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Intraarterial injection of muscle-derived CD34(+)Sca-1(+) stem cells restores dystrophin in mdx mice.

Torrente Y, Tremblay JP, Pisati F, Belicchi M, Rossi B, Sironi M, Fortunato F, El Fahime M, D'Angelo MG, Caron NJ, Constantin G, Paulin D, Scarlato G, Bresolin N - J. Cell Biol. (2001)

Bottom Line: One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions.Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message.Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale Maggiore Policlinico, 20122 Milan, Italy.

ABSTRACT
Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.

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Sections of the muscles of mdx/mdx mice injected intraarterially with muscle-derived pp6 cells showing dystrophin and des-LacZ gene expression. LacZ staining of muscle sections from donor des-LacZ mice was performed as a positive control (A and B). Some LacZ-positive myofibers (C and E) were found in the intraarterially injected mdx muscles that colocalized with dystrophin-positive myofibers of the adjacent sections (D and F). Bars, 50 μm.
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Figure 8: Sections of the muscles of mdx/mdx mice injected intraarterially with muscle-derived pp6 cells showing dystrophin and des-LacZ gene expression. LacZ staining of muscle sections from donor des-LacZ mice was performed as a positive control (A and B). Some LacZ-positive myofibers (C and E) were found in the intraarterially injected mdx muscles that colocalized with dystrophin-positive myofibers of the adjacent sections (D and F). Bars, 50 μm.

Mentions: To identify the ability of intraarterially injected tissues to transcribe normal dystrophin and LacZ mRNAs, histochemical staining on cryostat sections of muscles, brain, liver, lungs, kidneys, and bones were performed. β-Gal–positive nuclei were detected only in striated muscles of the injected hindlimb. Positive LacZ myofibers were found in restricted areas. Muscle fibers scoring positive had peripheral nuclei and fiber diameters varied from 20 to 60 μm. 10 different fibers having β-gal–positive nuclei were counted in the soleus, corresponding to ∼1% of total fibers per cross sectional area, and 15 positive fibers, corresponding to 0.5% of total fibers in a given cross section, were counted in the quadriceps. The pattern of LacZ gene expression was compared with that of dystrophin, and typical immunofluorescence for dystrophin was found in the same fibers that contained β-gal–positive nuclei (Fig. 8). The dystrophin antibody did not cross-react with autosomal dystrophin-related proteins such as utrophin. Positive fiber always seemed to exist as isolated small groups (one to five positive fibers), suggesting that there might have been clonal proliferation of some donor cells, which contributed to the formation of the fibers in that region of the muscle. Control mdx muscles contained rare revertant fibers that were positive for dystrophin (Hoffman et al. 1990), but also β-gal negative.


Intraarterial injection of muscle-derived CD34(+)Sca-1(+) stem cells restores dystrophin in mdx mice.

Torrente Y, Tremblay JP, Pisati F, Belicchi M, Rossi B, Sironi M, Fortunato F, El Fahime M, D'Angelo MG, Caron NJ, Constantin G, Paulin D, Scarlato G, Bresolin N - J. Cell Biol. (2001)

Sections of the muscles of mdx/mdx mice injected intraarterially with muscle-derived pp6 cells showing dystrophin and des-LacZ gene expression. LacZ staining of muscle sections from donor des-LacZ mice was performed as a positive control (A and B). Some LacZ-positive myofibers (C and E) were found in the intraarterially injected mdx muscles that colocalized with dystrophin-positive myofibers of the adjacent sections (D and F). Bars, 50 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199616&req=5

Figure 8: Sections of the muscles of mdx/mdx mice injected intraarterially with muscle-derived pp6 cells showing dystrophin and des-LacZ gene expression. LacZ staining of muscle sections from donor des-LacZ mice was performed as a positive control (A and B). Some LacZ-positive myofibers (C and E) were found in the intraarterially injected mdx muscles that colocalized with dystrophin-positive myofibers of the adjacent sections (D and F). Bars, 50 μm.
Mentions: To identify the ability of intraarterially injected tissues to transcribe normal dystrophin and LacZ mRNAs, histochemical staining on cryostat sections of muscles, brain, liver, lungs, kidneys, and bones were performed. β-Gal–positive nuclei were detected only in striated muscles of the injected hindlimb. Positive LacZ myofibers were found in restricted areas. Muscle fibers scoring positive had peripheral nuclei and fiber diameters varied from 20 to 60 μm. 10 different fibers having β-gal–positive nuclei were counted in the soleus, corresponding to ∼1% of total fibers per cross sectional area, and 15 positive fibers, corresponding to 0.5% of total fibers in a given cross section, were counted in the quadriceps. The pattern of LacZ gene expression was compared with that of dystrophin, and typical immunofluorescence for dystrophin was found in the same fibers that contained β-gal–positive nuclei (Fig. 8). The dystrophin antibody did not cross-react with autosomal dystrophin-related proteins such as utrophin. Positive fiber always seemed to exist as isolated small groups (one to five positive fibers), suggesting that there might have been clonal proliferation of some donor cells, which contributed to the formation of the fibers in that region of the muscle. Control mdx muscles contained rare revertant fibers that were positive for dystrophin (Hoffman et al. 1990), but also β-gal negative.

Bottom Line: One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions.Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message.Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale Maggiore Policlinico, 20122 Milan, Italy.

ABSTRACT
Duchenne muscular dystrophy is a lethal recessive disease characterized by widespread muscle damage throughout the body. This increases the difficulty of cell or gene therapy based on direct injections into muscles. One way to circumvent this obstacle would be to use circulating cells capable of homing to the sites of lesions. Here, we showed that stem cell antigen 1 (Sca-1), CD34 double-positive cells purified from the muscle tissues of newborn mice are multipotent in vitro and can undergo both myogenic and multimyeloid differentiation. These muscle-derived stem cells were isolated from newborn mice expressing the LacZ gene under the control of the muscle-specific desmin or troponin I promoter and injected into arterial circulation of the hindlimb of mdx mice. The ability of these cells to interact and firmly adhere to endothelium in mdx muscles microcirculation was demonstrated by intravital microscopy after an intraarterial injection. Donor Sca-1, CD34 muscle-derived stem cells were able to migrate from the circulation into host muscle tissues. Histochemical analysis showed colocalization of LacZ and dystrophin expression in all muscles of the injected hindlimb in all of five out of five 8-wk-old treated mdx mice. Their participation in the formation of muscle fibers was significantly increased by muscle damage done 48 h after their intraarterial injection, as indicated by the presence of 12% beta-galactosidase-positive fibers in muscle cross sections. Normal dystrophin transcripts detected enzymes in the muscles of the hind limb injected intraarterially by the mdx reverse transcription polymerase chain reaction method, which differentiates between normal and mdx message. Our results showed that the muscle-derived stem cells first attach to the capillaries of the muscles and then participate in regeneration after muscle damage.

Show MeSH
Related in: MedlinePlus