The Experts below are selected from a list of 28950 Experts worldwide ranked by ideXlab platform
Helen M. Blau - One of the best experts on this subject based on the ideXlab platform.
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transient production of α smooth muscle actin by skeletal Myoblasts during differentiation in culture and following intramuscular implantation
Cytoskeleton, 2002Co-Authors: Matthew L Springer, Clare R Ozawa, Helen M. BlauAbstract:alpha-smooth muscle actin (SMA) is typically not present in post-embryonic skeletal muscle Myoblasts or skeletal muscle fibers. However, both primary Myoblasts isolated from neonatal mouse muscle tissue, and C2C12, an established myoblast cell line, produced SMA in culture within hours of exposure to differentiation medium. The SMA appeared during the cells' initial elongation, persisted through differentiation and fusion into myotubes, remained abundant in early myotubes, and was occasionally observed in a striated pattern. SMA continued to be present during the initial appearance of sarcomeric actin, but disappeared shortly thereafter leaving only sarcomeric actin in contractile myotubes derived from primary Myoblasts. Within one day after implantation of primary Myoblasts into mouse skeletal muscle, SMA was observed in the Myoblasts; but by 9 days post-implantation, no SMA was detectable in Myoblasts or muscle fibers. Thus, both neonatal primary Myoblasts and an established myoblast cell line appear to similarly reprise an embryonic developmental program during differentiation in culture as well as differentiation within adult mouse muscles.
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purification of mouse primary Myoblasts based on alpha 7 integrin expression
Experimental Cell Research, 2001Co-Authors: William Blancobose, Randall H Kramer, Chungchen Yao, Helen M. BlauAbstract:Fundamental insights have come from the study of myogenesis. Primary Myoblasts isolated directly from muscle tissue more closely approximate myogenesis than established cell lines. However, contamination of primary muscle cultures with nonmyogenic cells can complicate the results. To overcome this problem, we previously described a method for myoblast purification based on novel culture conditions (T. A. Rando and H. M. Blau, 1994, J. Cell Biol. 125, 1275--1287). Here we report a refinement of this method that leads directly to an enriched population of mouse primary Myoblasts, within significantly fewer population doublings. The method described here avoids using adhesion as a criterion for selection. This advance capitalizes on the ability of the antibody CA5.5 to recognize alpha 7 integrin, a muscle-specific cell surface antigen. Enrichment of Myoblasts to greater than 95% of the cell population can be achieved by a single round of flow cytometry or magnetic bead separation. This is the first description of a mouse myoblast purification method based on a cell-type-specific antigen. The ease of this procedure for isolating primary Myoblasts should expand the opportunities for (1) using these cells in cell transplantation studies in animal models of human disease, (2) isolating and characterizing mutant Myoblasts from transgenic animals, and (3) allowing in vitro studies of molecules that regulate muscle cell growth, differentiation, and neoplasia.
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primary mouse myoblast purification characterization and transplantation for cell mediated gene therapy
Journal of Cell Biology, 1994Co-Authors: Thomas A Rando, Helen M. BlauAbstract: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.
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normal dystrophin transcripts detected in duchenne muscular dystrophy patients after myoblast transplantation
Nature, 1992Co-Authors: Emanuela Gussoni, Andrea M Lanctot, Grace K. Pavlath, Lawrence Steinman, Robert G Miller, Khema R. Sharma, Helen M. BlauAbstract:GENE delivery by transplantation of normal Myoblasts has been proposed as a treatment of the primary defect, lack of the muscle protein dystrophin, that causes Duchenne muscular dystrophy (DMD), a lethal human muscle degenerative disorder1,2. To test this possibility, we transplanted normal Myoblasts from a father or an unaffected sibling into the muscle of eight boys with DMD, and assessed their production of dystrophin. Three patients with deletions in the dystrophin gene expressed normal dystrophin transcripts in muscle biopsy specimens taken from the transplant site one month after myoblast injection. Using the polymerase chain reaction we established that the dystrophin in these biopsies derived from donor myoblast DNA. These results show that trans-planted Myoblasts persist and produce dystrophin in muscle fibres of DMD patients.
