HCT and myoblast transplantation protocol
Bone marrow and G-CSF mobilized peripheral blood mononuclear cells (PBMCs) were harvested from two normal donors and transplanted into two irradiated DLA-identical cxmd recipients (, Materials and Methods). The hematopoietic cells of one cxmd recipient were all donor derived (; G289; full chimera), whereas the second recipient had a mix of donor and recipient hematopoietic cells (; G604; mixed chimera). Skeletal muscle-derived mononuclear cells isolated from the same donors were processed for injection immediately after isolation or cultured for 14 days to specifically expand myogenic cells prior to injection (, Materials and Methods). G289 underwent the myeloablative HCT at 5.5 months of age, followed by myoblast transplantation at 32 months of age. G604 underwent the nonmyeloablative HCT at 7 months of age, and myoblast transplantation at 18 months of age.
Figure 1 (a) Myoblast transplantation in an immune tolerant xmd canine. Wild-type donor and xmd recipient dogs were matched by intrafamilial histocompatibility typing. Total body irradiation (TBI; 200 or 920 cGy) of the recipient was followed by intravenous infusion (more ...)
Chimeric xmd recipients used for muscle cell transplantation.
Injection of donor muscle-derived cells restored dystrophin expression in chimeric xmd canines
Enzymatic digestion of a skeletal muscle biopsy from the HCT donor released a mixed population of mononuclear cells that included, but was not limited to, satellite cells and fibroblasts. Freshly isolated muscle-derived cells were injected intramuscularly at several sites in the biceps femoris muscle of chimeric recipients. Cryosections from biopsies taken between four and twenty-four weeks post-injection were analyzed for dystrophin expression. It was expected that if cells injected into the skeletal muscle of the xmd chimeric dog were differentiation and fusion competent, then dystrophin expression would be restored.
Indeed, intramuscular injection of 1×106 fresh donor muscle derived cells into the fully chimeric cxmd recipient, G289, restored dystrophin expression to muscle fibers in muscle biopsies taken at five and ten weeks after injection (). Dystrophin expression was observed throughout the biopsy, extending approximately 0.5 cm from the site of injection (data not shown). Similarly, injection of 4.5×106 freshly isolated donor muscle-derived cells directly into the muscle of the mixed chimeric xmd canine, G604, resulted in clusters of dystrophin expressing fibers, grouped near the sites of injection in biopsies at 4, 8, and 24 weeks after injection (). Expression of dystrophin appeared to be maintained over at least 1000 μm of fiber length, as assessed by examination of serial sections (data not shown).
Figure 2 (a) Dystrophin expression is restored in full chimeric xmd canine. Myeloablative bone marrow transplantation in an xmd recipient (G289) was followed by intramuscular injection of muscle-derived cells from the marrow donor. Cryosections from donor muscle, (more ...)
Quantitation of donor contribution in injected cxmd recipient muscle
The amount of donor derived wild-type dystrophin mRNA was assessed by real-time RT-PCR using a probe that specifically recognizes the wild-type dystrophin splice junction and not the abnormal spliced form in cxmd muscle. The level of dystrophin mRNA observed in wild-type donor muscle was arbitrarily set to 100, and resulted in a relative level of dystrophin mRNA of 0.00035% in the fully chimeric cxmd recipient (G289) before muscle-derived cell transplantation. The level of dystrophin in G289 increased significantly to 6.48% (p =0.0007) ten weeks after injection of freshly isolated cells.
The relative level of skeletal muscle dystrophin mRNA in G604 was 0.0019% before muscle-derived cell transplantation (). This increased to 1.29% (p =0.003) 4 weeks after injection of fresh cells, and remained constant at 24 weeks after injection at 1.32% (p=0.01). Therefore, in both G289 and G604, the values for dystrophin expression were significantly higher after muscle-derived cell transplantation. Because mononuclear satellite cells and muscle stem cells do not express dystrophin, this indicates that the donor muscle-derived cells differentiated into dystrophin expressing myotubes.
Quantitation of wild-type dystrophin transcript in xmd recipient muscle injected with fresh cells.a
Genomic DNA (gDNA) isolated from groups of cryosections serially cut from biopsies of injected muscle and analyzed by VNTR-PCR confirmed the persistence of significant amounts of donor-derived cell DNA at the latest time points of analysis. The fully chimeric xmd dog, G289, displayed an average of 13.9% donor gDNA pre-injection, representing donor blood cell nuclei present as the result of hematopoietic cell transplantation (). Ten weeks after injection of freshly isolated cells, an average of 20.2% of gDNA was donor-derived (p=0.0017), and the highest value of donor contribution to gDNA was 23.1%. The mixed chimeric xmd dog, G604, displayed an average of 5.0% donor gDNA before injection of muscle-derived cells. Twenty-four weeks after injection of freshly isolated cells, an average of 9.3% was donor-derived (p =0.007), with the highest value of donor contribution to gDNA was 12.3%.
Genomic DNA (gDNA) was isolated from groups of serial cryosections from skeletal muscle biopsies and analyzed using VNTR-PCR.
