Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy

The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.     

The Fate of Myoblasts Following Transplantation into Mature Muscle

Cell transplantation has potential benefits for tissue replacement in the the enhancement of tissue regeneration and as cell-mediated gene therapy for systemic diseases. The transplantation of myoblasts into skeletal muscle also allows gene transfer into cells of the host since myoblasts fuse with host fibers thereby forming hybrid myofibers. The success of myoblast transplantation can be determined by a variety of measures, such as the percentage of myoblasts that fuse, the number of hybrid myofibers formed, or the level of transgene expression. Each measure is a reflection of the fate of the transplanted cells. In order to compare different measures of transplantation efficacy, we followed the fate of transplanted myoblasts expressing the marker enzyme β-galactosidase (β-gal) in two different assays. Two weeks after transplantation, the number of hybrid myofibers was determined histochemically, whereas transgene (β-gal) expression was measured biochemically. To control for variabilities of transplantation among different animals, we obtained both measurements from each muscle by using alternate cryosections in the two assays. Within each individual muscle, both hybrid fiber number and/β-gal expression were maximal at the site of implantation and diminished in parallel with distance from the site. However, for determining the success of transplantation among groups of muscles, these two measures of efficacy yielded discordant results: the transplants with the highest number of hybrid fibers were not the transplants with the greatest β-gal activity. Such discrepancies are likely due to regional variations at the transplantation site that arise when cells are introduced into a solid tissue. These results demonstrate the importance of multiple measures of cell fate and transplantation efficacy for studies of cell trans-plantation and for the application of such studies to cell therapy and cell-mediated gene therapy.     

Transplantation of normal or genetically modified myoblasts for the treatment of hereditary or acquired diseases

The clinical trials of myoblast transplantation in Duchenne Muscular Dystrophy (DMD) patients produced disappointing results. The main problems responsible for these poor results have since then been identified and partially resolved. One of them was related to the use of an inadequate immunosuppression and, since then, immunosuppression with FK506 has permitted successful myoblast transplantation not only in mice but also in monkeys. The requirement for a sustained immunosuppression may be eventually avoided by developing a state of tolerance to the allogeneic cells or by autologous transplantation of genetically corrected myoblasts or stem cells. The rapid death of 75-80% of the injected myoblasts during the first five days has also contributed to the limited success of the early trials. This death was due to an inflammatory reaction and has been compensated in animal experiments by the injection of a larger number of cells (30 millions per cc). Finally, the myoblasts migrated only 0.5 mm away from their site of injection. This problem is currently compensated in animal experiments by injecting the myoblasts at every mm. The number of injections required may eventually be reduced by transfecting myoblasts with one or several metalloproteinase genes. The very good results obtained during the last two years in primates permit us to undertake a new phase I clinical trial to verify that myoblast transplantation can lead to the formation of muscle fibers expressing normal dystrophin in muscles of DMD patients.     

Absence of MyoD Increases Donor Myoblast Migration into Host Muscle

Donor myoblast migration is a major limiting factor in the success of myoblast transfer therapy, a potential treatment for Duchenne muscular dystrophy. A possible strategy to promote the migration of donor myoblasts into host muscle is to enhance their proliferation and delay their fusion, two properties that are major characteristics of myoblasts in regenerating skeletal muscle in MyoD null (-/-) mice. Here we investigate whether the migration of MyoD (-/-) donor myoblasts into host muscle is enhanced in vivo. Sliced muscle grafts from male MyoD (-/-) or normal control (Balb/c) mice were transplanted into the muscles of female normal (Balb/c) host mice. Muscles were sampled at 1, 3, and 12 weeks after grafting, and the fate of male donor myoblasts within female host muscles determined by in situ hybridization with the mouse Y-chromosome-specific Y-1 probe. MyoD (-/-) donor myoblasts migrated into host muscle continuously over 1, 3, and 12 weeks after grafting, in contrast with Balb/c donor myoblasts, whose overall numbers and migratory distances did not increase significantly after 1 week. These results strongly support a role for elevated donor myoblast proliferation and/or their delayed fusion in enhancing migration into host muscle in vivo, and endorse the use of either genetically engineered donor myoblasts, or the administration of exogenous myoblast mitogens to improve donor myoblast migration in myoblast transfer therapy.     

