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D.I.4 Satellite cells and skeletal muscle regeneration
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Abstracts / Neuromuscular Disorders 21 (2011) 639–751
care likely preserves cardiac function. Similar cardiac involvement is also seen in limb girdle muscular dystrophy associated with sarcoglycan mutations. Mutations in LMNA or emerin can also have additional needs such as early pacemaker or ICD insertion since cardiac conduction system can be targeted. Therefore it is imperative to have the proper genetic diagnosis in order to manage the cardiac complications of muscular dystrophy. Ongoing research efforts are aimed at understanding genetic modifiers in mouse models. Genetic modifier loci which help predict the likelihood of developing cardiomyopathy have been identified. Genetic modifiers for skeletal muscle dysfunction may also impact cardiac function. Unique genetic modifiers that alter diaphragm muscle dysfunction may also play a role in right ventricular function (Supported by NIH, MDA and Doris Duke Charitable Foundation). doi:10.1016/j.nmd.2011.06.755
DUCHENNE AND BECKER MUSCULAR DYSTROPHY AND ASSOCIATED DYSTROPHINOPATHIES 2
D.I.4 Satellite cells and skeletal muscle regeneration J.E. Morgan UCL Institute of Child Health, Dubowitz Neuromuscular Centre, London, United Kingdom Skeletal muscle repair, maintenance and regeneration are mediated by satellite cells located under the basal lamina of muscle fibres. In muscular dystrophies such as Duchenne Muscular Dystrophy, muscle fibres are lost and endogenous satellite cells initially contribute to muscle regeneration. However, regeneration does not keep up with muscle fibre loss, either because the satellite cells are defective, or the dystrophic muscle environment is inhospitable. Cell therapy is a promising strategy for treating muscular dystrophies. However, there are hurdles to be overcome, in particular, delivery of cells to muscles throughout the body. Satellite cells and their progeny myoblasts are not systemically-deliverable and their capacity to regenerate skeletal muscle is reduced by even a short time in tissue culture. Other stem cells – mesoangioblasts or pericytes, or stem cells expressing CD133-contribute to muscle regeneration after systemic delivery in animal models of muscular dystrophies. However, satellite cells remain the canonical skeletal muscle stem cell, able to regenerate skeletal muscle and functionally reconstitute the satellite cell compartment. But the local or systemic environment has a major effect on donor stem cellderived muscle regeneration. Satellite cells only contribute extensively to muscle regeneration when the dystrophic host mouse muscle is irradiated prior to intra-muscular injection of donor satellite cells. In contrast, donor satellite cells contribute to significantly fewer muscle fibres when host muscle is injured by physical or chemical means, or by toxins, prior to grafting. We are currently elucidating factors in the irradiated host muscle environment that enhance donor satellite cell proliferation. doi:10.1016/j.nmd.2011.06.756
O.1 Pre-clinical studies with human adult mesenchymal stem-cells: What have we learned? M. Zatz a, N.M. Vieira a, M. Valadares a, M. Secco a, E. Zucconi a, C.J.R. Bueno a, V. Brandalise a, A. Assoni a, J. Gomes a, V. Landini a, T. Andrade a, M. Vainzof a, G.D. Shelton b a University of Sa˜o Paulo, Human Genome Research Center, Sa˜o Paulo, Brazil; b University of California, Dept. of Pathology, San Diego, La Jolla, United States
Before using stem-cells as a therapy for progressive muscular dystrophies many questions still need to be addressed. How to deliver: locally or systemically? What cells best express human muscle proteins? One or multiple injections? Is immunosuppression required? What directs homing? In order to address these questions we have injected human mesenchymal stem-cells (hMSCs) in two animal models: SJL mice and GRMD dogs, without immunosuppression. Systemic injections of hMSCs from both umbilical cord tissue (UCT) and adipose tissue (AT), into the SJL mice, showed that cells from both sources were able to reach the muscle. However only cells from AT expressed human muscle proteins and promoted significant clinical improvement in injected animals. Systemic injections of human AT and UC MSCs into the cephalic vein of GRMD dogs, showed results comparable to those observed in SJL mice. Biopsies from biceps femoris revealed that only hMSC from AT were able to express human muscle proteins. A dystrophin band was seen through WB up to 6 months after the last injection but not after 12 months suggesting that multiple injections are required. No human cells were found in the injected muscles after local injections. In order to assess if the dystrophic process interferes with homing we repeated the same experiment with AT-hMSCs in normal mice. No cells were found after two months which suggests that factors released during muscle degeneration direct homing. In GRMD dogs it is very difficult to assess any clinical impact taking into account the enormous variability seen among them. How much dystrophin is required to rescue the phenotype is an open question since a mild phenotype may occur, in GRMD and Labrador retriever muscular dystrophy, without any muscle dystrophin. Therefore, other parameters should be analyzed in order to evaluate the clinical impact of cell therapy in the dog model. doi:10.1016/j.nmd.2011.06.757
