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E.C. Pacheco–Pinedo, M.T. Budak, S. Bogdanovich, N.A. Rubinstein, T.S. Khurana; Extraocular Muscles (EOM): Identification of an Anatomical Niche With Greater Stem Cell Content . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5059.
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© ARVO (1962-2015); The Authors (2016-present)
Duchenne's Muscular Dystrophy (DMD) is a fatal genetic disorder resulting in widespread muscle damage. Enigmatically, EOM are spared even in advanced cases. A number of hypotheses have been proposed to explain this sparing, however, independent tests of these hypotheses have been equivocal. Based on previous expression profiling and metabolic labeling experiments, (Fischer et al. 2002, McLoon et al. 2002 and Fischer et al. 2005) we propose that enhanced myogenesis and/or tissue stem (Side–population or SP) cell content may contribute to EOM sparing.
In this study we fractionated and analyzed SP cells from EOM and tibialis anterior (TA) as first steps to test this novel hypothesis.
SP cells from TA and EOM were quantified using Hoechst–33342 exclusion and verapamil gating–strategy (Montanaro et al. 2004). Briefly, muscles from adults male C57BL/10 mice were dissected and digested with 1.2 U/ml dispase–5mg/ml collagenase–IV at 37oC/45 min. Digestion was stopped with 20% FBS/Ham's–F10 medium. Cells were filtered through 70um and 40um cell strainers. After RBC lysis, cells were re–suspended at 1 million cells/ml in PBS/BSA and incubated with 5ug/ml of Hoechst–33342 60 min/37oC in presence or absence of 100 uM verapamil. For analysis of Sca–1/CD45 expression, cells were incubated for 15min with 2 ug/million cells of primary or isotype control monoclonal antibodies right after Hoechst staining. Finally cells were re–suspended in cold PBS/BSA and 2ug/ml Propidium iodide was added to the samples before FACS analysis using BD LSRII.
The majority of SP cells using a selection strategy of three logs in the density plot are Sca–1+ve (68% of TA and 67% of EOM) /CD45–ve cells (92% of TA and 96% of EOM). The SP cells abundance ranged from 0.33 – 0.63% for TA and from 0.7 – 1.1% for EOM (n=8).
Consistent with our hypothesis EOM contains c. 15.7x more SP cells per gram than TA (p<.02; n=8).
This study identifies EOM as an anatomical niche enriched in stem cells with potential use for DMD therapy.
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