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IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors.

Sacco A, Doyonnas R, LaBarge MA, Hammer MM, Kraft P, Blau HM - J. Cell Biol. (2005)

Bottom Line: One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC).However, the molecular components responsible for this injury-related response remain largely unknown.These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Muscle damage has been shown to enhance the contribution of bone marrow-derived cells (BMDCs) to regenerating skeletal muscle. One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC). However, the molecular components responsible for this injury-related response remain largely unknown. In this paper, we show that delivery of insulin-like growth factor I (IGF-I) to adult skeletal muscle by three different methods-plasmid electroporation, injection of genetically engineered myoblasts, and recombinant protein injection-increases the integration of BMDCs up to fourfold. To investigate the underlying mechanism, we developed an in vitro fusion assay in which co-cultures of MMCs and myotubes were exposed to IGF-I. The number of fusion events was substantially augmented by IGF-I, independent of its effect on cell survival. These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

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Myoblast-mediated delivery of IGF-I increases the contribution of BMDCs to skeletal muscle. (a) Western blot analysis of IGF-I expression by retrovirally transduced myoblasts isolated based on CD8 expression using FACS. IGF-IEa migrates as a 14-kD protein and MGF as a 12-kD protein. (b) 5 ×105 myoblasts were injected into the TA muscles of mice that had received a BMT 8 wk earlier, and muscles were harvested 4 wk after cell injection. The percentage of GFP+β-gal+ myofibers relative to total β-gal+ myofibers is shown (± SEM). P value was determined with a t test. *, P < 0.02. (c) Muscle sections were analyzed for β-gal expression (top), and serial sections were analyzed by immunofluorescence staining for GFP and laminin (bottom). Arrows indicate examples of β-gal+ myofibers that were also GFP+. Bars, 15 μm.
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fig2: Myoblast-mediated delivery of IGF-I increases the contribution of BMDCs to skeletal muscle. (a) Western blot analysis of IGF-I expression by retrovirally transduced myoblasts isolated based on CD8 expression using FACS. IGF-IEa migrates as a 14-kD protein and MGF as a 12-kD protein. (b) 5 ×105 myoblasts were injected into the TA muscles of mice that had received a BMT 8 wk earlier, and muscles were harvested 4 wk after cell injection. The percentage of GFP+β-gal+ myofibers relative to total β-gal+ myofibers is shown (± SEM). P value was determined with a t test. *, P < 0.02. (c) Muscle sections were analyzed for β-gal expression (top), and serial sections were analyzed by immunofluorescence staining for GFP and laminin (bottom). Arrows indicate examples of β-gal+ myofibers that were also GFP+. Bars, 15 μm.

Mentions: Genetically engineered myoblasts provide a potent means for delivering proteins to skeletal muscle tissues. After injection into adult skeletal muscle, these cells fuse with the preexisting muscle fibers of the host. Myoblast-mediated gene delivery to skeletal or heart muscle was first shown to lead to physiological levels of secreted proteins in the circulation (e.g., human growth hormone; Barr and Leiden, 1991; Dhawan et al., 1991). Subsequently, localized secretion of proteins such as VEGF-A within heart and skeletal muscle tissues has been achieved by myoblast-mediated delivery, leading to novel insights into its mechanism of action in angiogenesis (Springer et al., 1998; Lee et al., 2000; Ozawa et al., 2004). Primary myoblasts previously engineered to express β-galactosidase (β-gal; Rando and Blau, 1994) were transduced with a retroviral vector encoding IGF-IEa.CD8, MGF.CD8, or an empty control vector (CD8 cassette only; Springer and Blau, 1997; Springer et al., 1998). All transduced cells were isolated by flow cytometry (FACS) using an antibody to CD8. IGF-I expression in these myoblast populations was confirmed by Western blot (Fig. 2 a).


IGF-I increases bone marrow contribution to adult skeletal muscle and enhances the fusion of myelomonocytic precursors.

