Nonfunctional mutant Wrn protein leads to neurological deficits, neuronal stress, microglial alteration, and immune imbalance in a mouse model of Werner syndrome.

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter lifespan. Yet, little is known about the impact of WRN mutations on the central nervous system in both humans and mouse models of WS. In the current study, we have performed a longitudinal behavioral assessment on mice bearing a Wrn helicase deletion. Behavioral tests demonstrated a loss of motor activity and coordination, reduction in perception, increase in repetitive behavior, and deficits in both spatial and social novelty memories in Wrn mutant mice compared to age-matched wild type mice. These neurological deficits were associated with biochemical and histological changes in the brain of aged Wrn mutant mice. Microglia, resident immune cells that regulate neuronal plasticity and function in the brain, were hyper-ramified in multiple regions involved with the behavioral deficits of Wrn mutant mice. Furthermore, western analyses indicated that Wrn mutant mice exhibited an increase of oxidative stress markers in the prefrontal cortex. Supporting these findings, electron microscopy studies revealed increased cellular aging and oxidative stress features, among microglia and neurons respectively, in the prefrontal cortex of aged Wrn mutant mice. In addition, multiplex immunoassay of serum identified significant changes in the expression levels of several pro- and anti-inflammatory cytokines. Taken together, these findings indicate that microglial dysfunction and neuronal oxidative stress, associated with peripheral immune system alterations, might be important driving forces leading to abnormal neurological symptoms in WS thus suggesting potential therapeutic targets for interventions.

Human MuStem cells are competent to fuse with nonhuman primate myofibers in a clinically relevant transplantation context: A proof-of-concept study.

We previously reported that human muscle-derived stem cells (hMuStem cells) contribute to tissue repair after local administration into injured skeletal muscle or infarcted heart in immunodeficient rodent models. However, extrapolation of these findings to a clinical context is problematic owing to the considerable differences often seen between in vivo findings in humans versus rodents. Therefore, we investigated whether the muscle regenerative behavior of hMuStem cells is maintained in a clinically relevant transplantation context. Human MuStem cells were intramuscularly administered by high-density microinjection matrices into nonhuman primates receiving tacrolimus-based immunosuppression thereby reproducing the protocol that has so far produced the best results in clinical trials of cell therapy in myopathies. Four and 9 weeks after administration, histological analysis of cell injection sites revealed large numbers of hMuStem cell-derived nuclei in all cases. Most graft-derived nuclei were distributed in small myofiber groups in which no signs of a specific immune response were observed. Importantly, hMuStem cells contributed to simian tissue repair by fusing mainly with host myofibers, demonstrating their capacity for myofiber regeneration in this model. Together, these findings obtained in a valid preclinical model provide new insights supporting the potential of hMuStem cells in future cell therapies for muscle diseases.

Laminin-111: a potential therapeutic agent for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) still needs effective treatments, and myoblast transplantation (MT) is considered as an approach to repair damaged skeletal muscles. DMD is due to the complete loss of dystrophin from muscles. The lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins in the extracellular matrix. Laminin-111 is normally present in skeletal and cardiac muscles in mice and humans but only during embryonic development. In this study, we showed that intramuscular injection of laminin-111 increased muscle strength and resistance in mdx mice. We also used laminin-111 as a coadjuvant in MT, and we showed this protein decreased considerably the repetitive cycles of degeneration, inflammatory reaction, and regeneration. Moreover, MT is significantly improved. To explain the improvement, we confirmed with the same myoblast cell batch that laminin-111 improves proliferation and drastically increases migration in vitro. These results are extremely important because DMD could be treated only by the injection of a recombinant protein, a simple and safe therapy to prevent loss of muscle function. Moreover, the improvement in MT would be significant to treat the muscles of DMD patients who are already weak.

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.

Expression of dog microdystrophin in mouse and dog muscles by gene therapy.

