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S. S. Tjoe, A. B. Young, H. J. Kaminski, L. L. Kusner; Differential Role of Orbital and Hindlimb Fibroblasts in Maintaining the Extraocular Muscle Cell Line. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5280.
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© ARVO (1962-2015); The Authors (2016-present)
Extraocular muscle (EOM) is a phenotypically unique skeletal muscle whose development is affected by a number of extrinsic environmental factors. In this study, we hypothesize that fibroblasts are one such factor that supports the development and maintenance of the unique EOM phenotype.
The extraocular muscle (EOM) cell line and the C2C12 cell line were obtained from neonatal (4-6 day old) C57BL/6J mice and the ATCC, respectively. EOM (EOM-Fibro) and hindlimb (LM-Fibro) fibroblasts were derived from adult (3-5 week) C57BL/6J mice skeletal muscle. Co-cultures of the EOM and C2C12 cell lines were established on a confluent monolayer of fibroblast cells and allowed to differentiate for up to 12 days. Maturity of the myotubes was assessed by morphological evaluation and RNA analysis. Myotube counts were performed by determining the percentage of myotubes containing 4 or more nuclei per co-culture. Immunohistochemistry and qPCR were used to determine myosin expression.
EOM/EOM-Fibro and EOM/LM-Fibro co-cultures yield highly differentiated, contractile myotubes. Addition of condition media obtained from EOM-Fibro produced between 5-10% more myotubes containing 4 or more nuclei in EOM differentiating cultures than in defined differentiation media (F10C supplemented with 2% Horse serum). Analysis of myosin expression shows that the EOM/EOM-Fibro co-culture significantly expresses higher levels of myosin than the EOM/LM-Fibro co-culture. This myosin expression pattern is corroborated in the C2C12/EOM-Fibro and C2C12/LM-Fibro co-cultures but to a lesser extent.
While both orbital and hindlimb fibroblasts support the development and maintenance of the extraocular muscle, orbital fibroblasts are seen to significantly bolster the maturation of extraocular muscle myotubes. Extrinsic factors and unique cell-to-cell interaction between the extraocular muscle and the orbital fibroblasts may influence the unique extraocular muscle phenotype.
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