A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells

Yasin, Rose; Beers, Gisela Van; Nurse, K.C.E.; Alani, S. M.; Landon, D. N.; Thompson, Edward J.

doi:10.1016/0022-510x(77)90018-1

Cited by 135 publications

(51 citation statements)

References 20 publications

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“…Similarly, myotubes derived from human fetal dermal fibroblasts show a well organized initial sarcomerogenesis, with aligned sarcomeres and patterned Z lines (Fig. 2), as reported for myotubes derived from primary myogenic cells, which never complete sarcomagenesis in vitro (29). To verify whether the introduction of MyoD in primary fibroblasts would also activate transcription of genes responsible for membrane and metabolic muscle-specific functions, we measured by Northern blotting the expression of RNAs coding muscle-specific proteins such as the ACh ␣ -subunit, MCK, as well as MyoD, myogenin, and MLC1F chosen as positive controls.…”

Section: Converted Fibroblasts Express a Myogenic Phenotype In Vitrosupporting

confidence: 76%

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Lattanzi¹,

Salvatori²,

Coletta³

et al. 1998

J. Clin. Invest.

129

103

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Ex vivo gene therapy of primary myopathies, based on autologous transplantation of genetically modified myogenic cells, is seriously limited by the number of primary myogenic cells that can be isolated, expanded, transduced, and reimplanted into the patient's muscles. We explored the possibility of using the MyoD gene to induce myogenic conversion of nonmuscle, primary cells in a quantitatively relevant fashion. Primary human and murine fibroblasts from skin, muscle, or bone marrow were infected by an E1-deleted adenoviral vector carrying a retroviral long terminal repeat-promoted MyoD cDNA. Expression of MyoD caused irreversible withdrawal from the cell cycle and myogenic differentiation in the majority (from 60 to 90%) of cultured fibroblasts, as defined by activation of muscle-specific genes, fusion into contractile myotubes, and appearance of ultrastructurally normal sarcomagenesis in culture. 24 h after adenoviral exposure, MyoD -converted cultures were injected into regenerating muscle of immunodeficient (severe combined immunodeficiency/beige) mice, where they gave rise to ␤ -galactosidase positive, centrally nucleated fibers expressing human myosin heavy chains. Fibers originating from converted fibroblasts were indistinguishable from those obtained by injection of control cultures of lacZ -transduced satellite cells. MyoD -converted murine fibroblasts participated to muscle regeneration also in immunocompetent, syngeneic mice. Although antibodies from these mice bound to adenoviral infected cells in vitro, no inflammatory infiltrate was present in the graft site throughout the 3-wk study period. These data support the feasibility of an alternative approach to gene therapy of primary myopathies, based on implantation of large numbers of genetically modified primary fibroblasts massively converted to myogenesis by adenoviral delivery of MyoD ex vivo. ( J. Clin. Invest.

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Section: Converted Fibroblasts Express a Myogenic Phenotype In Vitrosupporting

confidence: 76%

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Lattanzi¹,

Salvatori²,

Coletta³

et al. 1998

J. Clin. Invest.

129

103

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“…Epstein -Barr virus-transformed lymphoblastoid cells were cultured in RPMI 1640 with 10% FCS. Human pectoral myoblasts (kindly provided by Olli Carpén, University of Helsinki, Finland) were isolated as described before, 24 cultured in DMEM with 15% FCS and 2% Ultroser G (Life Technologies) and differentiated into myotubes for 4 days in DMEM with 0.4% Ultroser G.…”

Section: Expression Constructsmentioning

confidence: 99%

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations

et al. 2004

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Loss-of-function mutations in the cystatin B (CSTB), a cysteine protease inhibitor, gene underlie progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1), characterized by myoclonic and tonic -clonic seizures, ataxia and a progressive course. A minisatellite repeat expansion in the promoter region of the CSTB gene is the most common mutation in EPM1 patients and leads to reduced mRNA levels. Seven other mutations altering the structure of CSTB, or predicting altered splicing, have been described. Using a novel monoclonal CSTB antibody and organelle-specific markers in human primary myoblasts, we show here that endogenous CSTB localizes not only to the nucleus and cytoplasm but also associates with lysosomes. Upon differentiation to myotubes, CSTB becomes excluded from the nucleus and lysosomes, suggesting that the subcellular distribution of CSTB is dependent on the differentiation status of the cell. Four patient mutations altering the CSTB polypeptide were transiently expressed in BHK-21 cells. The p.Lys73fsX2-truncated mutant protein shows diffuse cytoplasmic and nuclear distribution, whereas p.Arg68X is rapidly degraded. Two missense mutations, the previously described p.Gly4Arg affecting the highly conserved glycine, critical for cathepsin binding, and a novel mutation, p.Gln71Pro, fail to associate with lysosomes. These data imply an important lysosome-associated physiological function for CSTB and suggest that loss of this association contributes to the molecular pathogenesis of EPM1.

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“…Primary cell cultures were prepared by dissociation of hind and fore limb muscle tissues of new born C57/B10 mice as previously described. 29 These cells were plated in 15% FCS DMEM plus 2 mm glutamine, on 35 mm dishes (Nunclon, from Life Technologies, Paisley, UK) that had been pre-coated in Matrigel (Becton Dickinson, Oxford, UK). Cultures were grown to 50-80% confluence and transfected without further passage.…”

Section: Methodsmentioning

confidence: 99%

Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER

et al. 1998

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A quantitative technique for growing human adult skeletal muscle in culture starting from mononucleated cells

Cited by 135 publications

References 20 publications

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

High efficiency myogenic conversion of human fibroblasts by adenoviral vector-mediated MyoD gene transfer. An alternative strategy for ex vivo gene therapy of primary myopathies.

Loss of lysosomal association of cystatin B proteins representing progressive myoclonus epilepsy, EPM1, mutations

Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER

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