O.2 Dystrophin-deficient muscular dystrophy in a pedigree of Labrador retrievers without obvious clinical manifestations G.D. Shelton a, N. Vieira b, L.T. Guo a, R. Bennett c, L. Kunkel c, M. Zatz b a University of California, San Diego, Department of Pathology, La Jolla, California, United States; b University of Sa˜o Paulo, Human Genome Research Center, Sa˜o Paulo, Brazil; c Harvard Medical School, Childrens Hospital, Boston, MA, United States Different animal models for progressive muscular dystrophies (PMDs) are available, including fish, mouse, cat and dog models. The best animal model for Duchenne MD (DMD) is the canine model as it reproduces the full spectrum of human pathology. Available canine models include the Golden Retriever (GRMD; naturally occurring mutation that has been bred), German Shorthair Pointer (dystrophin knockout), Welsh Corgis (naturally occurring mutation that has been bred), and beagle (CXMDJ; artificially inseminated with spermatozoa derived from GRMD). All canine dystrophin negative dogs show a severe clinical course; however, two mildly affected GRMD were reported by the group of Dr. Zatz as an exception to the mitigating effect of the dystrophin mutation. Recently we have identified a pedigree including 12 young male Labrador retrievers without obvious clinical signs of muscle weakness, atrophy or exercise intolerance. Markedly elevated creatine kinase activities (30,000– 40,000 IU/L) were identified in all dogs on pre-neuter blood evaluations. Muscle biopsies collected at the time of neutering (at 6–9 months of age) showed pathological changes consistent with a dystrophic phenotype in all cases. Immunostaining showed markedly decreased staining for dystrophin and increased staining for utrophin as typically found in severely affected dogs. Staining for sarcoglycan was variably decreased among the affected dogs.These findings were confirmed by immunoblotting. We have followed the clinical course in the first 2 dogs identified, and by 3 years of age, mild exercise intolerance was observed. Mutational analysis
Abstracts / Neuromuscular Disorders 21 (2011) 639–751
care likely preserves cardiac function. Similar cardiac involvement is also seen in limb girdle muscular dystrophy associated with sarcoglycan mutations. Mutations in LMNA or emerin can also have additional needs such as early pacemaker or ICD insertion since cardiac conduction system can be targeted. Therefore it is imperative to have the proper genetic diagnosis in order to manage the cardiac complications of muscular dystrophy. Ongoing research efforts are aimed at understanding genetic modifiers in mouse models. Genetic modifier loci which help predict the likelihood of developing cardiomyopathy have been identified. Genetic modifiers for skeletal muscle dysfunction may also impact cardiac function. Unique genetic modifiers that alter diaphragm muscle dysfunction may also play a role in right ventricular function (Supported by NIH, MDA and Doris Duke Charitable Foundation). doi:10.1016/j.nmd.2011.06.755
DUCHENNE AND BECKER MUSCULAR DYSTROPHY AND ASSOCIATED DYSTROPHINOPATHIES 2
D.I.4 Satellite cells and skeletal muscle regeneration J.E. Morgan UCL Institute of Child Health, Dubowitz Neuromuscular Centre, London, United Kingdom Skeletal muscle repair, maintenance and regeneration are mediated by satellite cells located under the basal lamina of muscle fibres. In muscular dystrophies such as Duchenne Muscular Dystrophy, muscle fibres are lost and endogenous satellite cells initially contribute to muscle regeneration. However, regeneration does not keep up with muscle fibre loss, either because the satellite cells are defective, or the dystrophic muscle environment is inhospitable. Cell therapy is a promising strategy for treating muscular dystrophies. However, there are hurdles to be overcome, in particular, delivery of cells to muscles throughout the body. Satellite cells and their progeny myoblasts are not systemically-deliverable and their capacity to regenerate skeletal muscle is reduced by even a short time in tissue culture. Other stem cells – mesoangioblasts or pericytes, or stem cells expressing CD133-contribute to muscle regeneration after systemic delivery in animal models of muscular dystrophies. However, satellite cells remain the canonical skeletal muscle stem cell, able to regenerate skeletal muscle and functionally reconstitute the satellite cell compartment. But the local or systemic environment has a major effect on donor stem cellderived muscle regeneration. Satellite cells only contribute extensively to muscle regeneration when the dystrophic host mouse muscle is irradiated prior to intra-muscular injection of donor satellite cells. In contrast, donor satellite cells contribute to significantly fewer muscle fibres when host muscle is injured by physical or chemical means, or by toxins, prior to grafting. We are currently elucidating factors in the irradiated host muscle environment that enhance donor satellite cell proliferation. doi:10.1016/j.nmd.2011.06.756