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muscle fiber pattern is independent of cell lineage in postnatal rodent development
Cell, 1992Co-Authors: Simon M Hughes, Helen M. BlauAbstract:Abstract Muscle fibers specialized for fast or slow contraction are arrayed in characteristic patterns within developing limbs. Clones of Myoblasts analyzed in vitro express fast and slow myosin isoforms typical of the muscle from which they derive. As a result, it has been suggested that distinct myoblast lineages generate and maintain muscle fiber pattern. We tested this hypothesis in vivo by using a retrovirus to label Myoblasts genetically so that the fate of individual clones could be monitored. Both myoblast clones labeled in muscle in situ and clones labeled in tissue culture and then injected into various muscles contribute progeny to all fiber types encountered. Thus, extrinsic signals override the intrinsic commitment of myoblast nuclei to particular programs of gene expression. We conclude that in postnatal development, pattern is not dictated by myoblast lineage.
Marica Catinozzi - One of the best experts on this subject based on the ideXlab platform.
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the drosophila fus ortholog cabeza promotes adult founder myoblast selection by xrp1 dependent regulation of fgf signaling
PLOS Genetics, 2020Co-Authors: Marica Catinozzi, Moushami Mallik, Marie Frickenhaus, Marije Been, Celine SijlmansAbstract:The number of adult myofibers in Drosophila is determined by the number of founder Myoblasts selected from a myoblast pool, a process governed by fibroblast growth factor (FGF) signaling. Here, we show that loss of cabeza (caz) function results in a reduced number of adult founder Myoblasts, leading to a reduced number and misorientation of adult dorsal abdominal muscles. Genetic experiments revealed that loss of caz function in both adult Myoblasts and neurons contributes to caz mutant muscle phenotypes. Selective overexpression of the FGF receptor Htl or the FGF receptor-specific signaling molecule Stumps in adult Myoblasts partially rescued caz mutant muscle phenotypes, and Stumps levels were reduced in caz mutant founder Myoblasts, indicating FGF pathway deregulation. In both adult Myoblasts and neurons, caz mutant muscle phenotypes were mediated by increased expression levels of Xrp1, a DNA-binding protein involved in gene expression regulation. Xrp1-induced phenotypes were dependent on the DNA-binding capacity of its AT-hook motif, and increased Xrp1 levels in founder Myoblasts reduced Stumps expression. Thus, control of Xrp1 expression by Caz is required for regulation of Stumps expression in founder Myoblasts, resulting in correct founder myoblast selection.
Moushami Mallik - One of the best experts on this subject based on the ideXlab platform.
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the drosophila fus ortholog cabeza promotes adult founder myoblast selection by xrp1 dependent regulation of fgf signaling
PLOS Genetics, 2020Co-Authors: Marica Catinozzi, Moushami Mallik, Marie Frickenhaus, Marije Been, Celine SijlmansAbstract:The number of adult myofibers in Drosophila is determined by the number of founder Myoblasts selected from a myoblast pool, a process governed by fibroblast growth factor (FGF) signaling. Here, we show that loss of cabeza (caz) function results in a reduced number of adult founder Myoblasts, leading to a reduced number and misorientation of adult dorsal abdominal muscles. Genetic experiments revealed that loss of caz function in both adult Myoblasts and neurons contributes to caz mutant muscle phenotypes. Selective overexpression of the FGF receptor Htl or the FGF receptor-specific signaling molecule Stumps in adult Myoblasts partially rescued caz mutant muscle phenotypes, and Stumps levels were reduced in caz mutant founder Myoblasts, indicating FGF pathway deregulation. In both adult Myoblasts and neurons, caz mutant muscle phenotypes were mediated by increased expression levels of Xrp1, a DNA-binding protein involved in gene expression regulation. Xrp1-induced phenotypes were dependent on the DNA-binding capacity of its AT-hook motif, and increased Xrp1 levels in founder Myoblasts reduced Stumps expression. Thus, control of Xrp1 expression by Caz is required for regulation of Stumps expression in founder Myoblasts, resulting in correct founder myoblast selection.
