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"The Role of Pluripotent Stem Cells in the Correction of Muscular Dystrophy"

Joseph M. Vitale
Cell Biology and Molecular Medicine Program
B.A. 2005, Montclair State University

Thesis Advisor: Diego Fraidenraich, Ph.D.
Assistant Professor
Department of Cell Biology and Molecular Medicine

Tuesday, December 20, 2011
12:00 P.M., MSB G609b


Muscular dystrophy represents a large group of incurable muscular hereditary and non-hereditary diseases. Overall, the muscular dystrophies result in progressive muscle weakness as a consequence of contraction-induced damage. This damage is characterized by inflammation and fiber necrosis, which is then followed by fibrosis and adiposis within the muscle tissue.

Duchenne muscular dystrophy (DMD) is a lethal X-linked disease that occurs in 1 out of 3500 people. It is characterized by the loss of the cytoskeleton protein dystrophin. The absence of dystrophin destabilizes the dystrophin-glycoprotein complex (DGC) and results in skeletal muscle extremely vulnerable to contraction-induced injury. Utrophin, a dystrophin-homolog protein, is then upregulated in the absence of dystrophin and helps compensate for the absence of dystrophin, although inefficiently. The DMD phenotype is recapitulated in both a mild phenotype, mdx mice, and a severe phenotype, mdx:utrophin double knockout (KO) mice. In order to elucidate the mechanisms involved in the rescue of DMD, chimeric mice were produced. This involved injecting pluripotent stem cells (PSCs), such as wild type (WT) mouse embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), into either mdx or mdx:utrophin double KO mutant blastocysts. The resultant mosaic mice were then analyzed for corrections in DMD.

Mdx chimeric (or mosaic) mice, with just 10-30% PSC-incorporation, showed that PSC-derived dystrophin was supplied to the muscle sarcolemma to effect corrections at morphological and functional levels. Interestingly, dystrophin was observed at WT levels and utrophin was upregulated in areas with no dystrophin in the mosaic mice. In the mdx:utrophin mutant chimeras, PSC-derived dystrophin was also supplied to the muscle sarcolemma, however the chimeras still displayed poor skeletal muscle histopathology. These results suggest that the presence of utrophin is required for the PSC-corrections in skeletal muscle.
In addition, it was discovered in the DMD mouse models that non-dystrophin expressing tissues, such as the fat, are also affected by PSC injection. Mdx and mdx:utrophin mutant mice normally have a reduced fat/body weight ratio, but PSC injection normalized this parameter in both mdx and mdx:utrophin chimeric fat. This effect was not recapitulated by the injection of PSCs derived from mdx mice. The results highlight a potential non-cell autonomous role, which is independent of utrophin, for the PSC-derived dystrophin in the corrections of non-muscle tissues like fat, which is intimately related to the muscle.

In order to test the previous rescue paradigm in another model of a DGC-related muscular dystrophy, WT PSCs were injected into sarcoglycan- (SG) KO blastocysts, which are predisposed to limb-girdle muscular dystrophy-2F (LGMD-2F). LGMD-2F, a sarcoglycanopathy, is a progressive muscle wasting disease that occurs in 2.3 out of 100,000 people. It primarily affects proximal muscles due to the loss of the sarcoglycan complex (SGC) from the DGC. In the SG chimeras, PSC-derived SG was supplied to the muscle sarcolemma, although not overproduced like dystrophin in the rescue of the mdx mosaic mice. In line with this observation, the SG chimeras with low PSC incorporation still displayed poor skeletal and cardiac histopathology. In correlation, these chimeric animals did not gain any significant fat mass. Indeed, over 60% incorporation was required for the histological recovery of both skeletal and cardiac tissue. However, the diaphragm shows the least recovery and suffers from low levels of both the SGC and dystrophin along with no upregulation of compensatory utrophin, which is likely attributed to the residual dystrophin staining observed in all fibers.

Therefore, both the WT ESCs and the WT iPSCs were successful in incorporating with widespread tissue distribution when injected into mdx, mdx:utrophin double KO and SG KO blastocysts. Unlike mdx, the SG KO model is not amenable to PSC-treatment, particularly the diaphragm. Collectively, these results indicate that only specific types of muscular dystrophy may be easily treatable with PSCs.

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