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M.T. Budak, N.A. Rubinstein, T.S. Khurana; Identification and Characterization of Stem Cells in Extraocular Muscle (EOM) . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5726.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: EOMs remain clinically and pathologically spared even in advanced cases of Duchenne's muscular dystrophy (DMD). Recent profiling (Fischer et al. 2002 Physiol Genomics) and metabolic labeling studies (McLoon et al. 2002 Muscle & Nerve) have demonstrated active regeneration/ myogenesis in EOM, suggesting that the greater regeneration may play a role in the sparing of EOM in DMD. In this study we have fractionated and analyzed the stem cell population (SP cells) of EOM as first steps toward testing our hypothesis that EOM stem cells contribute to sparing of EOM noted in DMD. Methods:Fractionation & quantification of Stem cell fractions (SP cells) was performed based on methods described in Gussoni et al. Nature 1999. Briefly, normal adult C57BL/10ScSn mice were sacrificed using CO2 asphyxia. EOMs and Tibialis anterior (TA) muscles were dissected rapidly and digested in dispase/collagenase solution. Cells were resuspended at 106 cell/ml concentration, incubated with 5µg/ml Hoechst 33342 and 2µg/ml propidium iodide (PI) and sorted using FACS using dye–exclusion crieteria to define side population (SP) cell fractions that are enriched in stem cells. Aliquots of cells were stained in the presence of 100µM Verapamil to ensure that the dye–efflux can be inhibited by this drug. Results:Consistent with previous reports, stem cells are rarely present in adult TA muscle; their abundance ranged from 0.06–0.2%. Interestingly stem cell proportion was far greater (5–20 fold increased) in EOMs compared to TA. Conclusions: EOM represent a rare anatomical niche that contains a high proportion of stem cells in adult tissue. These data support our hypothesis that EOM stem cells may contribute to EOM sparing in DMD and may prove to be a viable source for therapeutic harvesting/ development for DMD therapy. (Supported in part by R21 AR051696 from NIAMS "Extraocular muscle stem cells for DMD therapy" to TSK)
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