Macrophages Improve Survival,Proliferation and Migration of Engrafted Myogenic Precursor Cells into MDX Skeletal Muscle

Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i) myoblast survival by limiting their massive death, ii) myoblast expansion within the tissue and iii) myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.     

Tubulyzine, a novel tri-substituted triazine, prevents the early cell death of transplanted myogenic cells and improves transplantation success.

Myoblast transplantation (MT) is a potential therapeutic approach for several muscular dystrophies. A major limiting factor is that only a low percentage of the transplanted myoblasts survives the procedure. Recent advances regarding how and when the myoblasts die indicate that events preceding actual tissue implantation and during the first days after the transplantation are crucial. Myoseverin, a recently identified tri-substituted purine, was shown to induce in vitro the fission of multinucleated myotubes and affect the expression of a variety of growth factors, and immunomodulation, extracellular matrix-remodeling, and stress response genes. Since the effects of myoseverin are consistent with the activation of pathways involved in wound healing and tissue regeneration, we have investigated whether pretreatment and co-injection of myoblasts with Tubulyzine (microtubule lysing triazine), an optimized myoseverin-like molecule recently identified from a triazine library, could reduce myoblast cell death following their transplantation and consequently improves the success of myoblast transplantation. In vitro, using annexin-V labeling, we showed that Tubulyzine (5 microM) prevents normal myoblasts from apoptosis induced by staurosporine (1 microM). In vivo, the pretreatment and co-injection of immortal and normal myoblasts with Tubulyzine reduced significantly cell death (assessed by the radio-labeled thymidine of donor DNA) and increased survival of myoblasts transplanted in Tibialis anterior (TA) muscles of mdx mice, thus giving rise to more hybrid myofibers compared to transplanted untreated cells. Our results suggest that Tubulyzine can be used as an in vivo survival factor to improve the myoblast-mediated gene transfer approach.     

Induction of Macrophage-Like Immunosuppressive Cells from Mouse ES Cells That Contribute to Prolong Allogeneic Graft Survival

Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) as a donor resource for transplantation. However, immune suppression is still needed when the donor-recipient combination is allogeneic. Protection of ESCs-derived grafts from host immune response might be achieved thought the utilization of immunosuppressive cells generated from ESCs. In the present study, we show that a certain fraction of immunosuppressive cells can be generated from ESCs and help to suppress immune response against allogeneic grafts. ESCs-derived suppressor cells (ES-SCs) resembled macrophages in terms of cell surface molecule and gene expressions. Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response. Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule. Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.     

Development of Approaches to Improve Cell Survival in Myoblast Transfer Therapy

Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle–derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.     

Mass and functional capacity of regenerating muscle is enhanced by myoblast transfer

Morphology and functional capacity of homotopically transplanted extensor digitorum longus muscles (EDL) of adult SCID mice that received 1 × 10⁶ myoblasts [stably transfected to express nuclear localizing β-galactosidase under the control of the myosin light-chain 3F promoter/enhancer] 2 days posttransplantation were evaluated 9 weeks after transplantation, to determine whether the injection of exogenous myoblasts had an effect on muscle regeneration. Regenerated muscles that received exogenous myoblasts were compared to similarly transplanted muscles that received (a) no further treatment, or (b) sham injection of the vehicle (without myoblasts) and to unoperated EDL. Nine weeks after myoblast transfer, myofibers containing donor-derived nuclei could be identified after staining with X-gal solution. Judging from its size and poor functional performance compared to muscles subjected to transplantation only, sham injection provided a secondary trauma to the regenerating muscle from which it failed to fully recover. In comparison to the sham-injected muscle, the myoblast-injected muscles weighed 61% more and had 50% more myofibers and 82% more cross-sectional area occupied by myofibers at the muscles' widest girths. Their absolute twitch and tetanic tensions were threefold and twofold greater, respectively, and their specific twitch and tetanic tensions were 71% and 50% greater, respectively, than those of sham-injected muscles. In many parameters, the regenerating muscle subjected to myoblast transfer equaled or exceeded those of muscles that were transplanted only received only one trauma). Absolute twitch and tetanic tensions were 73% and 65% greater, respectively, and specific twitch tensions of the muscles receiving myoblasts were 50% greater than forces generated by muscles subjected to whole-muscle transplantation only. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 185–198, 1997     

