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

Abstract

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.

Figures

Figure 1

Characterization, myogenic differentiation in vitro and migrating capacity in vivo of hIDPSC. (A) Fibroblast-like morphology of hIDPSC. (B) Proliferating potential of these cells during 16 successive passages. (C-E) Flow cytometry showing expression of CD105 (SH2), CD73 (SH3 and SH4), respectively. (F) Negative control for respective isotype. (G,H) Myogenic differentiation in vitro: fused hIDPSC forming myotubes. Positive immunostaining with α-actinin (G) and myosin (H): insets in (G and H) show details of anti-bodies localization within myotubes, higher magnification. (I) RT-PCR analysis of the expression of human dystrophin (MyoD1) observed hIDPSC and human muscles, used as a positive control. (J-M). Migrating capacity of hIDPSC visualized 30 days after their intraperitoneal injection into normal mice (J-M): (J) Cells stained with DiI-Vybrant (red) in mouse, (K) positive reaction with primary human anti-IDPSC antibody in mice (secondary antibody FITC-conjugated was used (green), (L) Morphology of mouse cardiac muscles. (M) merged image of J-L. A= light microscopy, phase contrast, G-H = epi-fluorescence (EF), J-M= confocal microscopy: J, K = fluorescent microscopy (Fcm), L = DIC (Differential interference contrast) M = Fcm+DIC. Scale bars: G,H = 10 μm, A,J-M = 100 μm, N-P = 50 μm

Figure 2

DMD genotyping. GRMD puppies from a colony of dogs with X-linked muscular dystrophy were genotyped within 48 hs after birth. The genomic PCR product digested with Sau96I produces the wild type band (310 pb) and the mutant band (150 pb) labelled with arrows. ■ = Affected male. ● = Affected female. ◉ = Carrier female.

Figure 3

Representative figures of hIDPSC engraftment observed within canine skeletal muscles (GRMD). (A) FT2-IM, after 107 days of single transplantation: Positive immunostaining with anti-hIDPSC antibody (green) was observed in several muscle fibers (white arrowhead) and in the nuclei (white arrows, blue, DAPI stained superposition with anti-IDPSC antibody, green) of hIDPSC localized in the periphery of canine muscle fibers. (B-I) One year after multiple hIDPSC transplantation. Positive immunostaining with anti-IDPSC antibody was observed in MT1-S muscles: (B-D) transversal and (E-H) longitudinal sections. Inset in (F) demonstrate (higher magnification) skeletal muscle Z-bands (red arrowhead) observed in the local of positive immunostaining with anti-hIDPSC antibody (DIC). (H) Higher magnification of (G) demonstrating positive reaction of hIDPSC antibody (green) with skeletal muscle Z-bands (red arrowhead). (I) Chimeric human/canine muscle fiber only a half of which presents positive green fluorescent immunostaining (green). (J) Control: affected male without hIDPSC transplantation immunostained with anti-hIDPSC antibody did not present any labeling. (K) FISH analysis of dystrophic male's muscles using specific human probe for chromosome: Y (red) and in inset X (yellow, as a result of merged images of PI (red) stained nucleus and probe of chromosome X (green) are presented). (L-P) Immunostaining using FITC conjugated anti-human nucleus (anti-HN) antibody (green). (L) Positive control. Merged image of positive staining with anti-HN antibody and nuclei stained with DAPI in normal human muscles. (M-O) Positive immunostaining with anti-HN antibody observed in the nuclei of hIDPSC (green) engrafted into canine muscle fibers of MT1-S. They (white arrowhead) can be seen within canine muscle fibers and in perimysium (white arrows). Canine nuclei (group of 4 nuclei indicated by red arrow) did not present any reaction with anti-HN antibody. (P) Negative control. Muscles of normal dog did not react with anti-HN antibody. Only nuclei stained with DAPI can be observed. Confocal microscopy, A,D,G-L,P = Fcm+DIC; C,F,M,N = Fcm, B,E, Inset in (F) = DIC, Scale bars: A-H, L,P = 50 μm, K = 100 μm, and M-O = 20 μm, Inset in (K) = 10 μm

Figure 4

Immunofluorescence analysis using the specific human anti-dystrophin monoclonal antibodies: Mandys106 2C6 (A-D) and C-terminal (E-J) antibodies one year after the hIDPSC transplantation. (A) Positive control: expression of Mandys106 2C6 antibody in normal human muscles. (B) Negative control: lack of expression of same antibody in the muscles of normal dog. (C, D) MT1-S shows positive staining with Mandys106 2C6 in large dystrophic fibers (white arrows). (E) Positive control: expression of C-terminal in normal human muscles. (F) C-terminal antibody in the muscles of normal dog presents weak labeling. (G) Negative control: lack of its expression in the muscles of affected dog. (H-J) MT1-S shows positive staining with C-terminal antibody in some large dystrophic fibers (arrow). K) Toluidine blue and L) HE staining shows very large fibers with multiple centrally located nuclei and splitting. A, C, E-H = Fcm + DIC, B = DIC, D,I,= EF, J,K = Light microscopy, Scale bars = 50 μm