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. 2010 Aug 5;1(8):e61.
doi: 10.1038/cddis.2010.35.

Partial Dysferlin Reconstitution by Adult Murine Mesoangioblasts Is Sufficient for Full Functional Recovery in a Murine Model of Dysferlinopathy

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Partial Dysferlin Reconstitution by Adult Murine Mesoangioblasts Is Sufficient for Full Functional Recovery in a Murine Model of Dysferlinopathy

J Díaz-Manera et al. Cell Death Dis. .
Free PMC article

Abstract

Dysferlin deficiency leads to a peculiar form of muscular dystrophy due to a defect in sarcolemma repair and currently lacks a therapy. We developed a cell therapy protocol with wild-type adult murine mesoangioblasts. These cells differentiate with high efficiency into skeletal muscle in vitro but differ from satellite cells because they do not express Pax7. After intramuscular or intra-arterial administration to SCID/BlAJ mice, a novel model of dysferlinopathy, wild-type mesoangioblasts efficiently colonized dystrophic muscles and partially restored dysferlin expression. Nevertheless, functional assays performed on isolated single fibers from transplanted muscles showed a normal repairing ability of the membrane after laser-induced lesions; this result, which reflects gene correction of an enzymatic rather than a structural deficit, suggests that this myopathy may be easier to treat with cell or gene therapy than other forms of muscular dystrophies.

Figures

Figure 1
Figure 1
Characterization of C57-J1 cells. Phase-contrast microscopy of adult-derived C57-J1 MABs revealing a small, refractile, triangular shape (a). Once confluent, cells progressively differentiated into multinucleated myotubes (b shows cells after 8 days in differentiation medium). At this stage, IF reveals that expression of striated myosin (c, top) and dysferlin (c, middle) colocalized in myotubes (c, bottom). Differentiation index, calculated as the proportion of total nuclei expressing MHC, ranged between 25 and 40% (d). Western blot analysis performed at different points during differentiation to striated muscle showing concomitant presence of MyoD, myogenin and dysferlin (e). During proliferation status C57-J1 cells did not express MyoD and Pax7 as in satellite cells (e and f)
Figure 2
Figure 2
Inflammatory infiltrates surrounded transplanted cells in BlAJ mice. Hematoxylin-eosin staining of transplanted muscles, showing intense inflammatory reaction after intramuscular injection of 5 × 105 nLacZ-positive C57-J1 cells into tibialis anterior of 5-month-old BlAJ mice (a, left column). Note almost normal muscle architecture and infiltrate resolution 21 days after the injection. X-gal staining on transplanted muscles showing a progressive loss of LacZ-positive cells (a, middle left column). Immune staining with anti-CD3 and anti-CD68 antibodies detected a mixed population of T lymphocytes and macrophages composing the infiltrate (a, middle right and right column). Numerous X-gal-positive muscle fibers invaded by CD3- and CD68-positive cells were detected (b, yellow arrow) whereas X-gal negative fibers maintained its basal lamina intact (b, white arrow)
Figure 3
Figure 3
C57-J1 cells successfully colonized muscles of SCID/BlAJ mice. X-gal staining of SCID/BlAJ tibialis anterior 1 month after a single intramuscular or intra-arterial injection of 5 × 105 C57-J1 cells, previously labeled with nLacZ. At 1 month after the injection numerous LacZ-positive muscle fibers were observed (Figure 3a), localized throughout the entire area of the muscle section. Quantification of LacZ-positive fibers in tibialis anterior and gastrocnemius (Figure 3b): dysferlin mRNA was detected at significant higher levels in transplanted than in nontransplanted muscles in all conditions performed (c, tibialis anterior, quadriceps and gastrocnemius). Differences were analyzed using Student's t-test and considered significant if P<0.05. Bars show mean values, with standard error. Im, intramuscular; Ia, intra-arterial; Ctx, cardiotoxin
Figure 4
Figure 4
Transplantation of C57-J1cells into the SCID/BlAJ murine model successfully restored the expression of dysferlin. Immunofluorescence with anti-dysferlin antibodies detecting the protein only on the membrane of β-galactosidase-positive fibers (a). Western blot analysis of muscle lysates showing the presence of dysferlin protein in all conditions studied (b)
Figure 5
Figure 5
Functional assays. GFP expression pattern of dystrophic tibialis anterior of 5-month-old SCID/BlAJ 1 month after the intramuscular injection of 5 × 105 GFP-positive C57-J1 cells (a). Immunofluorescence analysis showing dysferlin expression on the membrane of GFP-positive single fibers in a patchy distribution (b, white arrows). Membrane repair assay was performed in isolated single fibers from WT, SCID (c, top panel), nontransplanted SCID/BlAJ (c, middle panel) and transplanted SCID/BlAJ (c, bottom panel) mice. Note a larger area of dye staining in the nontreated SCID/BlAJ fibers after laser-induced lesions (c). Measurement of fluorescence intensity versus time is shown in d for WT (blue line), nontransplanted SCID/BlAJ (red line) and transplanted SCID/BlAJ (green line) showing significant higher levels in the nontransplanted SCID/BlAJ. There were no differences between transplanted SCID/BlAJ and WT mice. Data are mean±S.E. (WT n:10 fibers, nontreated SCID/BlAJ n:15 fibers, transplanted SCID/BlAJ n:20 fibers); statistical analysis was carried out with Student's t-test and ANOVA test for repeated measurements. P<0.05 was considered significant

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