Résumé :
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Communication n° 53. Introduction : Differentiation of mammalian cells is thought to be a terminal and irreversible process. The in vitro differentiation of myocytes into myotubes is a well characterised example of terminal differentiation. Apart from the msx1 gene, no other genes have been shown to cause myotube dedifferentiation when overexpressed. Twist is a basic helix-loop-helix (bHLH) transcription factor, whose expression has been implicated in the inhibition of differentiation of multiple cell lineages, including muscle, cartilage and bone cells. Objectives : To prove that overexpression of the Twist gene in differentiated mouse muscle cells induces myotube dedifferentiation and production of proliferating mononucleated precursor cells, capable of redifferentiation or transdiferentiation to other cell types. Methods : A mouse Twist cDNA adenoviral vector was produced and transfected multinucleated myotubes. Investigations were carried out to detect muscle differentiation marker levels by immmunocytochemistry, initiation of DNA synthesis by BrdU and myotube cleavage, by light microscopy. Adipogenesis, myogenesis and osteogenesis were detected by alkaline phosphatase, myosin heavy chain and oil Red O staining, respectively. Results : Overexpression of the Twist cDNA in the presence of growth factors caused a reduction in muscle differentiation markers, followed by an initiation in DNA synthesis, indicating that myotube differentiation has been reversed. Multinucleated myotubes underwent cleavage into mononucleated cells, capable of proliferation. Moreover, overexpression of Twist was enough to drive myotube cleavage in the absence of growth factors. Finally, these product cells had the ability to redifferentiate into myotubes or trans-differentiate into adipocytes or osteocytes, proving that Twist can cause dedifferentiation and cleavage into pluripotent cells. Conclusions : Twist-mediated dedifferentiation and cleavage of multinucleated mammalian myotubes into stem-like proliferating mononucleated cells provides evidence of cell dedifferentiation in mammalian cells, as means to regenerate tissue. The exploitation of such technology would be important to study mammalian cellular dedifferentiation and also develop therapeutic approaches for tissue regeneration.
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