Neonatal induction of tolerance to skeletal tissue allografts without immunosuppression

Vascularized allogeneic skeletal tissue transplantation without the need for host immunosuppression would increase reconstructive options for treating congenital and acquired defects. Because the immune system of a fetus or neonate is immature, it may be possible to induce tolerance to allogeneic skeletal tissues by alloantigen injection during this permissive period. Within 12 hours after birth, 17 neonatal Lewis rats were injected through the superficial temporal vein with 3.5 to 5 million Brown Norway bone marrow cells in 0.1 ml normal saline. Ten weeks after the injection, peripheral blood from the Lewis rats was analyzed for the presence of Brown Norway cells to determine hemopoietic chimerism. The Lewis rats then received a heterotopic, vascularized limb tissue transplant (consisting of the knee, the distal femur, the proximal tibia, and the surrounding muscle on a femoral vascular pedicle) from Brown Norway rat donors to determine their tolerance to the allogeneic tissue. A positive control group (n = 6) consisted of syngeneic transplants from Lewis rats into naive Lewis rats to demonstrate survival of transplants. A negative control group (n = 6) consisted of Brown Norway transplants into naive Lewis rats not receiving bone marrow or other immunosuppressive treatment. The animals were assessed for transplant viability 30 days after transplantation using histologic and bone fluorochrome analysis. All the syngeneic controls (Lewis to Lewis) remained viable throughout the experiment, whereas all the Brown Norway to Lewis controls had rejected. Ten of the 17 allografts transplanted into bone marrow recipients were viable at 30 days, with profuse bleeding from the ends of the bone graft and the surrounding graft muscle. The percent of chimerism correlated with survival, with 3.31 percent (SD = 1.9) of peripheral blood, Brown Norway chimerism present in the prolonged survival groups and 0.75 percent (SD = 0.5) of Brown Norway chimerism in the rejected graft group. This study demonstrated prolonged survival of allogeneic skeletal tissue without immunosuppression after early neonatal injection of allogeneic bone marrow in a rat model.     

Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treatment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and massive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42°C for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and nontreated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analysis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treatment.     

Heat shock pretreatment enhances porcine myoblasts survival after autotransplantation in intact skeletal muscle

Myoblast transplantation (MT) is a cell-based gene therapy treatment, representing a potential treat-ment for Duchenne muscular dystrophy (DMD), cardiac failure and muscle trauma. The rapid and mas-sive death of transplanted cells after MT is considered as a major hurdle which limits the efficacy of MT treatment. Heat shock proteins (HSPs) are overexpressed when cells undergo various insults. HSPs have been described to protect cells in vivo and in vitro against diverse insults. The aim of our study is to investigate whether HSP overexpression could increase myoblast survival after autotransplantation in pig intact skeletal muscle. HSP expression was induced by warming the cells at 42℃ for 1 h. HSP70 expression was quantified by Western blot and flow cytometry 24 h after the treatment. To investigate the myogenic characteristics of myoblasts, desmin and CD56 were analysed by Western blot and flow cytometry; and the fusion index was measured. We also quantified cell survival after autologous transplantation in pig intact skeletal muscle and followed cell integration. Results showed that heat shock treatment of myoblasts induced a significative overexpression of the HSP70 (P < 0.01) without loss of their myogenic characteristics as assessed by FACS and fusion index. In vivo (n=7), the myoblast survival rate was not significantly different at 24 h between heat shock treated and non- treated cells (67.69% ± 8.35% versus 58.79% ± 8.35%, P > 0.05). However, the myoblast survival rate in the heat shocked cells increased by twofold at 48 h (53.32% ± 8.22% versus 28.27% ± 6.32%, P < 0.01) and more than threefold at 120 h (26.33% ± 5.54% versus 8.79% ± 2.51%, P < 0.01). Histological analy-sis showed the presence of non-heat shocked and heat shocked donor myoblasts fused with host myoblasts. These results suggested that heat shock pretreatment increased the HSP70 expression in porcine myoblasts, and improved the survival rate after autologous transplantation. Therefore, heat shock pretreatment of myoblast in vitro is a simple and effective way to enhance cell survival after transplantation in pig. It might represent a potential method to overcome the limitations of MT treat-ment.     

