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. 2008 Jul;16(7):1340-6.
doi: 10.1038/mt.2008.102. Epub 2008 May 27.

Hematopoietic cell transplantation provides an immune-tolerant platform for myoblast transplantation in dystrophic dogs

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Hematopoietic cell transplantation provides an immune-tolerant platform for myoblast transplantation in dystrophic dogs

Maura H Parker et al. Mol Ther. 2008 Jul.

Abstract

Duchenne Muscular Dystrophy (DMD) is the most common and severe form of muscular dystrophy in humans. The goal of myogenic stem cell transplant therapy for DMD is to increase dystrophin expression in existing muscle fibers and to provide a source of stem cells for future muscle generation. Although syngeneic myogenic stem cell transplants have been successful in mice, allogeneic transplants of myogenic stem cells were ineffective in several human trials. To determine whether allogeneic muscle progenitor cells can be successfully transplanted in an immune-tolerant recipient, we induced immune tolerance in two DMD-affected (cxmd) dogs through hematopoietic cell transplantation (HCT). Injection of freshly isolated muscle-derived cells from the HCT donor into either fully or partially chimeric xmd recipients restored dystrophin expression up to 6.48% of wild-type levels, reduced the number of centrally located nuclei, and improved muscle structure. Dystrophin expression was maintained for at least 24 weeks. Taken together, these data indicate that immune tolerance to donor myoblasts provides an important platform from which to further improve myoblast transplantation, with the goal of restoring dystrophin expression to patients with DMD.

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Figures

Figure 1
Figure 1
Outline of hematopoietic cell transplantation, donor muscle cell transplant, and cell culture protocols. (a) Myoblast transplantation in an immune-tolerant cxmd canine. Wild-type donor and cxmd recipient dogs were matched by intrafamilial histocompatibility typing. Total body irradiation (TBI; 200 or 920 cGy) of the recipient was followed by intravenous infusion of donor bone marrow (BM), and peripheral blood mononuclear cells were harvested after G-CSF mobilization of hematopoietic stem cells from the BM (G-PBMC). The recipient was ready for donor myoblast transplantation once stable hematopoietic chimerism was reached. For each experiment, the wild-type donor provided two skeletal muscle biopsies, normally harvested from the biceps femoris muscle. The first skeletal muscle biopsy was used to isolate mononuclear cells for culture before injection. The second skeletal muscle biopsy was used to isolate cells that were to be directly injected into the recipient. (b) Cultured cells were separated into distinct cell populations based on differential adherence. Donor muscle–derived cells were plated on culture dishes. After 1 hour, non-adherent cells were transferred to a new plate, and the adherent cells of the original plate were refed with fresh medium. This was repeated at the time intervals indicated until six distinct populations were obtained—PP1 through PP6. Cells were cultured for 14 days and maintained at, or below, 70% confluence. G-CSF, granulocyte colony-stimulating factor; PP, preplate.
Figure 2
Figure 2
Donor muscle-derived cells engraft into cxmd muscle. (a) Dystrophin expression is restored in full-chimeric cxmd canine. Myeloablative bone marrow transplantation in an cxmd recipient (G289) was followed by intramuscular injection of muscle-derived cells from the marrow donor. Cryosections from donor muscle, recipient muscle preinjection, and recipient muscle 5 and 10 weeks after injection of 1 × 106 freshly isolated cells, were stained using an antibody specific for dystrophin (green). 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei (blue). (b) Dystrophin expression is restored in mixed chimeric cxmd canine. Nonmyeloablative bone marrow transplantation in an cxmd recipient (G604) was followed by intramuscular injection of muscle-derived cells from the marrow donor. Cryosections from donor muscle, recipient muscle preinjection, and recipient muscle 4, 8, and 24 weeks after injection of 4.5 × 106 freshly isolated cells were stained using an antibody specific for dystrophin (green). DAPI was used to visualize nuclei (blue). (c,d) Lack of immune cell infiltration in cxmd recipient muscle injected with donor muscle–derived cells. Cryosections from donor muscle, recipient muscle preinjection, and recipient muscle 10 weeks after injection of fresh or cultured cells were stained with antibodies specific to CD45 (hematopoietic cells; green, c) or CD14 (macrophages; green, d). DAPI (blue) was used to visualize nuclei.
Figure 3
Figure 3
Donor muscle-derived cells restore structure to cxmd muscle. (a) Muscle structure is restored in cxmd recipient muscle injected with donor muscle–derived cells. Cryosections from donor muscle, recipient muscle preinjection, and recipient muscle 10 weeks after injection of freshly isolated cells were stained with hematoxylin and eosin. (b) The number of centrally located nuclei is reduced in cxmd recipient muscle injected with donor muscle–derived cells. The number of centrally located nuclei was counted in four randomly selected fields of view from serial stained sections in a. Bars represent the average percentage of centrally located nuclei in a field of view. Error bars represent SD. The percentage of centrally located nuclei after injection was compared to the percentage preinjection using the Student's t-test (*P < 0.01).
Figure 4
Figure 4
Cultured donor muscle-derived cells engraft into cxmd muscle. (a) Injection of cultured cells into full-chimeric cxmd canine. Myeloablative bone marrow transplantation in an cxmd recipient (G289) was followed by intramuscular injection of muscle-derived cells from the marrow donor. Cryosections from donor muscle, recipient muscle preinjection, and recipient muscle 10 weeks after injection of 5 × 105 cultured cells were stained using an antibody specific for dystrophin (green). 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize nuclei (blue). (b) Injection of cultured cells into mixed chimeric cxmd canine. Nonmyeloablative bone marrow transplantation in an cxmd recipient (G604) was followed by intramuscular injection of muscle-derived cells from the marrow donor. Cryosections from donor muscle, recipient muscle preinjection, and recipient muscle 24 weeks after injection of 3.0 × 106 cultured cells were stained using an antibody specific for dystrophin (green). DAPI was used to visualize nuclei (blue).

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