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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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To determine whether individual muscle-derived cells (i.e., pp6) exhibit stem cell characteristics of self-renewal and pluripotentiality, individual clones were analyzed. To establish clones, pp6 were plated at ∼10 cells/cm2. Single cells replicated as typical muscle-derived stem cells (A) and differentiated after 15 (B), 30 (C), and 45 (D) d of culture. Undifferentiated, rounded (indicated by arrows) muscle-derived stem cells were evident in each clonal line. Therefore, clones derived from single cells give rise to both muscle-derived stem cells and differentiated cells, indicating stem cell characteristics. Bar, 120 μm.
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Figure 5: To determine whether individual muscle-derived cells (i.e., pp6) exhibit stem cell characteristics of self-renewal and pluripotentiality, individual clones were analyzed. To establish clones, pp6 were plated at ∼10 cells/cm2. Single cells replicated as typical muscle-derived stem cells (A) and differentiated after 15 (B), 30 (C), and 45 (D) d of culture. Undifferentiated, rounded (indicated by arrows) muscle-derived stem cells were evident in each clonal line. Therefore, clones derived from single cells give rise to both muscle-derived stem cells and differentiated cells, indicating stem cell characteristics. Bar, 120 μm.

Mentions: To verify whether the Sca-1 and CD34 double-positive cells (i.e., pp6 cells) or muscle-derived cells contained hematopoietic progenitor cells, a colony-forming unit in culture assay in methylcellulose was performed (Delassus and Cumano 1996). Cells were cultured under conditions that promote myeloid cell development (i.e., presence of IL-3 and SCF). The cloning efficiency was 22% (∼1 cell in 5; Fig. 4) and two types of clones derived from Sca-1, CD34 positive cells were identified: (1) clones with a restricted myeloid potential that were able to differentiate into either macrophages or mast cells; (2) multipotent clones that displayed a multimyeloid potentiality (mainly macrophages, megakaryocytes, and granular polymorphonuclear cells). Verification of the multimyeloid nature of the resulting cells was performed by May-Grunwald-Giemsa staining (Fig. 4). In some experiments, cultures of pp0–5 in methylcellulose supplemented with IL-3 and SCF showed no colonies, even after 28 d of culture. These data suggest that there is a strong relationship between the expression of CD34 and the in vitro myeloid differentiation. To determine whether muscle-derived cells exhibit stem cell characteristics of self-renewal, pp6 were plated at ∼10 cells/cm2, isolated with cloning cylinders, and transferred to separate wells and finally to individual flasks. Single cells replicated as typical muscle-derived stem cells and underwent morphologic differentiation (Fig. 5). Using immunohistochemistry, these cells were found to be positive for the expression of several markers, such as PECAM (endothelial and hemopoietic cells), desmin (myoblasts and smooth muscle cells), and Mac-1 (macrophages). Undifferentiated muscle-derived stem cells were evident in each clonal line. Therefore, clones derived from single cells give rise to both muscle-derived stem cells and differentiated cells, indicating stem cell characteristics.


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)

To determine whether individual muscle-derived cells (i.e., pp6) exhibit stem cell characteristics of self-renewal and pluripotentiality, individual clones were analyzed. To establish clones, pp6 were plated at ∼10 cells/cm2. Single cells replicated as typical muscle-derived stem cells (A) and differentiated after 15 (B), 30 (C), and 45 (D) d of culture. Undifferentiated, rounded (indicated by arrows) muscle-derived stem cells were evident in each clonal line. Therefore, clones derived from single cells give rise to both muscle-derived stem cells and differentiated cells, indicating stem cell characteristics. Bar, 120 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: To determine whether individual muscle-derived cells (i.e., pp6) exhibit stem cell characteristics of self-renewal and pluripotentiality, individual clones were analyzed. To establish clones, pp6 were plated at ∼10 cells/cm2. Single cells replicated as typical muscle-derived stem cells (A) and differentiated after 15 (B), 30 (C), and 45 (D) d of culture. Undifferentiated, rounded (indicated by arrows) muscle-derived stem cells were evident in each clonal line. Therefore, clones derived from single cells give rise to both muscle-derived stem cells and differentiated cells, indicating stem cell characteristics. Bar, 120 μm.
Mentions: To verify whether the Sca-1 and CD34 double-positive cells (i.e., pp6 cells) or muscle-derived cells contained hematopoietic progenitor cells, a colony-forming unit in culture assay in methylcellulose was performed (Delassus and Cumano 1996). Cells were cultured under conditions that promote myeloid cell development (i.e., presence of IL-3 and SCF). The cloning efficiency was 22% (∼1 cell in 5; Fig. 4) and two types of clones derived from Sca-1, CD34 positive cells were identified: (1) clones with a restricted myeloid potential that were able to differentiate into either macrophages or mast cells; (2) multipotent clones that displayed a multimyeloid potentiality (mainly macrophages, megakaryocytes, and granular polymorphonuclear cells). Verification of the multimyeloid nature of the resulting cells was performed by May-Grunwald-Giemsa staining (Fig. 4). In some experiments, cultures of pp0–5 in methylcellulose supplemented with IL-3 and SCF showed no colonies, even after 28 d of culture. These data suggest that there is a strong relationship between the expression of CD34 and the in vitro myeloid differentiation. To determine whether muscle-derived cells exhibit stem cell characteristics of self-renewal, pp6 were plated at ∼10 cells/cm2, isolated with cloning cylinders, and transferred to separate wells and finally to individual flasks. Single cells replicated as typical muscle-derived stem cells and underwent morphologic differentiation (Fig. 5). Using immunohistochemistry, these cells were found to be positive for the expression of several markers, such as PECAM (endothelial and hemopoietic cells), desmin (myoblasts and smooth muscle cells), and Mac-1 (macrophages). Undifferentiated muscle-derived stem cells were evident in each clonal line. Therefore, clones derived from single cells give rise to both muscle-derived stem cells and differentiated cells, indicating stem cell characteristics.

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