Sacco A, Doyonnas R, LaBarge MA, Hammer MM, Kraft P, Blau HM - J. Cell Biol. (2005)

Myoblast-mediated delivery of IGF-I increases the contribution of BMDCs to skeletal muscle. (a) Western blot analysis of IGF-I expression by retrovirally transduced myoblasts isolated based on CD8 expression using FACS. IGF-IEa migrates as a 14-kD protein and MGF as a 12-kD protein. (b) 5 ×105 myoblasts were injected into the TA muscles of mice that had received a BMT 8 wk earlier, and muscles were harvested 4 wk after cell injection. The percentage of GFP+β-gal+ myofibers relative to total β-gal+ myofibers is shown (± SEM). P value was determined with a t test. *, P < 0.02. (c) Muscle sections were analyzed for β-gal expression (top), and serial sections were analyzed by immunofluorescence staining for GFP and laminin (bottom). Arrows indicate examples of β-gal+ myofibers that were also GFP+. Bars, 15 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171272&req=5

fig2: Myoblast-mediated delivery of IGF-I increases the contribution of BMDCs to skeletal muscle. (a) Western blot analysis of IGF-I expression by retrovirally transduced myoblasts isolated based on CD8 expression using FACS. IGF-IEa migrates as a 14-kD protein and MGF as a 12-kD protein. (b) 5 ×105 myoblasts were injected into the TA muscles of mice that had received a BMT 8 wk earlier, and muscles were harvested 4 wk after cell injection. The percentage of GFP+β-gal+ myofibers relative to total β-gal+ myofibers is shown (± SEM). P value was determined with a t test. *, P < 0.02. (c) Muscle sections were analyzed for β-gal expression (top), and serial sections were analyzed by immunofluorescence staining for GFP and laminin (bottom). Arrows indicate examples of β-gal+ myofibers that were also GFP+. Bars, 15 μm.
Mentions: Genetically engineered myoblasts provide a potent means for delivering proteins to skeletal muscle tissues. After injection into adult skeletal muscle, these cells fuse with the preexisting muscle fibers of the host. Myoblast-mediated gene delivery to skeletal or heart muscle was first shown to lead to physiological levels of secreted proteins in the circulation (e.g., human growth hormone; Barr and Leiden, 1991; Dhawan et al., 1991). Subsequently, localized secretion of proteins such as VEGF-A within heart and skeletal muscle tissues has been achieved by myoblast-mediated delivery, leading to novel insights into its mechanism of action in angiogenesis (Springer et al., 1998; Lee et al., 2000; Ozawa et al., 2004). Primary myoblasts previously engineered to express β-galactosidase (β-gal; Rando and Blau, 1994) were transduced with a retroviral vector encoding IGF-IEa.CD8, MGF.CD8, or an empty control vector (CD8 cassette only; Springer and Blau, 1997; Springer et al., 1998). All transduced cells were isolated by flow cytometry (FACS) using an antibody to CD8. IGF-I expression in these myoblast populations was confirmed by Western blot (Fig. 2 a).

Bottom Line: One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC).However, the molecular components responsible for this injury-related response remain largely unknown.These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Baxter Laboratory in Genetic Pharmacology, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Muscle damage has been shown to enhance the contribution of bone marrow-derived cells (BMDCs) to regenerating skeletal muscle. One responsible cell type involved in this process is a hematopoietic stem cell derivative, the myelomonocytic precursor (MMC). However, the molecular components responsible for this injury-related response remain largely unknown. In this paper, we show that delivery of insulin-like growth factor I (IGF-I) to adult skeletal muscle by three different methods-plasmid electroporation, injection of genetically engineered myoblasts, and recombinant protein injection-increases the integration of BMDCs up to fourfold. To investigate the underlying mechanism, we developed an in vitro fusion assay in which co-cultures of MMCs and myotubes were exposed to IGF-I. The number of fusion events was substantially augmented by IGF-I, independent of its effect on cell survival. These results provide novel evidence that a single factor, IGF-I, is sufficient to enhance the fusion of bone marrow derivatives with adult skeletal muscle.

Show MeSH
Related in: MedlinePlus