Injection of donor muscle-derived cells did not elicit an immune response in HCT Transplanted Recipients
To determine if injection of donor muscle-derived cells induced an immune response and infiltration of host immune cells in the recipient, cryosections were immunostained using antibodies directed against CD45, a hematopoietic cell marker; CD3, a T-cell specific marker; and CD14, a monocyte/macrophage-specific marker. Minimal hematopoietic cell infiltration was observed in donor and dystrophic recipient skeletal muscle pre-transplantation, as evidenced by occasional CD45 expressing cells ( – donor, recipient pre-injection). There was an absence of CD3-positive T cells (data not shown), yet CD14-positive macrophages were clearly observed in small patches within the recipient DMD-affected muscle pre-injection ( – recipient pre-injection), confirming the mild inflammatory process associated with DMD.
At 10 weeks after cell transplantation, a persistent minimal lymphocytic infiltration was observed ( – CD45). Careful examination of immunostained cryosections uncovered no CD3-positive cells (data not shown); however, a small region of CD14-positive cells was detected, indicative of monocytes/macrophages, which normally infiltrate degenerating skeletal muscle ( – recipient after injection). Therefore, injection of donor muscle-derived cells into cxmd muscle did not induce immune cell infiltration.
Intramuscular injection of donor muscle-derived cells restored muscle structure and reduced regeneration in chimeric xmd canine
For stem cell therapy to be effective and clinically relevant, function must be restored to skeletal muscle. However, the localized injections used in this study precluded testing for functional improvement. Therefore, we evaluated improvement in muscle structure and stability through histological examination. Hematoxylin and eosin staining of cryosections from the G289 recipient muscle pre-transplantation revealed a striking loss of muscle structure, considerable variation in muscle fiber size, and increased fatty and connective tissue as compared to normal donor muscle ( – donor, recipient pre-injection). Injection of freshly isolated muscle-derived cells markedly improved muscle structure at 10 weeks after transplantation and resulted in less connective and fatty tissue infiltration and reduced size variation in fiber diameter when compared to the pre-cell injection biopsy ().
Figure 3 (a) Muscle structure is restored in xmd recipient muscle injected with donor muscle-derived cells. Cryosections from donor muscle, recipient muscle pre-injection, and recipient muscle 10 weeks after injection of freshly isolated cells were stained with (more ...)
Newly formed skeletal muscle fibers or fibers that have acquired differentiating myocytes as a result of regeneration are characterized by nuclei located in the center of the muscle fiber. In donor wild-type muscle, less than 1% of muscle nuclei were centrally located. However, approximately 10% of muscle nuclei in the G289 DMD-affected recipient were centrally located, indicating that a portion of dystrophic muscle was regenerating (). Injection of fresh muscle-derived cells reduced the number of centrally located myonuclei in the G289 DMD-affected recipient muscle to less than 5%, which was statistically significant as compared to the recipient before injection (p<0.01). Together, these findings suggested that engraftment of donor muscle-derived cells and restoration of dystrophin expression stabilized cxmd muscle, preventing repeated attempts to regenerate.
Cultured donor muscle-derived cells restore dystrophin expression
Our data demonstrate that direct injection of freshly isolated donor-derived muscle cells engraft without immune rejection in chimeric recipients. However, many muscle stem cell transplant procedures require expansion of cells in culture, which might alter the immunogenic status of the donor cells. Therefore, to determine whether similar engraftment could be obtained by cells expanded in culture, donor muscle-derived cells were cultured before injection into biceps femoris muscle of the chimeric cxmd
recipients. We used a standard serial pre-plating technique to enrich for myoblasts and analyzed cells from pre-plates 2 (PP2) and 6 (PP6) (). In order to ensure that donor muscle-derived mononuclear cells were myogenic, adherent PP2 and PP6 cells were fixed in proliferation (Supplemental Figure 1a
) and differentiation (Supplemental Figure 1b
) conditions. Immunostaining with antibodies specific to desmin and Pax7, two markers of myoblasts in culture, established that more than 90% of proliferating cultured cells were myogenic.
To assess differentiation potential in vitro
, PP2 and PP6 cells were cultured until confluent, and fixed after an additional 72 hours. The PP2 population differentiated to form multinucleated myotubes that expressed myogenin and myosin heavy chain (MyHC), two markers of myogenic differentiation (Supplemental Figure 1b
– PP2). The PP6 population also expressed myogenin and MyHC, yet remained primarily mononuclear (Supplemental Figure 1b
– PP6). Mouse muscle-derived cells cultured in a comparable manner exhibited similar results [28
Similar to direct injection of freshly isolated muscle-derived cells, injection of cultured PP6 cells did not induce immune cell infiltration in the injected muscle (data not shown). In addition, the injection of cultured PP6 cells restored dystrophin expression to skeletal muscle of chimeric cxmd recipients (). The level of dystrophin reached a maximum of 1.45% of wild-type levels in the fully chimeric dog, G289, and 0.05% in the mixed chimeric dog, G604 (). Although these were significantly higher relative to the recipient levels before muscle cell transplantation (p =0.0026 and p =0.009, respectively), the maximum levels of wild-type dystrophin was less than that reached after injection of freshly isolated cells ().
Figure 4 (a) Injection of cultured cells into full chimeric xmd canine. Myeloablative bone marrow transplantation in an xmd recipient (G289) was followed by intramuscular injection of muscle-derived cells from the marrow donor. Cryosections from donor muscle, (more ...)
Quantitation of wild-type dystrophin transcript in xmd recipient muscle injected with cultured cells.a