Kodimangalam S. Ravichandran - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylserine receptor bai1 and apoptotic cells as new promoters of myoblast fusion
Nature, 2013Co-Authors: Amelia Hochreiterhufford, Jason M. Kinchen, Jennifer D Sokolowski, Jarrod A. Call, Alexander L. Klibanov, James Mandell, Sanja Arandjelovic, Kodimangalam S. RavichandranAbstract:The apoptotic event of phosphatidylserine exposure and its recognition by the receptor BAI1 has an unexpected new role as a signal enhancing mouse myoblast fusion, an insight with relevance to some congenital muscle diseases and muscle injury treatments. Apoptotic cell death occurs throughout development and homeostasis in healthy tissues, including skeletal muscle. This study questions previous assumptions that the resulting dead cells have no beneficial effects. Kodi Ravichandran and colleagues show that during skeletal muscle differentiation in mice, a fraction of precursor muscle cells undergoes apoptosis, and that these cells provide a key signal — phosphatidylserine — that promotes muscle development. The idea that the body may use cell death not only to rid itself of unwanted cells, but also to regulate differentiation adds an intriguing dimension to cell turnover within tissues. Skeletal muscle arises from the fusion of precursor Myoblasts into multinucleated myofibres1,2. Although conserved transcription factors and signalling proteins involved in myogenesis have been identified, upstream regulators are less well understood. Here we report an unexpected discovery that the membrane protein BAI1, previously linked to recognition of apoptotic cells by phagocytes3, promotes myoblast fusion. Endogenous BAI1 expression increased during myoblast fusion, and BAI1 overexpression enhanced myoblast fusion by means of signalling through ELMO/Dock180/Rac1 proteins4. During myoblast fusion, a fraction of Myoblasts within the population underwent apoptosis and exposed phosphatidylserine, an established ligand for BAI1 (ref. 3). Blocking apoptosis potently impaired myoblast fusion, and adding back apoptotic Myoblasts restored fusion. Furthermore, primary human Myoblasts could be induced to form myotubes by adding apoptotic Myoblasts, even under normal growth conditions. Mechanistically, apoptotic cells did not directly fuse with the healthy Myoblasts, rather the apoptotic cells induced a contact-dependent signalling with neighbours to promote fusion among the healthy Myoblasts. In vivo, myofibres from Bai1−/− mice are smaller than those from wild-type littermates. Muscle regeneration after injury was also impaired in Bai1−/− mice, highlighting a role for BAI1 in mammalian myogenesis. Collectively, these data identify apoptotic cells as a new type of cue that induces signalling via the phosphatidylserine receptor BAI1 to promote fusion of healthy Myoblasts, with important implications for muscle development and repair.
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Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion
Nature, 2013Co-Authors: Amelia E. Hochreiter-hufford, James W. Mandell, Jason M. Kinchen, Jennifer D Sokolowski, Jarrod A. Call, Alexander L. Klibanov, Sanja Arandjelovic, Zhen Yan, Chang Sup Lee, Kodimangalam S. RavichandranAbstract:Skeletal muscle arises from the fusion of precursor Myoblasts into multinucleated myofibres. Although conserved transcription factors and signalling proteins involved in myogenesis have been identified, upstream regulators are less well understood. Here we report an unexpected discovery that the membrane protein BAI1, previously linked to recognition of apoptotic cells by phagocytes, promotes myoblast fusion. Endogenous BAI1 expression increased during myoblast fusion, and BAI1 overexpression enhanced myoblast fusion by means of signalling through ELMO/Dock180/Rac1 proteins. During myoblast fusion, a fraction of Myoblasts within the population underwent apoptosis and exposed phosphatidylserine, an established ligand for BAI1 (ref. 3). Blocking apoptosis potently impaired myoblast fusion, and adding back apoptotic Myoblasts restored fusion. Furthermore, primary human Myoblasts could be induced to form myotubes by adding apoptotic Myoblasts, even under normal growth conditions. Mechanistically, apoptotic cells did not directly fuse with the healthy Myoblasts, rather the apoptotic cells induced a contact-dependent signalling with neighbours to promote fusion among the healthy Myoblasts. In vivo, myofibres from Bai1(-/-) mice are smaller than those from wild-type littermates. Muscle regeneration after injury was also impaired in Bai1(-/-)mice, highlighting a role for BAI1 in mammalian myogenesis. Collectively, these data identify apoptotic cells as a new type of cue that induces signalling via the phosphatidylserine receptor BAI1 to promote fusion of healthy Myoblasts, with important implications for muscle development and repair.
