43: Use of Nonhuman Primates as a Preclinical Model to Test In Vitro Treatments that Were Reported to Improve the Outcome of Muscle Precursor Cell Transplantation in Mice

Introduction Transplantation of muscle precursor cells (MPCs) is a major strategy for the potential future treatment of several skeletal muscle diseases. The protocol of MPC transplantation that we developed in non-human primates (NHPs) allowed the restoration of the genetically missing protein (dystrophin) with better efficacy than previously in muscle fibers of patients with Duchenne muscular dystrophy. However, our protocol needs improvements to obtain a broader cell engraftment and thus increase dystrophin restoration. Studies in mice reported methods that substantially increased the MPC engraftment, based on in vitro treatments and sometimes co-injection of the cells with growth factors or other molecules. Since these methods would be easily applicable in the clinic, we wanted to verify them in NHPs, given that this animal model is crucial in preclinical transplantation research before translating protocols to humans. Methods We allotransplanted CD56+ MPCs labeled with ß-galactosidase (ß-Gal) in muscle regions of 1 cm3 in 14 cynomolgus monkeys. Approximately 20 x 106 cells were injected per site, always following the same pattern: matrices of 25 injections per cm2, delivering 25 μl of cell suspension per injection. Tacrolimus, combined to dexamethasone, was administered daily to control acute rejection. The strategies to improve the MPC engraftment consisted in the in vitro treatment of the cells (and sometimes co-injection of them) with the following molecules: insulin growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF), IGF-1 + bFGF + plasmin, concanavalin A and SB203580 (a p38 MAP kinase inhibitor). One of two cell-grafted regions was treated by electroporation to induce broad muscle fiber regeneration concomitant to the graft. Cell grafted regions were sampled one to two months after cell transplantation, snap frozen in liquid nitrogen and sectioned in a cryostat. Muscle cross-sections were stained with hematoxylin and eosin, and for histochemical demonstration of ß-Gal. ß-Gal+ muscle fibers were quantified as an indication of engraftment success. Results The only factor that substantially increased the number of ß-Gal+ muscle fibers was the extensive muscle fiber regeneration produced by the muscle electroporation. Otherwise, for the same cell transplantation conditions (injection with or without electroporation) none of the cell treatments increased the amount of ß-Gal+ muscle fibers and therefore the success of the cell transplantation in terms of genetic complementation. Conclusion Cell treatments reported to increase the MPC engraftment in mice had no incidence in NHPs.The only strategy capable of increasing the MPC engraftment in NHPs remained the induction of extensive muscle regeneration concomitant with the cell transplantation. As in other areas of preclinical research, this study also questions the clinical validity of results reported only in mice. This work was supported by a grant of the Jesse’s Journey Foundation for Gene and Cell Therapy of Canada to D.S. and a grant of the Canadian Institutes of Health Research to J.P.T.

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