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. Several previous studies demonstrated the feasibility of delivering microdystrophin complementary DNA (cDNA) into mouse and normal nonhuman primate muscles by ex vivo gene therapy. However, these animal models do not reproduce completely the human DMD phenotype, while the dystrophic dog model does. To progress toward the use of the best animal model of DMD, a dog microdystrophin was transduced into human and dystrophic dog muscle precursor cells (MPCs) with a lentivirus before their transplantation into mouse muscles. One month following MPC transplantation, myofibers expressing the dog microdystrophin were observed. We also used another approach to introduce this transgene into myofibers, i.e., the electrotransfer of a plasmid coding for the dog microdystrophin. The plasmid was injected into mouse and dog muscles, and brief electric pulses were applied in the region of injection. Two weeks later, the transgene was detected in both animals. Therefore, ex vivo gene therapy and electrotransfer are two possible methods to introduce a truncated version of dystrophin into myofibers of animal models and eventually into myofibers of DMD patients.

A Comment on "Muscle Xenografts Reproduce Key Molecular Features of Facioscapulohumeral Muscular Dystrophy": What Is New and What Has Already been Done and Reported but Was Not Quoted?

A study was recently published that sought to develop an in vivo model of facioscapulohumeral muscular dystrophy by transplanting muscle precursor cells from a patient into immunodeficient mice. The study largely applied the methodology used by our team in a study published more than two decades ago with a similar objective, albeit for another muscular dystrophy. However, our study is not cited, leaving the wrong idea that the concept, methodology, and part of the results are original to this recent study. Although the recent study is of interest, the omission of our publication, as well as other relevant references, deprives it of an adequate scientific context. We, therefore, want to point out the importance of a careful bibliographic search in any scientific work.

Human Muscle Precursor Cells Form Human-Derived Myofibers in Skeletal Muscles of Nonhuman Primates: A Potential New Preclinical Setting to Test Myogenic Cells of Human Origin for Cell Therapy of Myopathies.

This study aimed to verify if human myogenic cells could participate in muscle regeneration in macaques. This experimental setting would grant researchers a model that could better evaluate the effects of cell therapies in myopathies with a better translation to human patients. Human muscle precursor cells (MPCs) were cultured in vitro and transduced with ß-galactosidase. The cells were subsequently injected into 1-cm3 muscle regions of 6 macaques immunosuppressed with tacrolimus and dexamethasone. Allogeneic ß-galactosidase+ MPCs were injected in other regions as positive controls. Some cell-grafted regions were electroporated to induce extensive muscle regeneration. MPC-grafted regions were sampled 1 month later and analyzed by histology. There were ß-galactosidase+ myofibers in both the regions grafted with human and macaque MPCs. Electroporation increased the engraftment of human MPCs in the same way as in macaque allografts. The histological analysis (hematoxylin and eosin, CD8, and CD4 immunodetection) demonstrated an absence of cellular rejection in most MPC-grafted regions, as well as minimal lymphocytic infiltration in the regions transplanted with human MPCs in the individual with the lowest tacrolimus levels. Circulating de novo anti-donor antibodies were not detected. In conclusion, we report the successful engraftment of human myogenic cells in macaques, which was possible using tacrolimus-based immunosuppression.

Sarcolemmal Complement Membrane Attack Complex Deposits During Acute Rejection of Myofibers in Nonhuman Primates.

We have previously studied in nonhuman primates several aspects of the acute rejection of myofibers, including the histological characteristics, the mechanisms of myofiber elimination by the T cells, and the development of anti-donor antibodies. Here, we report the participation of the complement membrane attack complex (MAC) in this context. We used muscle sections of macaques from experiments of allogeneic muscle precursor cell transplantation with confirmed rejection of the graft-derived myofibers. Sections were stained with hematoxylin and eosin, alizarin red and for immunodetection of MAC, CD8, CD4, C3, C4d, and immunoglobulins. The prominent finding was the presence of sarcolemmal MAC (sMAC) deposits in biopsies with ongoing acute rejection or with recent acute rejection. The numbers of sMAC-positive myofibers were variable, being higher when there was an intense lymphocyte infiltration. Few sMAC-positive myofibers were necrotic or had evidence of sarcolemma permeation. The immunodetection of C3, C4d, and immunoglobulins did not provide significant elements. In conclusion, sMAC deposits were related to myofiber rejection. The fact that the vast majority of sMAC-positive myofibers had no signs of necrosis or sarcolemmal permeation suggests that MAC would not be harmful to myofibers by itself.