O.1 Pre-clinical studies with human adult mesenchymal stem-cells: What have we learned? M. Zatz a, N.M. Vieira a, M. Valadares a, M. Secco a, E. Zucconi a, C.J.R. Bueno a, V. Brandalise a, A. Assoni a, J. Gomes a, V. Landini a, T. Andrade a, M. Vainzof a, G.D. Shelton b a University of Sa˜o Paulo, Human Genome Research Center, Sa˜o Paulo, Brazil; b University of California, Dept. of Pathology, San Diego, La Jolla, United States
Before using stem-cells as a therapy for progressive muscular dystrophies many questions still need to be addressed. How to deliver: locally or systemically? What cells best express human muscle proteins? One or multiple injections? Is immunosuppression required? What directs homing? In order to address these questions we have injected human mesenchymal stem-cells (hMSCs) in two animal models: SJL mice and GRMD dogs, without immunosuppression. Systemic injections of hMSCs from both umbilical cord tissue (UCT) and adipose tissue (AT), into the SJL mice, showed that cells from both sources were able to reach the muscle. However only cells from AT expressed human muscle proteins and promoted significant clinical improvement in injected animals. Systemic injections of human AT and UC MSCs into the cephalic vein of GRMD dogs, showed results comparable to those observed in SJL mice. Biopsies from biceps femoris revealed that only hMSC from AT were able to express human muscle proteins. A dystrophin band was seen through WB up to 6 months after the last injection but not after 12 months suggesting that multiple injections are required. No human cells were found in the injected muscles after local injections. In order to assess if the dystrophic process interferes with homing we repeated the same experiment with AT-hMSCs in normal mice. No cells were found after two months which suggests that factors released during muscle degeneration direct homing. In GRMD dogs it is very difficult to assess any clinical impact taking into account the enormous variability seen among them. How much dystrophin is required to rescue the phenotype is an open question since a mild phenotype may occur, in GRMD and Labrador retriever muscular dystrophy, without any muscle dystrophin. Therefore, other parameters should be analyzed in order to evaluate the clinical impact of cell therapy in the dog model. doi:10.1016/j.nmd.2011.06.757
O.2 Dystrophin-deficient muscular dystrophy in a pedigree of Labrador retrievers without obvious clinical manifestations G.D. Shelton a, N. Vieira b, L.T. Guo a, R. Bennett c, L. Kunkel c, M. Zatz b a University of California, San Diego, Department of Pathology, La Jolla, California, United States; b University of Sa˜o Paulo, Human Genome Research Center, Sa˜o Paulo, Brazil; c Harvard Medical School, Childrens Hospital, Boston, MA, United States Different animal models for progressive muscular dystrophies (PMDs) are available, including fish, mouse, cat and dog models. The best animal model for Duchenne MD (DMD) is the canine model as it reproduces the full spectrum of human pathology. Available canine models include the Golden Retriever (GRMD; naturally occurring mutation that has been bred), German Shorthair Pointer (dystrophin knockout), Welsh Corgis (naturally occurring mutation that has been bred), and beagle (CXMDJ; artificially inseminated with spermatozoa derived from GRMD). All canine dystrophin negative dogs show a severe clinical course; however, two mildly affected GRMD were reported by the group of Dr. Zatz as an exception to the mitigating effect of the dystrophin mutation. Recently we have identified a pedigree including 12 young male Labrador retrievers without obvious clinical signs of muscle weakness, atrophy or exercise intolerance. Markedly elevated creatine kinase activities (30,000– 40,000 IU/L) were identified in all dogs on pre-neuter blood evaluations. Muscle biopsies collected at the time of neutering (at 6–9 months of age) showed pathological changes consistent with a dystrophic phenotype in all cases. Immunostaining showed markedly decreased staining for dystrophin and increased staining for utrophin as typically found in severely affected dogs. Staining for sarcoglycan was variably decreased among the affected dogs.These findings were confirmed by immunoblotting. We have followed the clinical course in the first 2 dogs identified, and by 3 years of age, mild exercise intolerance was observed. Mutational analysis