C2C12 Co‐culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression

Abstract

We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development.

Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration

“…In particular, we found in vitro that myoblasts cultured in the presence of Tcf4 + fibroblasts formed larger, more multinucleated myofibers than myoblasts cultured alone (Mathew et al, 2011). In addition, other experiments have shown in vitro that fibroblasts are a source of trophic signals for myogenic cells (Cooper et al, 2004;Joe et al, 2010;Kusner et al, 2010;Melone et al, 2000). However, this is the first demonstration in vivo that fibroblasts regulate muscle regeneration.…”

Connective tissue fibroblasts and Tcf4 regulate myogenesis

“…Innervation and thyroid hormones have been identified as important extrinsic regulators of fiber type development (Schiaffino and Reggiani, 1996). In addition, previous in vitro studies and avian surgical manipulations had hinted that the connective tissue might modulate fiber type (Cooper et al, 2004;Kusner et al, 2010;Robson et al, 1994), but the role of connective tissue has never been rigorously established by loss-of-function or in vivo experiments.…”

Fibroblasts Can Be Genetically Modified to Produce Excitable Cells Capable of Electrical Coupling

“…Feeder layers were produced on circular 22-mm glass coverslips (Matsunami) as described. 10 Fibroblasts for genetic modification were transduced with LVMyoD alone or in combination with LVCx43GFP and plated onto the feeder layer at 2ϫ10 5 cells per coverslip. Growth medium was changed to differentiation medium after 2 to 3 days and subsequently changed every third day.…”