Blastocyst Injection of Wild Type Embryonic Stem Cells Induces Global Corrections in Mdx Mice

Elizabeth Stillwell,Genie Elson,Farah Khadim,Joel Schneider,Ghassan Yehia,Joseph Vitale,Robert Grange,Diego Fraidenraich,Willie Mark,Aneela Altaf,Mantu Bhaumik,Jing Liu,Amanda Beck,Jia-Hui Dong,Qingshi Zhao,Maurizio Capogrossi

doi:10.1371/journal.pone.0004759

Elizabeth Stillwell, Genie Elson + Show 14 more

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https://doi.org/10.1371/journal.pone.0004759

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Abstract

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology and function. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection increases the amount of fat in the chimeras to reach WT levels. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally in a dose-dependent manner.

Highlights

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder affecting 1 in 3500 newborn males
We report that dystrophin supplied by WT embryonic stem cells (ESCs) stabilizes the skeletal muscle in areas with high dystrophin and in areas with low dystrophin, and corrects tissues that are affected in mdx and in DMD outside of the muscle, such as fat
We described that WT ROSA26 (R26) LacZ-marked ESCs (WT ESCs) rescue the lethality of Id knockout embryos, a mouse model of the thin myocardial syndrome, and that ESC-derived secretion factors accounted for the reversion of the phenotypes in the embryonic heart and brain [41,42,43]

Summary

Introduction

DMD is an X-linked recessive disorder affecting 1 in 3500 newborn males. DMD is characterized by progressive muscle weakness associated with necrosis of muscle fibers and fibrosis of muscle tissue. DMD is caused by defects in the dystrophin gene. The full isoform (427 kD) of dystrophin localizes to the sarcolemma of the skeletal muscle. Dystrophin forms a complex with other proteins including dystroglycans, sarcoglycans, dystrobrevin and syntrophin called the dystrophin-glycoprotein complex (DGC) [15]. The DGC provides stability to the membrane and protects the skeletal muscle from mechanically induced damage. The C terminus of dystrophin is dispensable, partly because dystrobrevin (a distant dystrophin-related protein) can substitute for some of the functions of dystrophin, like binding to syntrophin. Syntrophin is responsible for localizing neuronal nitric oxide synthase (nNOS) to the sarcolemma. In the absence of dystrophin, the DGC is unstable and is destroyed [16,17,18,19,20,21]

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