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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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Intramuscular transplantation of muscle-derived stem cells. (A) A small number of β-gal myofibers surrounding the injected area were observed in muscles injected with pp6, labeled with a LacZ transgene, and investigated 30 d after intramuscular transplantation. We also verified whether Sca-1, CD34+ stem cells remain in the muscle after intraarterial injection without differentiation and participate actively to muscle formation only after muscle damage. Thus, muscle damage with needle insertions was induced 48 h after intraarterial injection of muscle-derived pp6 cells in the TA muscle of injected hindlimb. The myogenic potentialities of the injected cells was determined by LacZ staining. TA muscle damaged with needle insertions showed a higher increase of LacZ expression (B, C, and D) compared with intramuscular injections (A). In these experiments, fibers of the same section exhibited different levels of LacZ activity, some fibers being more intensely stained than others. Most of the labeling was restricted to regions near the three needle tracks made in the muscles where most of the muscle regeneration was present.
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Figure 9: Intramuscular transplantation of muscle-derived stem cells. (A) A small number of β-gal myofibers surrounding the injected area were observed in muscles injected with pp6, labeled with a LacZ transgene, and investigated 30 d after intramuscular transplantation. We also verified whether Sca-1, CD34+ stem cells remain in the muscle after intraarterial injection without differentiation and participate actively to muscle formation only after muscle damage. Thus, muscle damage with needle insertions was induced 48 h after intraarterial injection of muscle-derived pp6 cells in the TA muscle of injected hindlimb. The myogenic potentialities of the injected cells was determined by LacZ staining. TA muscle damaged with needle insertions showed a higher increase of LacZ expression (B, C, and D) compared with intramuscular injections (A). In these experiments, fibers of the same section exhibited different levels of LacZ activity, some fibers being more intensely stained than others. Most of the labeling was restricted to regions near the three needle tracks made in the muscles where most of the muscle regeneration was present.

Mentions: 21 d after intraarterial injection of Sca-1, CD34 positive muscle-derived cells derived from the pp6 of TnILacZ, clusters of β-gal–positive myofibers (12% ± 0.8 per cross section) of 20–60-μm diameter were observed (Fig. 9, B–D). These fibers had peripherally located nuclei and were along the needle tracks made in the TA 2 d after the intraarterial injection of the pp6 cells. Fibers of the same section exhibited different levels of LacZ activity, some fibers being more intensely stained than others. Most of the labeling was restricted to regions near the three needle tracks done in the muscles where most of the muscle regeneration was present. TA controlateral muscle did not show any positive cells. In a second similar experiment, the damage to the muscle fibers 48 h after the intraarterial injection of the same type of cells was achieved by a prolonged (60-min) swimming exercise. A preliminary experiment has demonstrated that this type of activity damaged 25% of the muscle fibers in the TA muscle, as indicated by the detection of intracellular calcium with alizarin red staining (data not shown). The histology of muscles of these mice presented foci of degenerating and regenerating fibers 24 h after the swimming exercise. In fact, a significant number of basophilic myotubes with vesicular myonuclei and numerous myotubes of small diameters with central nuclei were evident (data not shown). An inflammatory cellular infiltrate was also present. The cell infiltration decreased rapidly and disappeared starting 3 d after exercise. As in the first experiment, the animals were killed 21 d after the intraarterial injection. Quite surprisingly, this type of exercise-induced damage did not increase the percentage of β-gal–positive muscle fibers, as only 1% of them were positive.


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)

Intramuscular transplantation of muscle-derived stem cells. (A) A small number of β-gal myofibers surrounding the injected area were observed in muscles injected with pp6, labeled with a LacZ transgene, and investigated 30 d after intramuscular transplantation. We also verified whether Sca-1, CD34+ stem cells remain in the muscle after intraarterial injection without differentiation and participate actively to muscle formation only after muscle damage. Thus, muscle damage with needle insertions was induced 48 h after intraarterial injection of muscle-derived pp6 cells in the TA muscle of injected hindlimb. The myogenic potentialities of the injected cells was determined by LacZ staining. TA muscle damaged with needle insertions showed a higher increase of LacZ expression (B, C, and D) compared with intramuscular injections (A). In these experiments, fibers of the same section exhibited different levels of LacZ activity, some fibers being more intensely stained than others. Most of the labeling was restricted to regions near the three needle tracks made in the muscles where most of the muscle regeneration was present.
© Copyright Policy
Related In: Results  -  Collection

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Figure 9: Intramuscular transplantation of muscle-derived stem cells. (A) A small number of β-gal myofibers surrounding the injected area were observed in muscles injected with pp6, labeled with a LacZ transgene, and investigated 30 d after intramuscular transplantation. We also verified whether Sca-1, CD34+ stem cells remain in the muscle after intraarterial injection without differentiation and participate actively to muscle formation only after muscle damage. Thus, muscle damage with needle insertions was induced 48 h after intraarterial injection of muscle-derived pp6 cells in the TA muscle of injected hindlimb. The myogenic potentialities of the injected cells was determined by LacZ staining. TA muscle damaged with needle insertions showed a higher increase of LacZ expression (B, C, and D) compared with intramuscular injections (A). In these experiments, fibers of the same section exhibited different levels of LacZ activity, some fibers being more intensely stained than others. Most of the labeling was restricted to regions near the three needle tracks made in the muscles where most of the muscle regeneration was present.
Mentions: 21 d after intraarterial injection of Sca-1, CD34 positive muscle-derived cells derived from the pp6 of TnILacZ, clusters of β-gal–positive myofibers (12% ± 0.8 per cross section) of 20–60-μm diameter were observed (Fig. 9, B–D). These fibers had peripherally located nuclei and were along the needle tracks made in the TA 2 d after the intraarterial injection of the pp6 cells. Fibers of the same section exhibited different levels of LacZ activity, some fibers being more intensely stained than others. Most of the labeling was restricted to regions near the three needle tracks done in the muscles where most of the muscle regeneration was present. TA controlateral muscle did not show any positive cells. In a second similar experiment, the damage to the muscle fibers 48 h after the intraarterial injection of the same type of cells was achieved by a prolonged (60-min) swimming exercise. A preliminary experiment has demonstrated that this type of activity damaged 25% of the muscle fibers in the TA muscle, as indicated by the detection of intracellular calcium with alizarin red staining (data not shown). The histology of muscles of these mice presented foci of degenerating and regenerating fibers 24 h after the swimming exercise. In fact, a significant number of basophilic myotubes with vesicular myonuclei and numerous myotubes of small diameters with central nuclei were evident (data not shown). An inflammatory cellular infiltrate was also present. The cell infiltration decreased rapidly and disappeared starting 3 d after exercise. As in the first experiment, the animals were killed 21 d after the intraarterial injection. Quite surprisingly, this type of exercise-induced damage did not increase the percentage of β-gal–positive muscle fibers, as only 1% of them were positive.

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