Early transplantation of human immature dental pulp stem cells from baby teeth to golden retriever muscular dystrophy (GRMD) dogs: Local or systemic?

Background:The golden retriever muscular dystrophy (GRMD) dogs represent the best available animal model for therapeutic trials aiming at the future treatment of human Duchenne muscular dystrophy (DMD). We have obtained a rare litter of six GRMD dogs (3 males and 3 females) born from an affected male and a carrier female which were submitted to a therapeutic trial with adult human stem cells to investigate their capacity to engraft into dogs muscles by local as compared to systemic injection without any immunosuppression. Methods Human Immature Dental Pulp Stem Cells (hIDPSC) were transplanted into 4 littermate dogs aged 28 to 40 days by either arterial or muscular injections. Two non-injected dogs were kept as controls. Clinical translation effects were analyzed since immune reactions by blood exams and physical scores capacity of each dog. Samples from biopsies were checked by immunohistochemistry (dystrophin markers) and FISH for human probes. Results and Discussion We analyzed the cells' ability in respect to migrate, engraftment, and myogenic potential, and the expression of human dystrophin in affected muscles. Additionally, the efficiency of single and consecutive early transplantation was compared. Chimeric muscle fibers were detected by immunofluorescence and fluorescent in situ hybridisation (FISH) using human antibodies and X and Y DNA probes. No signs of immune rejection were observed and these results suggested that hIDPSC cell transplantation may be done without immunosuppression. We showed that hIDPSC presented significant engraftment in GRMD dog muscles, although human dystrophin expression was modest and limited to several muscle fibers. Better clinical condition was also observed in the dog, which received monthly arterial injections and is still clinically stable at 25 months of age. Conclusion Our data suggested that systemic multiple deliveries seemed more effective than local injections. These findings open important avenues for further researches.

This research was supported by FAPESP, CEPID Process n°: 1998/14254-2 and CNPq Process n°: 55.2210/2005–6. FAPESP support other two projects: 2004/15370-9 and 2005/50082-7. We would like to thank Dr. Glenn Morris from Center for Inherited Neuromuscular Disease (CIND), RJAH Orthopaedic Hospital, Oswestry, Shropshire, UK for providing anti-human dystrophin antibodies and Dr. Mariz Vainzof for helpful suggestions. Also, to Prof. Joe N. Kornegay and Dr. Janet Bogan from Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, and the Dalton Cardiovascular Research Center, University of Missouri, Columbia, USA who were responsible for the donation of the first GRMD carrier female and the establishment of the Brazilian colony of GRMD dogs. We are very grateful also to Marta Canovas, for technical support for dystrophin analysis, Alex S.Souza for an excellent technical assistance with Confocal Microscopy, to T. Kawano chief of the Laboratory of Parasitology of Butantan Institute, São Paulo, Brasil and to Constancia Gotto for secretarial assistance and invaluable support. Finally to Dr. Marcia Triunfol for final English review.