Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy

Abstract

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin–deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 106) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

Cardiac Protection after Systemic Transplant of Dystrophin Expressing Chimeric (DEC) Cells to the mdx Mouse Model of Duchenne Muscular Dystrophy

“…Myoblast Isolation DEC cells of myoblast origin were created after myoblast isolation from the mdx and snj wild type (wt) animals as reported before [32]. Briefly, hind limb muscles were harvested, minced and incubated with 1.5 U/l Collagenase type D (Roche-ThermoFischer, Waltham, MA, USA) and 2.4 U/l Dispase II (Sigma, St. Louis, MO, USA) in 2.5 mM CaCl 2 solution at 37°C for 30 min.…”

“…Our standard polyethylene glycol (PEG) fusion protocol was applied as described before [31,32]. Parent snj (wt) and mdx myoblasts (MB wt and MB mdx ) as well as mdx MSC (MSC mdx ) were harvested and washed in serum-free DMEM culture media supplemented with 1% Antibiotic-Antimycotic solution (Gibco-ThermoFischer, Waltham, MA, USA).…”

“…Stem cell transplants based on the delivery of either autologous or allogenic stem cells have shown promise as an alternative method for DMD treatment, but limited or short-term cell engraftment, allogenic immune response, and side effects of immunosuppressive therapy have all been challenges that these treatments have faced in their path to changing the course of the disease for DMD patients [26][27][28][29][30][31][32][33][34][35][36][37][38][39].…”

“…Previous in vitro studies have shown that autologous multipotent stem cells from wild type mice are able to differentiate into reconstituted skeletal muscle cells when injected into mdx mice [32]. Gussoni et al demonstrated that bone marrow transplantation via intravenous injection of hematopoietic stem cells can reconstitute expression of dystrophin in affected animals [40].…”

“…We have previously reported in both the mdx and mdx/scid mouse models of DMD that local transplantation of Dystrophin Expressing Chimeric (DEC) cells significantly improved dystrophin expression which correlated with significant improvement of strength and functional outcomes [31,32]. Thus, in order to assess the effect of DEC therapy on cardiac function, we applied our established protocol of systemic-intraosseous administration of both DEC lines (MB wt /MB mdx and MB wt /MSC mdx ) into mdx mice [31,32,45].…”