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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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Immunophenotyping of the muscle-derived cells using FACScan™ analysis. Muscle-derived cells were tested for CD34 and Sca-1 single (A) and double (B) expression. A distinct population of Sca-1+ cells was found in all seven preplates, but CD34+ cells were observed only in pp6 (A). The morphology distribution of muscle-derived cells after flow cytometry showed different subpopulations within the Sca-1+ cell population (B). The profile of these cells varied from cells with large diameters (pp0–4) to small diameters (pp5–6). The majority of cells derived from pp3 (B, top left quadrant) and pp5 (B, top right quadrant) highly express Sca-1, but were also poorly CD34+. Muscle-derived cells from pp6 showed 69% double-positive cells for CD34 and Sca-1 markers (B, bottom left quadrant). However, all Sca-1+ cells were not CD34+ and a fraction of the population of pp6 was single-positive for CD34 (7%). The Sca-1, CD34 double-positive muscle-derived cell population was gated in a region of small cell diameter, a phenotype characteristic of hematopoietic progenitors. Labeling of cells with antibodies against Sca-1 and the lymphoid CD4 and CD8 markers was also performed (B, bottom right quadrant). Dead cells were excluded from analysis. Calculation of Sca-1 and CD34 single (C, left) and double (C, right) positive cells as the percentage of cells contained within all preplates is shown graphically.
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Figure 3: Immunophenotyping of the muscle-derived cells using FACScan™ analysis. Muscle-derived cells were tested for CD34 and Sca-1 single (A) and double (B) expression. A distinct population of Sca-1+ cells was found in all seven preplates, but CD34+ cells were observed only in pp6 (A). The morphology distribution of muscle-derived cells after flow cytometry showed different subpopulations within the Sca-1+ cell population (B). The profile of these cells varied from cells with large diameters (pp0–4) to small diameters (pp5–6). The majority of cells derived from pp3 (B, top left quadrant) and pp5 (B, top right quadrant) highly express Sca-1, but were also poorly CD34+. Muscle-derived cells from pp6 showed 69% double-positive cells for CD34 and Sca-1 markers (B, bottom left quadrant). However, all Sca-1+ cells were not CD34+ and a fraction of the population of pp6 was single-positive for CD34 (7%). The Sca-1, CD34 double-positive muscle-derived cell population was gated in a region of small cell diameter, a phenotype characteristic of hematopoietic progenitors. Labeling of cells with antibodies against Sca-1 and the lymphoid CD4 and CD8 markers was also performed (B, bottom right quadrant). Dead cells were excluded from analysis. Calculation of Sca-1 and CD34 single (C, left) and double (C, right) positive cells as the percentage of cells contained within all preplates is shown graphically.

Mentions: Flow cytometric analysis of muscle-derived cell preplates from hindlimb muscles was performed. As shown in Fig. 3, a distinct population of Sca-1+ cells was found in all seven preplates, but CD34+ cells were observed only in pp6 (Fig. 3A and Fig. C). The morphological distribution of muscle-derived cells after flow cytometry showed different subpopulations within the Sca-1+ cell population. The profile of these cells varied from cells with large diameters (pp0–4) to cells with small diameters (pp5–6). Double staining of muscle-derived cells with Sca-1 and CD34 (Fig. 3B and Fig. C) showed that 69% of cells at pp6 coexpressed both markers. However, all Sca-1+ cells were not CD34+ and a fraction of the population of pp6 was only positive for CD34 (7%). The Sca-1, CD34 double-positive muscle-derived cell population was gated in a region of small cell diameter, a phenotype characteristic of hematopoietic progenitors. Since it had been demonstrated previously that Sca-1 is expressed on a high percentage of activated T lymphoid cells, a double staining with antibodies against Sca-1 and the lymphoid CD4 and CD8 markers was performed. As expected, no lymphoid markers were detected in any preplates (Fig. 3 B).


