 x 10(6), 3 x 10(6), 10 x 10(6), and 20 x 10(6) 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.

Autologous transplantation of muscle precursor cells modified with a lentivirus for muscular dystrophy: human cells and primate models.

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. We tested the ability of lentiviral vectors to deliver a transgene into myogenic cells before their transplantation. Enhanced green fluorescent protein (eGFP) transgene was efficiently transferred into cells and eGFP-positive fibers were generated following transplantation. An eGFP-micro-dystrophin transgene under the control of a cytomegalovirus promoter was then transferred with the same viral vector but caused some toxicity to the mono-nucleated cells. We then used instead a muscle creatine kinase promoter. Dystrophin expression was observed in the muscle fibers after the transplantation of such genetically modified cells into mdx and severe combined immunodeficient mice. Micro-dystrophin expression was also observed in monkey muscles a month after allogenic or autologous transplantation of genetically modified myoblasts. Therapeutic exon skipping was induced by infecting myoblasts of a DMD patient, deleted for dystrophin exons 49 and 50, with a lentivirus expressing a U7 small nuclear RNA containing antisense sequences against exon 51. The modification led to correct exon skipping and to the expression of a quasi-dystrophin in vitro and in vivo. These results demonstrate the feasibility of lentiviral-based ex vivo gene therapy for DMD.

First test of a "high-density injection" protocol for myogenic cell transplantation throughout large volumes of muscles in a Duchenne muscular dystrophy patient: eighteen months follow-up.

A 26-years old Duchenne muscular dystrophy (DMD) patient received normal muscle-precursor cells, proliferated in vitro and implanted in a thenar eminence, biceps brachii, and in a portion of a gastrocnemius by injections placed 1mm from each other or less. Saline was injected in the contralateral gastrocnemius. The patient was immunosuppressed with tacrolimus. The protocol of cell transplantation was well tolerated and did not cause permanent sequels. Some injected sites were biopsied at 1, 14 and 18 months post-transplantation. Muscles were replaced by fat and fibrosis. In the cell-grafted site of the gastrocnemius, 27.5% of the myofiber profiles expressed donor-derived dystrophin 1 month post-transplantation and 34.5% 18 months post-transplantation. The contralateral gastrocnemius was dystrophin-negative. Myofibers were virtually absent in the biceps brachii, where only two dystrophin-positive myofibers were observed. In conclusion, a "high-density injection" protocol was feasible for intramuscular cell-transplantation in a DMD patient and long-term expression of donor-derived dystrophin was observed.

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 beta-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 beta-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.

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 patient's parents were implanted in 0.25 or 1 cm 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 donor's dystrophin in 8 patients. The percentage of myofibers expressing donor's 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.

Dystrophin expression in myofibers of Duchenne muscular dystrophy patients following intramuscular injections of normal myogenic cells.

Three Duchenne muscular dystrophy (DMD) patients received injections of myogenic cells obtained from skeletal muscle biopsies of normal donors. The cells (30 x 10 (6)) were injected in 1 cm3 of the tibialis anterior by 25 parallel injections. We performed similar patterns of saline injections in the contralateral muscles as controls. The patients received tacrolimus for immunosuppression. Muscle biopsies were performed at the injected sites 4 weeks later. We observed dystrophin-positive myofibers in the cell-grafted sites amounting to 9 (patient 1), 6.8 (patient 2), and 11% (patient 3). Since patients 1 and 2 had identified dystrophin-gene deletions these results were obtained using monoclonal antibodies specific to epitopes coded by the deleted exons. Donor dystrophin was absent in the control sites. Patient 3 had exon duplication and thus specific donor-dystrophin detection was not possible. However, there were fourfold more dystrophin-positive myofibers in the cell-grafted than in the control site. Donor-dystrophin transcripts were detected by RT-PCR (using primers reacting with a sequence int eh deleted exons) only in the cell-grafted sites in patients 1 and 2. Dystrophin transcripts were more abundant in the cell-grafted than in the control site in patient 3. Therefore, significant dystrophin expression can be obtained in teh skeletal muscles of DMD patients following specific conditions of cell delivery and immunosuppression.

Resetting the problem of cell death following muscle-derived cell transplantation: detection, dynamics and mechanisms.

We conducted a study in mice to reevaluate and clarify many aspects of the early survival of muscle cells following transplantation. Male mouse muscle cells (primary-cultures and T-antigen-immortalized clones) labeled with [14C]thymidine and beta-galactosidase were injected into female muscles. Each label was detected in the muscles after different time periods. TUNEL, alizarin red, and immunodetection of active caspase-3 were done in muscle sections. The donor cell labels disappeared from the muscles following donor cell death, but this was not instantaneous and even if the donor cells were killed before transplantation, the first 6 hours were not enough to clear [14C]thymidine and Y chromosome. Using the cell pellet before injection as the 100% baseline for cells injected to evaluate cell death can lead to misinterpretations: the Y-chromosome band was 5-fold stronger than that of a muscle injected with cells, irrespective of whether the cells were previously killed or not. There was no evidence of an immediate massive donor cell death. Necrosis (detected by alizarin red) and apoptosis (detected by active caspase-3) were present among the donor myoblasts following transplantation. Necrosis seemed to be the most important mechanism during the first hours. T-antigen immortalized cells died earlier and more massively than primary-cultured cells, but the surviving cells proliferated more. Indeed, they seemed to exhibit more apoptosis and they triggered a more rapid CD8+ cell infiltration. As a result of our findings, many concepts concerning the early donor cell death following myoblast transplantation must be reconsidered.

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.

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.