Transplantation of Dystrophin Expressing Chimeric Human Cells of Myoblast/Mesenchymal Stem Cell Origin Improves Function in Duchenne Muscular Dystrophy Model

Abstract

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder caused by mutations in dystrophin gene. Currently, there is no cure for DMD. Cell therapies are challenged by limited engraftment and rejection. Thus, more effective and safer therapeutic approaches are needed for DMD. We previously reported increased dystrophin expression correlating with improved function after transplantation of dystrophin expressing chimeric (DEC) cells of myoblast origin in the mdx mouse models of DMD. This study established new DEC cell line of myoblasts and mesenchymal stem cells (MSC) origin and tested its efficacy and therapeutic potential in mdx/scid mouse model of DMD. Fifteen ex vivo cell fusions of allogenic human myoblast [normal myoblasts (MB^N)] and normal human bone marrow-derived MSC (MSC^N) from normal donors were performed using polyethylene glycol. Flow cytometry, confocal microscopy, polymerase chain reaction (PCR)-short tandem repeats, polymerase chain reaction-reverse sequence-specific oligonucleotide probe assessed chimeric state of fused MB^N/MSC^N DEC cells, whereas Comet assay assessed fusion procedure safety testing genotoxicity. Immunofluorescence and real-time PCR assessed dystrophin expression and myogenic differentiation. Mixed lymphocyte reaction (MLR) evaluated DEC's immunogenicity. To test MB^N/MSC^N DEC efficacy in vivo, gastrocnemius muscle of mdx/scid mice were injected with vehicle (n = 12), nonfused MB^N and MSC^N (n = 9, 0.25 × 10⁶/each) or MB^N/MSC^N DEC (n = 9, 0.5 × 10⁶). Animals were evaluated for 90 days using ex vivo and in vivo muscle strength tests. Histology and immunofluorescence staining assessed dystrophin expression, centrally nucleated fibers and scar tissue formation. Post-fusion, MB^N/MSC^N DEC chimeric state, myogenic differentiation, and dystrophin expression were confirmed. MLR reveled reduced DEC's immune response compared with controls (P < 0.05). At 90 days post-DEC transplant, increase in dystrophin expression (20.26% ± 2.5%, P < 0.05) correlated with improved muscle strength and function in mdx/scid mice. The created human MB^N/MSC^N DEC cell line introduces novel therapeutic approach combining myogenic and immunomodulatory properties of MB and MSC, and as such may open a universal approach for muscle regeneration in DMD.

Keywords: DEC therapy; Duchenne muscular dystrophy; cell fusion; immunomodulation; mesenchymal stem cells; myoblasts.