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.

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.

The Process of Engraftment of Myogenic Cells in Skeletal Muscles of Primates: Understanding Clinical Observations and Setting Directions in Cell Transplantation Research.

We studied in macaques the evolution of the intramuscular transplantation of muscle precursor cells between the time of administration and the time at which the graft is considered stable. Satellite cell-derived myoblasts labeled with ß-galactosidase were transplanted into 1 cm3 muscle regions following cell culture and transplantation protocols similar to our last clinical trials. These regions were biopsied 1 h, 1, 3, 7 d, and 3 wk later and analyzed by histology. We observed that the cell suspension leaks from the muscle bundles during injection toward the epimysium and perimysium, where most cells accumulate after transplantation. We observed evidence of necrosis, apoptosis, and mitosis in the accumulations of grafted cells, and of potential migration to participate in myofiber regeneration in the surrounding muscle bundles. After 3 wk, the compact accumulations of grafted cells left only some graft-derived myotubes and small myofibers in the perimysium. Hybrid myofibers were abundant in the muscle fascicles at 3 wk posttransplantation, and they most likely occur by grafted myoblasts that migrated from the peripheral accumulations than by the few remaining within the fascicles immediately after injection. These observations explain the findings in clinical trials of myoblast transplantation and provide information for the future research in cell therapy in myology.

Cell Therapy in Myology: Dynamics of Muscle Precursor Cell Death after Intramuscular Administration in Non-human Primates.

Cell therapy could be useful for the treatment of myopathies. A problem observed in mice, with different results and interpretations, is a significant death among the transplanted cells. We analyzed this problem in non-human primates, the animal model more similar to humans. Autologous or allogeneic myoblasts (with or without a reporter gene) were proliferated in vitro, labeled with [14C]thymidine, and intramuscularly injected in macaques. Some monkeys were immunosuppressed for long-term follow-up. Cell-grafted regions were biopsied at different intervals and analyzed by radiolabel quantification and histology. Most radiolabel was lost during the first week after injection, regardless of whether the cells were allogeneic or autologous, the culture conditions, and the use or not of immunosuppression. There was no significant difference between 1 hr and 1 day post-transplantation, a significant decrease between days 1 and 3 (45% to 83%), a significant decrease between days 3 and 7 (80% to 92%), and no significant differences between 7 days and 3 weeks. Our results confirmed in non-human primates a progressive and significant death of the grafted myoblasts during the first week after administration, relatively similar to some observations in mice but with different kinetics.

De Novo Circulating Antidonor's Cell Antibodies During Induced Acute Rejection of Allogeneic Myofibers in Myogenic Cell Transplantation: A Study in Nonhuman Primates.

BACKGROUND: Transplantation of myogenic cells has potential applications in the treatment of muscle pathologies. Excluding purely autologous cell transplantation, graft viability depends on an adequate control of acute rejection (AR). To contribute in understanding AR in this context, we analyzed whether de novo circulating antibodies against donor's cells are detected during induced AR of graft-derived myofibers in nonhuman primates. METHODS: We allotransplanted satellite cell-derived myoblasts in macaques immunosuppressed with tacrolimus. To induce AR of graft-derived myofibers, we administered tacrolimus for 4 weeks to allow complete myofiber formation, and then we stopped tacrolimus administration. Cell-grafted sites were biopsied at tacrolimus withdrawal and then every 2 weeks and analyzed by histology until AR completion. Blood samples were taken before immunosuppression, at tacrolimus withdrawal and then every 2 weeks to detect antibodies against the donor's cells by flow cytometry. RESULTS: There was an increase of antibodies against the donor's cells related to AR in all monkeys. This increase was variable in intensity, and preceded, coincided or followed the histological evidence of AR (focal accumulations of lymphocytes) and/or the loss of myofibers of donor origin, and remained until the end of the follow-up (up to 8 weeks after tacrolimus withdrawal). CONCLUSIONS: Flow cytometry detection of de novo circulating antibodies against the donor's cells was consistently associated with AR. A clear increase in this antibody detection indicated current or recent AR. Smaller increases in comparison to the preimmunosuppression values were not associated with AR.

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.

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.