Limits...
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.

Show MeSH

Related in: MedlinePlus

Detection by PCR of donor cells in the tissues of mdx mice. (A) Muscle-derived cells from Des-LacZ transgenic mice were injected intraarterially into mdx/mdx mice and the animals were examined 21 d later. Genomic DNA was extracted and analyzed by PCR for the des-LacZ transgene and the normal (25 bp) and mdx (50 bp) dystrophin gene. (B) Actin, the normal (25 bp) and mdx (50 bp) dystrophin, and des-LacZ expression was detected by RT-PCR. The 450-bp band of the des-LacZ transgene, indicating injected cells in mdx mice, was found in the analyzed muscle tissues. LacZ expression, indicated by the 272-bp band in RT-PCR, was also found in the same tissues showing the myogenic program. Actin expression was detected in all tissues. Normal dystrophin expression was indicated by the 25-bp band for wild-type dystrophin. Lane 1, treated muscle tissues; lane 2, control des-LacZ; lane 3, control mdx/mdx.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199616&req=5

Figure 7: Detection by PCR of donor cells in the tissues of mdx mice. (A) Muscle-derived cells from Des-LacZ transgenic mice were injected intraarterially into mdx/mdx mice and the animals were examined 21 d later. Genomic DNA was extracted and analyzed by PCR for the des-LacZ transgene and the normal (25 bp) and mdx (50 bp) dystrophin gene. (B) Actin, the normal (25 bp) and mdx (50 bp) dystrophin, and des-LacZ expression was detected by RT-PCR. The 450-bp band of the des-LacZ transgene, indicating injected cells in mdx mice, was found in the analyzed muscle tissues. LacZ expression, indicated by the 272-bp band in RT-PCR, was also found in the same tissues showing the myogenic program. Actin expression was detected in all tissues. Normal dystrophin expression was indicated by the 25-bp band for wild-type dystrophin. Lane 1, treated muscle tissues; lane 2, control des-LacZ; lane 3, control mdx/mdx.

Mentions: To assess the in vivo ability of pp6 cells to differentiate through the myogenic lineage, RT-PCR for dystrophin and LacZ mRNAs analysis was performed on mdx mice which were injected intraarterially with Sca-1, CD34 positive cells obtained from the Des-LacZ donor. Tissues which were shown by standard PCR to contain donor cells were evaluated for desmin-LacZ and dystrophin transcripts. Total RNA extracted from untreated and treated mdx mice was used as a template for RT-PCR. Digestion of the dystrophin RT-PCR reaction products by MaeIII allowed us to distinguish between normal and mutated dystrophin mRNAs. The normal dystrophin gene gave a 207-bp band plus two 25-bp fragments, whereas the mutated dystrophin gave a 207-bp band plus a single 50-bp fragment. Normal dystrophin and LacZ mRNAs were found in the soleus, TA, gastrocnemius, and quadriceps muscles of treated mice (Table and Fig. 7), suggesting that transplanted Sca-1, CD34 positive cells participated in myogenesis. Neither LacZ or dystrophin transcriptional activity was detected in the controlateral hindlimb muscles (Table ).


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)

Detection by PCR of donor cells in the tissues of mdx mice. (A) Muscle-derived cells from Des-LacZ transgenic mice were injected intraarterially into mdx/mdx mice and the animals were examined 21 d later. Genomic DNA was extracted and analyzed by PCR for the des-LacZ transgene and the normal (25 bp) and mdx (50 bp) dystrophin gene. (B) Actin, the normal (25 bp) and mdx (50 bp) dystrophin, and des-LacZ expression was detected by RT-PCR. The 450-bp band of the des-LacZ transgene, indicating injected cells in mdx mice, was found in the analyzed muscle tissues. LacZ expression, indicated by the 272-bp band in RT-PCR, was also found in the same tissues showing the myogenic program. Actin expression was detected in all tissues. Normal dystrophin expression was indicated by the 25-bp band for wild-type dystrophin. Lane 1, treated muscle tissues; lane 2, control des-LacZ; lane 3, control mdx/mdx.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Detection by PCR of donor cells in the tissues of mdx mice. (A) Muscle-derived cells from Des-LacZ transgenic mice were injected intraarterially into mdx/mdx mice and the animals were examined 21 d later. Genomic DNA was extracted and analyzed by PCR for the des-LacZ transgene and the normal (25 bp) and mdx (50 bp) dystrophin gene. (B) Actin, the normal (25 bp) and mdx (50 bp) dystrophin, and des-LacZ expression was detected by RT-PCR. The 450-bp band of the des-LacZ transgene, indicating injected cells in mdx mice, was found in the analyzed muscle tissues. LacZ expression, indicated by the 272-bp band in RT-PCR, was also found in the same tissues showing the myogenic program. Actin expression was detected in all tissues. Normal dystrophin expression was indicated by the 25-bp band for wild-type dystrophin. Lane 1, treated muscle tissues; lane 2, control des-LacZ; lane 3, control mdx/mdx.
Mentions: To assess the in vivo ability of pp6 cells to differentiate through the myogenic lineage, RT-PCR for dystrophin and LacZ mRNAs analysis was performed on mdx mice which were injected intraarterially with Sca-1, CD34 positive cells obtained from the Des-LacZ donor. Tissues which were shown by standard PCR to contain donor cells were evaluated for desmin-LacZ and dystrophin transcripts. Total RNA extracted from untreated and treated mdx mice was used as a template for RT-PCR. Digestion of the dystrophin RT-PCR reaction products by MaeIII allowed us to distinguish between normal and mutated dystrophin mRNAs. The normal dystrophin gene gave a 207-bp band plus two 25-bp fragments, whereas the mutated dystrophin gave a 207-bp band plus a single 50-bp fragment. Normal dystrophin and LacZ mRNAs were found in the soleus, TA, gastrocnemius, and quadriceps muscles of treated mice (Table and Fig. 7), suggesting that transplanted Sca-1, CD34 positive cells participated in myogenesis. Neither LacZ or dystrophin transcriptional activity was detected in the controlateral hindlimb muscles (Table ).

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