Differences in the Developmental Fate of Cultured and Noncultured Myoblasts When Transplanted into Embryonic Limbs

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.     

Successful histocompatible myoblast transplantation in dystrophin- deficient mdx mouse despite the production of antibodies against dystrophin

Myoblast transplantation has been considered a potential treatment for some muscular disorders. It has proven very successful, however, only in immunodeficient or immunosuppressed mice. In this study, myoblasts from C57BL10J +/+ mice were transplanted, with no immunosuppressive treatment, in the tibialis anterior of fully histocompatible but dystrophin-deficient C57BL10J mdx/mdx mice. One to 9 months after transplantation, the success of the graft was evaluated by immunohistochemistry. All the transplanted mice (n = 24) developed dystrophin-positive fibers following transplantation. Depending on myoblast cultures, transplantations, and time of analysis, the mice presented 15 to 80% of dystrophin-positive fibers in transplanted muscles. These fibers were correctly oriented and they were either from donor or hybrid origin. The dystrophin-positive fibers remained stable up to 9 months. Possible humoral and cellular immune responses were investigated after grafting. Antibodies directed against dystrophin and/or muscle membrane were developed by 58% of the mice as demonstrated by immunohistochemistry and Western blotting. Despite the presence of these antibodies, dystrophin-positive fibers were still present in grafted muscles 9 months after transplantation. Moreover, the muscles did not show massive infiltration by CD4 cells, CD8 cells, or macrophages, as already described in myoblast allotransplantations. This lack of rejection was attributed to the sequestrated nature of dystrophin after fiber formation. These results indicate that myoblast transplantation leads to fiber formation when immunocompetent but fully histocompatible donors and recipients are used and that dystrophin incompatibility alone is not sufficient to induce an immunological rejection reaction.     

Enhanced fusion of myoblasts with myofibers for efficient gene delivery induced by a partially purified protein fraction from rat muscle extract

The biggest challenge to gene therapy is how to efficiently deliver the desired therapeutic gene into a sufficient number of recipient cells to achieve significant clinical efficacy. Here, we identified a partially purified extract from rat muscle probably containing myoblast specific fusion factor(s) (MSF), which significantly enhanced fusion of donor myoblast with host muscle fibers. Once incorporated, the introduced genetic construct could instruct the machinery of the hybrid cells to express the desired protein(s). Rat satellite cells containing a plasmid carrying a marker bone morphogenetic protein-4 (BMP-4) coding sequence were used as foreign gene delivery vehicle. BrdU labeling of the MSF-pretreated satellite cells allowed tracing the fate of the genetically modified satellite cells in the host muscles. Immunohistochemistry using anti-BMP-4 antibody demonstrated the translation of the introduced gene construct. It was demonstrated that in the presence of MSF, numerous BrdU positive nuclei and the expression of BMP-4 polypeptides could be observed in host hybrid fibers, while in the control group using rat serum to replace MSF containing fraction, only a few BrdU positive signals were detected. The expression of osteocalcin and the elevated alkaline phosphatase activity detected in the hybrid fibers indicated the proper folding, secretion and, post-translational modification of the expressed foreign protein. This strategy of enhanced myoblast-mediated gene transfer would break the major barrier in current practice of normal or engineered myoblast transplantation in the management of genetic muscle diseases or systemic genetic disorders.     

