Myogenesis involves the determination of progenitor cells to myoblasts, their fusion to yield multinuclear myotubes, and the maturation of myotubes to muscle fibres. This development is reflected in a time pattern of gene expression, e.g. of genes coding for desmin, the myogenic factors myogenin and myoD, the acetylcholine receptor alpha-subunit and the muscular chloride channel CIC-1. We attempted to improve yields and myogenic differentiation in culture by using three-dimensional microcarrier systems. Out of a variety of carriers tested in stationary cultures, collagen-coated dextran Cytodex3 beads proved optimal for the proliferation and differentiation of the murine myogenic cell line C2C12. With C2C12 myoblasts in stationary and stirred systems (Spinner- and SuperSpinner flasks), surface adherence, differentiation into myotubes and expression of muscle-specific mRNAs on Cytodex3 beads were the same as in conventional cultures. Other carriers tested (DEAE cellulose, glass, plastic, cellulose, polyester) did not support growth and differentiation of C2C12 cells. The secondary mouse myogenic stem cells M12 and M2.7-MDX proliferated and differentiated well in stationary Cytodex3 cultures, but no differentiation occurred in Spinner flasks. As indicated by light and scanning electron microscopy, C2C12 myotubes formed not only on but also in between Cytodex beads. The secondary cell lines may succumb to shear forces under these conditions.
The chloride channel CIC-1 is required to maintain a normal excitability of mature muscle fibers; its blockade leads to hyperexcitability, the hallmark of the disease myotonia. In mouse and rat myotubes, representing the embryonic stage of muscle, CIC-1 mRNA is not detectable by Northern blotting. From neonatal to adult, CIC-1 expression increases at least fourfold. Using RT-PCR and hybridization on cultured myotubes we found CIC-1 mRNA at a level of 0.4-1.1% of that in mature mouse muscle, and -<0.01% in myoblasts, at stages when desmin mRNA levels are already high. The level of CIC-1 mRNA is thus a sensitive and specific indicator of the maturation of skeletal muscle cells.
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