Randall H Kramer - One of the best experts on this subject based on the ideXlab platform.
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purification of mouse primary Myoblasts based on alpha 7 integrin expression
Experimental Cell Research, 2001Co-Authors: William Blancobose, Randall H Kramer, Chungchen Yao, Helen M. BlauAbstract:Fundamental insights have come from the study of myogenesis. Primary Myoblasts isolated directly from muscle tissue more closely approximate myogenesis than established cell lines. However, contamination of primary muscle cultures with nonmyogenic cells can complicate the results. To overcome this problem, we previously described a method for myoblast purification based on novel culture conditions (T. A. Rando and H. M. Blau, 1994, J. Cell Biol. 125, 1275--1287). Here we report a refinement of this method that leads directly to an enriched population of mouse primary Myoblasts, within significantly fewer population doublings. The method described here avoids using adhesion as a criterion for selection. This advance capitalizes on the ability of the antibody CA5.5 to recognize alpha 7 integrin, a muscle-specific cell surface antigen. Enrichment of Myoblasts to greater than 95% of the cell population can be achieved by a single round of flow cytometry or magnetic bead separation. This is the first description of a mouse myoblast purification method based on a cell-type-specific antigen. The ease of this procedure for isolating primary Myoblasts should expand the opportunities for (1) using these cells in cell transplantation studies in animal models of human disease, (2) isolating and characterizing mutant Myoblasts from transgenic animals, and (3) allowing in vitro studies of molecules that regulate muscle cell growth, differentiation, and neoplasia.
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laminins promote the locomotion of skeletal Myoblasts via the alpha 7 integrin receptor
Journal of Cell Science, 1996Co-Authors: Chungchen Yao, Barry L Ziober, Ann Sutherland, Donna L Mendrick, Randall H KramerAbstract:The alpha 7 beta 1 integrin is specifically expressed by skeletal and cardiac muscles, and its expression and alternative mRNA splicing at the cytoplasmic domain are developmentally regulated. We analyzed the role of alpha 7 integrin in mediating myoblast adhesion and motility on different laminin isoforms. Mouse C2C12 and MM14 myoblast cell lines were found by flow cytometry and immunoprecipitation to express high levels of the alpha 7 integrin. Overall expression of alpha 7 increased as the C2C12 Myoblasts differentiated; Myoblasts expressed only the alpha 7B cytoplasmic variant whereas in differentiating myotubes alpha 7A increased markedly. Function-perturbing monoclonal antibodies generated to alpha 7 integrin efficiently blocked both adhesion and migration of MM14 and C2C12 mouse Myoblasts on laminin 1. Other studies with MM14 Myoblasts showed that alpha 7 is also a receptor for laminin 2/4 (human placental merosins) but not for epithelial-cell-specific laminin 5. Blocking antibody to alpha 7 only partially inhibited adhesion to laminin 2/4 but almost completely blocked motility on this substrate. Finally, to assess the potential role of the alpha 7 cytoplasmic domain, CHO cells were stably transfected to expressed chimeric alpha 5 cDNA constructs containing the wild-type alpha 5 or the alpha 7A or alpha 7B cytoplasmic domain; all forms of the integrin showed identical activities for adhesion, migration, proliferation, and matrix assembly on fibronectin substrates. These results established that alpha 7 beta 1 receptor can promote myoblast adhesion and motility on a restricted number of laminin isoforms and may be important in myogenic precursor recruitment during regeneration and differentiation.