Complement and myoblast transfer therapy: donor myoblast survival is enhanced following depletion of host complement C3 using cobra venom factor, but not in the absence of C5

Myoblast transfer therapy (MTT) is a potential cell therapy for myopathies such as Duchenne Muscular Dystrophy and involves the injection of cultured muscle precursor cells ('myoblasts') isolated from normal donor skeletal muscles into dystrophic host muscle. The failure of donor myoblast survival following MTT is widely accepted as being due to the immune response of the host. The role of complement as one possible mechanism for the initial, very rapid death of myoblasts following MTT was investigated. Donor male myoblasts were injected into the tibialis anterior (TA) muscles of female host mice that were: (i) untreated; (ii) depleted of C3 complement (24 h prior to MTT) using cobra venom factor (CVF); and/or (iii) deficient in C5 complement. Quantification of surviving male donor myoblast DNA was performed using the Y-chromosome specific (Y1) probe on slot blots for samples taken at 0 h, 1 h, 24 h, 1 week and 3 weeks after MTT. Peripheral depletion of C3 was confirmed using double immunodiffusion, and local depletion of C3 in host TA muscles was confirmed by immunostaining of muscle samples. Cobra venom factor treatment significantly increased the initial survival of donor myoblasts, but there was a marked decline in myoblast numbers after 1 h and little long-term benefit by 3 weeks. Strain specific variation in the immediate survival of donor male myoblasts following MTT in untreated C57BL/10Sn, DBA-1 and DBA-2 (C5-deficient) female hosts was observed. Cobra venom factor depletion of C3 increased initial donor male myoblast survival (approximately twofold at 0 h) in C57BL/10Sn and DBA-1 host mice and approximately threefold in DBA-2 hosts at 0 h and 1 h after MTT. The rapid and extensive number (approximately 90%) of donor male myoblasts in untreated DBA-2 mice (that lack C5) indicates that activation of the membrane attack complex (MAC) plays no role in this massive initial cell death. The observation that myoblast survival was increased in all mice treated with CVF suggests that CVF may indirectly enhance donor myoblast survival by a mechanism possibly involving activated C3 fragments.     

Transplantable liver production plan

Organ grafts developed in the xenogeneic pig scaffold are expected to resolve most issues of donor safety and ethical concerns about living-donor liver transplantation in Japan. We have been working on so-called “Yamaton” projects to develop transplantable organs using genetically engineered pigs. Our goal is to produce chimeric livers with human parenchyma in such pigs. The Yamaton-Liver project demonstrated the proof of concept by showing that rat–mouse chimeric livers could develop in mice and be successfully transplanted into syngeneic or allogeneic rats. Under conventional immunosuppression, the transplanted livers showed long-term function and protection against rejection. Because chimeric liver grafts have xenogeneic components, additional strategies, such as humanization of pig genes, induction of hematopoietic chimeras in donors, and replacement of pig endothelial cells with human ones, might be required in clinical use. Our projects still need to overcome various hurdles but can bring huge benefits to patients in the future.     

Real-time imaging of myoblast transplantation using the human sodium iodide symporter

The quantification of the graft success is a key element to evaluate the efficiency of cellular therapies for several pathologies such as Duchenne muscular dystrophy. This study describes an approach to evaluate the success of myoblast transplantation (i.e., survival of the transplanted cells and the muscle fibers formed) by real-time imaging. C2C12 myoblasts were first transfected with a plasmid containing the human sodium iodide symporter (hNIS) gene. Specific uptake of the radioactive sodium pertechnetate (Na99mTcO4) by the hNIS-positive myoblasts was demonstrated in vitro, while only background level of Na99mTcO4 was observed within the control cells. The cells were then transplanted into the tibialis anterior (TA) muscle of mdx (X-linked dystrophic) mice. Following intraperitoneal administration of Na99mTcO4, scintigraphies were performed to detect hNIS-dependent Na99mTcO4 uptake within the TA. This approach permitted to evaluate the progression of the transplantation and the graft success without having to biopsy the animals during the follow-up period.