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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 injection of an endothelial cell line. These cells expressed vimentin and the β-gal placed under a vimentin promoter. 30 d after transplantation, intensely β-gal (A and B) and vimentin (E and F) positive mononuclear cells were observed in different areas of the cross sections of the injected TA muscle. Few injected cells still remained large T antigen positive (C and D). Few perithelial layers of muscle vessels in the injected area were positive to LacZ and vimentin staining, indicating an incorporation of the endothelial injected cells in these sides (arrowheads in B, D, and F). Bar, 50 μm.
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Figure 10: Intramuscular injection of an endothelial cell line. These cells expressed vimentin and the β-gal placed under a vimentin promoter. 30 d after transplantation, intensely β-gal (A and B) and vimentin (E and F) positive mononuclear cells were observed in different areas of the cross sections of the injected TA muscle. Few injected cells still remained large T antigen positive (C and D). Few perithelial layers of muscle vessels in the injected area were positive to LacZ and vimentin staining, indicating an incorporation of the endothelial injected cells in these sides (arrowheads in B, D, and F). Bar, 50 μm.

Mentions: In an attempt to investigate whether the Sca-1, CD34 positive cells have characteristics of bilineag-committed precursors (hemopoietic and endothelial), the intramuscular injection of pp6 was compared with the injection of an endothelial cell line. The ability of injected pp6, labeled with a LacZ transgene, to fuse with host muscle fibers was investigated 30 d after intramuscular transplantation. A small number of β-gal myofibers surrounding the injected area were observed in these muscles (∼3% per cross section; Fig. 9 A). The endothelial cell line was isolated as described in Materials and Methods. These cells expressed the vimentin and the β-gal placed under a vimentin promoter. It has been shown that myoblasts and regenerating myofibers express the vimentin protein (Tokuyasu et al. 1984; Sarnat 1992). The fibers which had been colonized by the endothelial cells expressed the LacZ. They also contained the large T antigen with a thermosensitive mutation to achieve inactivation of large T antigen by temperature shift when necessary. Thus, these cells were able to differentiate when injected in the TA muscles of animals having a body temperature of 37°C. 30 d after transplantation, intensely β-gal– and vimentin-positive mononuclear cells were observed in different areas of the cross section of the injected TA muscle (Fig. 10). The positive cells were spread throughout the muscle, but fused poorly with the host's fibers. The PECAM staining confirmed that injected cells were still endothelial cells (data not shown). Interestingly, endothelial injected cells remained as mononuclear cells close to the muscle fibers. Very few cells expressed the large T antigen in their nuclei. Few of the perithelial layer of muscle vessels in the injected area were positive to LacZ and vimentin staining, indicating an incorporation of the endothelial injected cells in these sides (Fig. 10B, Fig. D, and Fig. F).


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 injection of an endothelial cell line. These cells expressed vimentin and the β-gal placed under a vimentin promoter. 30 d after transplantation, intensely β-gal (A and B) and vimentin (E and F) positive mononuclear cells were observed in different areas of the cross sections of the injected TA muscle. Few injected cells still remained large T antigen positive (C and D). Few perithelial layers of muscle vessels in the injected area were positive to LacZ and vimentin staining, indicating an incorporation of the endothelial injected cells in these sides (arrowheads in B, D, and F). Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Intramuscular injection of an endothelial cell line. These cells expressed vimentin and the β-gal placed under a vimentin promoter. 30 d after transplantation, intensely β-gal (A and B) and vimentin (E and F) positive mononuclear cells were observed in different areas of the cross sections of the injected TA muscle. Few injected cells still remained large T antigen positive (C and D). Few perithelial layers of muscle vessels in the injected area were positive to LacZ and vimentin staining, indicating an incorporation of the endothelial injected cells in these sides (arrowheads in B, D, and F). Bar, 50 μm.
Mentions: In an attempt to investigate whether the Sca-1, CD34 positive cells have characteristics of bilineag-committed precursors (hemopoietic and endothelial), the intramuscular injection of pp6 was compared with the injection of an endothelial cell line. The ability of injected pp6, labeled with a LacZ transgene, to fuse with host muscle fibers was investigated 30 d after intramuscular transplantation. A small number of β-gal myofibers surrounding the injected area were observed in these muscles (∼3% per cross section; Fig. 9 A). The endothelial cell line was isolated as described in Materials and Methods. These cells expressed the vimentin and the β-gal placed under a vimentin promoter. It has been shown that myoblasts and regenerating myofibers express the vimentin protein (Tokuyasu et al. 1984; Sarnat 1992). The fibers which had been colonized by the endothelial cells expressed the LacZ. They also contained the large T antigen with a thermosensitive mutation to achieve inactivation of large T antigen by temperature shift when necessary. Thus, these cells were able to differentiate when injected in the TA muscles of animals having a body temperature of 37°C. 30 d after transplantation, intensely β-gal– and vimentin-positive mononuclear cells were observed in different areas of the cross section of the injected TA muscle (Fig. 10). The positive cells were spread throughout the muscle, but fused poorly with the host's fibers. The PECAM staining confirmed that injected cells were still endothelial cells (data not shown). Interestingly, endothelial injected cells remained as mononuclear cells close to the muscle fibers. Very few cells expressed the large T antigen in their nuclei. Few of the perithelial layer of muscle vessels in the injected area were positive to LacZ and vimentin staining, indicating an incorporation of the endothelial injected cells in these sides (Fig. 10B, Fig. D, and Fig. F).

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