New approaches to the study of human dystrophic muscle cells in culture

Yasin, Rose; Walsh, Frank S.; Landon, D. N.; Thompson, Edward J.

doi:10.1016/0022-510x(83)90092-8

Cited by 22 publications

(8 citation statements)

References 32 publications

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“…In culture of normal muscle, myoblasts begin to fuse after 48 h after the change from a proliferation into a differentiation medium. After 4–5 days, 80–90% of the myoblasts fused to form myotubes [19,23]. The present data confirm that myoblasts from different donors in cat and human, respectively, can also fuse and form myotubes within a similar time frame.…”

Section: Discussionsupporting

confidence: 79%

Restoration of dystrophin expression in cultured hybrid myotubes

Radojević

Gaschen

Burgunder

2002

Neuropathology Appl Neurobio

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Absence of dystrophin, as found in Duchenne boys, mdx mice and HFMD cats, leads to destabilization of the sarcolemmal-associated protein complex. Gene and cell therapy strategies aim to restore the dystrophin-associated protein complex. In order to better understand the cellular events involved in such therapy in feline and human muscular dystrophy, we asked whether dystrophin-deficient myoblasts would fuse with myoblasts expressing normal dystrophin, and whether the complex would be restored after such a fusion. Cat and human myoblasts were isolated from skeletal muscle of normal subjects and of patients with dystrophin deficiency and proliferated well. After co-culture with normal myoblasts, they fused to form hybrid myotubes. These hybrid myotubes expressed dystrophin, utrophin and dystrophin- associated proteins. Expression of these proteins were restored also in the vicinity of nuclei from dystrophin-deficient donors. These results demonstrate that dystrophin can be expressed and handled normally by hybrid myotubes. They show that myoblasts with a normal dystrophin gene can restore dystrophin expression in dystrophin-deficient myoblasts.

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Section: Discussionsupporting

confidence: 79%

Restoration of dystrophin expression in cultured hybrid myotubes

Radojević

Gaschen

Burgunder

2002

Neuropathology Appl Neurobio

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“…Cell culture and parameters are important tools for future genetic and biochemical studies comparing normal and diseased human muscle cells. (Vainzof et al 1995;Yasin et al 1983).…”

Section: Discussionmentioning

confidence: 98%

Differential expression of dystrophin, utrophin, and dystrophin-associated proteins in human muscle culture

Radojević

Lin

Burgunder

2000

Cell and Tissue Research

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The dystrophin-associated protein complex (DAP) plays an important role in sarcolemmal function. Mutations of DAP elements lead to diverse forms of muscular dystrophies, among them Duchenne muscular dystrophy, one of the most severe neuromuscular diseases. Strategies in gene therapy are being assessed to restore DAP stability. However, the relationship between DAP elements and time-course of the DAP formation are still not known in detail. In order to better understand the relationship among DAP proteins, we therefore studied their expression during development in human muscle culture in comparison with developmentally regulated muscle proteins. Desmin immunoreactivity (IR) was detected by 3 days in vitro (DIV3), IR for developmental heavy-chain myosin, vimentin, utrophin, and beta-dystroglycan, as well as alpha-, beta-, and gamma-sarcoglycan, a day later. delta-Sarcoglycan was found by DIV7; dystrophin could be detected only by DIV11. In general, DAP proteins were first located in the perinuclear region, later diffusely in the cytoplasm, and finally exclusively at the membrane. This sequence of events during muscle development gives further support to our suggestion that utrophin could be a precursor of dystrophin during development and regeneration. These data also suggest that utrophin alone is sufficient to anchor the complex, which is important when utrophin replacement strategies are being investigated for the treatment of dystrophinopathies. In this study we demonstrated the establishment of a culture technique that should allow the close study of DAP expression in diseased muscle, including its use after gene modulatory strategies.

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“…Previously, mechanisms of myoblast differentiation and fusion have been studied in two-dimensional (2D) monolayer culture (Miller 1990;Cossu et al 1995). Considerable research has focused on developing differentiated skeletal muscle models in 2D (Yasin et al 1983;Blau et al 1985;Ham et al 1988;Vandenburgh et al 1993). However, to understand the basic functions and regulation that occurs in vivo, threedimensional (3D) modeling of tissues in vitro is required (Cheema et al 2003).…”

Section: Introductionmentioning

confidence: 99%

A novel in vitro three-dimensional skeletal muscle model

Marquette

Byerly

Sognier

2007

In Vitro Cell.Dev.Biol.-Animal

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A novel three-dimensional (3D) skeletal muscle model composed of C2C12 mouse myoblasts is described. This model was generated by cultivating myoblasts in suspension using the rotary cell culture system (RCCS), a unique culture environment. Single-cell suspensions of myoblasts were seeded at 5 x 10(5)/ml in growth medium without exogenous support structures or substrates. Cell aggregation occurred in both RCCS and suspension control (SC) conditions within 12 h but occurred more rapidly in the SC at all time intervals examined. RCCS-cultured myoblasts fused and differentiated into a 3D construct without serum deprivation or alterations. Syncitia were quantified at 3 and 6+ d in stained thin sections. A significantly greater number of syncitia was found at 6+ d in the RCCS cultures compared to the SC. The majority of syncitia were localized to the periphery of the cell constructs for all treatments. The expression of sarcomeric myosin heavy chain (MHC) was localized at or near the periphery of the 3D construct. The majority of MHC was associated with the large cells (syncitia) of the 6+-d aggregates. These results show, for the first time, that myoblasts form syncitia and express MHC in the presence of growth factors and without the use of exogenous supports or substrates. This model test system is useful for investigating initial cell binding, myoblast fusion and syncitia formation, and differentiation processes.

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New approaches to the study of human dystrophic muscle cells in culture

Cited by 22 publications

References 32 publications

Restoration of dystrophin expression in cultured hybrid myotubes

Restoration of dystrophin expression in cultured hybrid myotubes

Differential expression of dystrophin, utrophin, and dystrophin-associated proteins in human muscle culture

A novel in vitro three-dimensional skeletal muscle model

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