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)

Immunophenotyping of the muscle-derived cells using FACScan™ analysis. Muscle-derived cells were tested for CD34 and Sca-1 single (A) and double (B) expression. A distinct population of Sca-1+ cells was found in all seven preplates, but CD34+ cells were observed only in pp6 (A). The morphology distribution of muscle-derived cells after flow cytometry showed different subpopulations within the Sca-1+ cell population (B). The profile of these cells varied from cells with large diameters (pp0–4) to small diameters (pp5–6). The majority of cells derived from pp3 (B, top left quadrant) and pp5 (B, top right quadrant) highly express Sca-1, but were also poorly CD34+. Muscle-derived cells from pp6 showed 69% double-positive cells for CD34 and Sca-1 markers (B, bottom left quadrant). However, all Sca-1+ cells were not CD34+ and a fraction of the population of pp6 was single-positive for CD34 (7%). The Sca-1, CD34 double-positive muscle-derived cell population was gated in a region of small cell diameter, a phenotype characteristic of hematopoietic progenitors. Labeling of cells with antibodies against Sca-1 and the lymphoid CD4 and CD8 markers was also performed (B, bottom right quadrant). Dead cells were excluded from analysis. Calculation of Sca-1 and CD34 single (C, left) and double (C, right) positive cells as the percentage of cells contained within all preplates is shown graphically.
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Related In: Results  -  Collection

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

Figure 3: Immunophenotyping of the muscle-derived cells using FACScan™ analysis. Muscle-derived cells were tested for CD34 and Sca-1 single (A) and double (B) expression. A distinct population of Sca-1+ cells was found in all seven preplates, but CD34+ cells were observed only in pp6 (A). The morphology distribution of muscle-derived cells after flow cytometry showed different subpopulations within the Sca-1+ cell population (B). The profile of these cells varied from cells with large diameters (pp0–4) to small diameters (pp5–6). The majority of cells derived from pp3 (B, top left quadrant) and pp5 (B, top right quadrant) highly express Sca-1, but were also poorly CD34+. Muscle-derived cells from pp6 showed 69% double-positive cells for CD34 and Sca-1 markers (B, bottom left quadrant). However, all Sca-1+ cells were not CD34+ and a fraction of the population of pp6 was single-positive for CD34 (7%). The Sca-1, CD34 double-positive muscle-derived cell population was gated in a region of small cell diameter, a phenotype characteristic of hematopoietic progenitors. Labeling of cells with antibodies against Sca-1 and the lymphoid CD4 and CD8 markers was also performed (B, bottom right quadrant). Dead cells were excluded from analysis. Calculation of Sca-1 and CD34 single (C, left) and double (C, right) positive cells as the percentage of cells contained within all preplates is shown graphically.
Mentions: Flow cytometric analysis of muscle-derived cell preplates from hindlimb muscles was performed. As shown in Fig. 3, a distinct population of Sca-1+ cells was found in all seven preplates, but CD34+ cells were observed only in pp6 (Fig. 3A and Fig. C). The morphological distribution of muscle-derived cells after flow cytometry showed different subpopulations within the Sca-1+ cell population. The profile of these cells varied from cells with large diameters (pp0–4) to cells with small diameters (pp5–6). Double staining of muscle-derived cells with Sca-1 and CD34 (Fig. 3B and Fig. C) showed that 69% of cells at pp6 coexpressed both markers. However, all Sca-1+ cells were not CD34+ and a fraction of the population of pp6 was only positive for CD34 (7%). The Sca-1, CD34 double-positive muscle-derived cell population was gated in a region of small cell diameter, a phenotype characteristic of hematopoietic progenitors. Since it had been demonstrated previously that Sca-1 is expressed on a high percentage of activated T lymphoid cells, a double staining with antibodies against Sca-1 and the lymphoid CD4 and CD8 markers was performed. As expected, no lymphoid markers were detected in any preplates (Fig. 3 B).

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