PURPOSE: Allogeneic myogenic stem cell transplantation aims to restore muscle tissue after traumatic loss or to treat muscular dystrophies. Limited engraftment due to allogeneic rejection is a major challenge. We propose a novel therapy with Dystrophin Expressing Chimeric Cells (DEC) created via ex vivo fusion of myoblasts (MB)-originating from transplant donor and recipient as a promising therapeutic option.The aim of this study was to develop the DEC therapy and characterize the phenotype, genotype, engraftment capability and myogenic potential in the DMD scid/mdx mouse model.

METHODS: Two DEC lines created by ex vivo fusion of human myoblasts (MB) derived from two normal donors (MB^N1/MB^N2), and normal and DMD donors (MB^N/MB^DMD) were developed. PKH26 or PKH67 labeled myoblasts from each donor (MB^N1/MB^N2 or MB^N/MB^DMD) were fused using polyethylene-glycol and then characterized by flow cytometry (phenotype: CD45^–, CD90+, CD56+ and proliferation assay), confocal microscopy, PCR-STR, PCR-rSSOP, immunostaining (alpha and beta sarcoglycan, desmin, dystrophin, skeletal myosin heavy chain), real-time PCR (dystrophin) and COMET assay. Finally, the DEC therapeutic potential was assessed in scid/mdx mice (n=6) receiving cell injections (0.5×10^6) to the gastrocnemius muscle (GM): Group 1–vehicle (control), Group 2-MB (control), Group 3–DEC. Dystrophin expression and DEC engraftment were evaluated in the GM by immunostaining 90 days post-injection. GM fibrosis and functional tests (grip strength, wire hanging test, in-vivo and ex-vivo muscle force) were performed.

Results: DEC's phenotype, genotype and proliferation were characterized in vitro. Furthermore, DEC engraftment was confirmed and correlated with increased local dystrophin expression (17.3% at 90 days) and improved functional outcomes.

Conclusion: DEC creation via ex vivo myoblast fusion was confirmed. DEC engraftment resulted in dystrophin expression in dystrophin-deficient muscles of scid/mdx mice. DEC represents a novel approach supporting vascularized composite allograft (VCA) transplantation, muscle regeneration, and treatment of muscular dystrophies.

CITATION INFORMATION: Siemionow M., Cwykiel J., Marchese E., Rafidi G., Futoma K., Heydemann A., Garcia-Martinez J., Szilagyi E. Enhancement of Muscle Regeneration and Function via Transplantation of Human Dystrophin Expressing Chimeric Cells as a Novel Myoblast Stem-Cell Based Therapy Am J Transplant. 2017;17 (suppl 3).

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