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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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Immunoblotting analysis of dystrophin and slow MHCs of different populations of muscle-derived cells when cultivated in fusion medium. The first lane of all immunoblotting corresponds to a homogenate of normal muscle as control (CTR). The populations (pp0–pp6) had a variable ability to differentiate into myotubes when cultivated in a fusion medium for 14 d, resulting in an increase in dystrophin and MHC synthesis in pp3–pp5 (A). In contrast, a significant reduction in the amount of this myogenic marker expression was observed in pp6. Utrophin (Utr) immunoblotting (B) indicated a presence in all preplatings including pp6. These data were also confirmed by sMHC immunostaining (A, top, corresponding to pp3–pp5). The enumeration of differentiated myoblasts containing two or more nuclei (i.e., fusion index) is shown graphically beside Western blotting condition. The error bars represent the SEM from three independently derived preplating cultures.
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Figure 2: Immunoblotting analysis of dystrophin and slow MHCs of different populations of muscle-derived cells when cultivated in fusion medium. The first lane of all immunoblotting corresponds to a homogenate of normal muscle as control (CTR). The populations (pp0–pp6) had a variable ability to differentiate into myotubes when cultivated in a fusion medium for 14 d, resulting in an increase in dystrophin and MHC synthesis in pp3–pp5 (A). In contrast, a significant reduction in the amount of this myogenic marker expression was observed in pp6. Utrophin (Utr) immunoblotting (B) indicated a presence in all preplatings including pp6. These data were also confirmed by sMHC immunostaining (A, top, corresponding to pp3–pp5). The enumeration of differentiated myoblasts containing two or more nuclei (i.e., fusion index) is shown graphically beside Western blotting condition. The error bars represent the SEM from three independently derived preplating cultures.

Mentions: Different populations of muscle-derived cells isolated from hindlimb muscles by different numbers of preplatings were observed. The adherent cells of the earlier preplates display a low desmin immunoreactivity ranging from 5 to 15%, whereas the sequential preplates were enriched in their content of desmin-positive cells (pp3 = 37%; pp4 = 78%; pp5 = 85%) (Fig. 1). These data suggested that the attached cells of pp0–2 are mostly fibroblasts, as confirmed by their typical morphology and the many desmin-positive cells remaining as floating cells in the first 48 h of preplating. Surprisingly, the muscle-derived cells isolated at pp6 contained only 10% desmin-positive cells (Fig. 1G and Fig. H). Since these cells were mostly desmin negative, we believe that our pp6 are different than the muscle-derived cells described by Qu et al. 1998. The morphology of these pp6 cells was predominantly similar to medium-sized blast cells. Endothelial-like elongated cells were also present at this preplate. A number of the pp6 cells (10%) detached from the plastic surface and appeared similar to small lymphocytes. Few M-cadherin–positive cells were found, especially in pp5. These cell populations (pp0–pp6) consequently had a variable ability to differentiate into myotubes when cultivated into a fusion medium for 14 d. Differentiated myoblasts were detected by immunostaining with antibody reactive with MHCs. The number of myotubes obtained in earlier preplates was much lower than in pp4 and pp5. In contrast, the population of pp6 displayed a poor ability to differentiate into myotubes (data not shown). The fusion index of differentiated preplate cultures showed an increased kinetics of differentiation as follows: pp0 = 10%, pp1 = 15%, pp2 = 17%, pp3 = 30%, pp4 = 50%, pp5 = 75%, and pp6 = 12% (graphical in Fig. 2). Few myotubes were also found in grown conditions (pp3 = 7% and pp4–pp5 = 8%). These data were confirmed by immunoblotting experiments after maintaining the cells in a fusion medium for 14 d (Fig. 2). Dystrophin and slow MHCs were threefold higher in pp4 and pp5 than in the other preplates (Fig. 2).


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)

Immunoblotting analysis of dystrophin and slow MHCs of different populations of muscle-derived cells when cultivated in fusion medium. The first lane of all immunoblotting corresponds to a homogenate of normal muscle as control (CTR). The populations (pp0–pp6) had a variable ability to differentiate into myotubes when cultivated in a fusion medium for 14 d, resulting in an increase in dystrophin and MHC synthesis in pp3–pp5 (A). In contrast, a significant reduction in the amount of this myogenic marker expression was observed in pp6. Utrophin (Utr) immunoblotting (B) indicated a presence in all preplatings including pp6. These data were also confirmed by sMHC immunostaining (A, top, corresponding to pp3–pp5). The enumeration of differentiated myoblasts containing two or more nuclei (i.e., fusion index) is shown graphically beside Western blotting condition. The error bars represent the SEM from three independently derived preplating cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199616&req=5

Figure 2: Immunoblotting analysis of dystrophin and slow MHCs of different populations of muscle-derived cells when cultivated in fusion medium. The first lane of all immunoblotting corresponds to a homogenate of normal muscle as control (CTR). The populations (pp0–pp6) had a variable ability to differentiate into myotubes when cultivated in a fusion medium for 14 d, resulting in an increase in dystrophin and MHC synthesis in pp3–pp5 (A). In contrast, a significant reduction in the amount of this myogenic marker expression was observed in pp6. Utrophin (Utr) immunoblotting (B) indicated a presence in all preplatings including pp6. These data were also confirmed by sMHC immunostaining (A, top, corresponding to pp3–pp5). The enumeration of differentiated myoblasts containing two or more nuclei (i.e., fusion index) is shown graphically beside Western blotting condition. The error bars represent the SEM from three independently derived preplating cultures.
Mentions: Different populations of muscle-derived cells isolated from hindlimb muscles by different numbers of preplatings were observed. The adherent cells of the earlier preplates display a low desmin immunoreactivity ranging from 5 to 15%, whereas the sequential preplates were enriched in their content of desmin-positive cells (pp3 = 37%; pp4 = 78%; pp5 = 85%) (Fig. 1). These data suggested that the attached cells of pp0–2 are mostly fibroblasts, as confirmed by their typical morphology and the many desmin-positive cells remaining as floating cells in the first 48 h of preplating. Surprisingly, the muscle-derived cells isolated at pp6 contained only 10% desmin-positive cells (Fig. 1G and Fig. H). Since these cells were mostly desmin negative, we believe that our pp6 are different than the muscle-derived cells described by Qu et al. 1998. The morphology of these pp6 cells was predominantly similar to medium-sized blast cells. Endothelial-like elongated cells were also present at this preplate. A number of the pp6 cells (10%) detached from the plastic surface and appeared similar to small lymphocytes. Few M-cadherin–positive cells were found, especially in pp5. These cell populations (pp0–pp6) consequently had a variable ability to differentiate into myotubes when cultivated into a fusion medium for 14 d. Differentiated myoblasts were detected by immunostaining with antibody reactive with MHCs. The number of myotubes obtained in earlier preplates was much lower than in pp4 and pp5. In contrast, the population of pp6 displayed a poor ability to differentiate into myotubes (data not shown). The fusion index of differentiated preplate cultures showed an increased kinetics of differentiation as follows: pp0 = 10%, pp1 = 15%, pp2 = 17%, pp3 = 30%, pp4 = 50%, pp5 = 75%, and pp6 = 12% (graphical in Fig. 2). Few myotubes were also found in grown conditions (pp3 = 7% and pp4–pp5 = 8%). These data were confirmed by immunoblotting experiments after maintaining the cells in a fusion medium for 14 d (Fig. 2). Dystrophin and slow MHCs were threefold higher in pp4 and pp5 than in the other preplates (Fig. 2).

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