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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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MMCs can fuse spontaneously in vitro with differentiated myotubes. (a) Schematic of the co-culture experiments. (b and c) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with wild-type primary myoblasts and differentiation induced (DM) for 4 d, according to scheme a (2). Phase-contrast (b) and laser-scanning confocal images of immunofluorescence for myogenin only (b') or for GFP and myogenin (b”) are shown. Arrows indicate an example of GFP+myogenin+ multinucleated myotube. Arrowheads indicate GFP+ MMCs that do not express myogenin. (c) MMCs were isolated by FACS from fresh bone marrow from R26R Cre-reporter transgenic mice and co-cultured with Cre-expressing myoblasts, according to scheme a (2). A β-gal+ multinucleated myotube provides evidence of fusion between MMCs and Cre myotubes.
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fig4: MMCs can fuse spontaneously in vitro with differentiated myotubes. (a) Schematic of the co-culture experiments. (b and c) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with wild-type primary myoblasts and differentiation induced (DM) for 4 d, according to scheme a (2). Phase-contrast (b) and laser-scanning confocal images of immunofluorescence for myogenin only (b') or for GFP and myogenin (b”) are shown. Arrows indicate an example of GFP+myogenin+ multinucleated myotube. Arrowheads indicate GFP+ MMCs that do not express myogenin. (c) MMCs were isolated by FACS from fresh bone marrow from R26R Cre-reporter transgenic mice and co-cultured with Cre-expressing myoblasts, according to scheme a (2). A β-gal+ multinucleated myotube provides evidence of fusion between MMCs and Cre myotubes.

Mentions: To elucidate the mechanism underlying the IGF-I–enhanced BMDC contribution to skeletal muscle in vivo, BMDCs were co-cultured with myogenic cells in vitro. Because we and others previously showed that the bone marrow derivatives of HSCs capable of fusing with myofibers in vivo are the MMCs (Camargo et al., 2003; Doyonnas et al., 2004; Ojima et al., 2004), experiments were designed to assess whether MMCs in co-culture with muscle cells were able to fuse with myotubes. Various experimental conditions were tested (Fig. 4 a). When freshly isolated MMCs were immediately co-cultured with myoblasts in low-serum myoblast differentiation medium (DM), fusion events were never observed, even after 6 d (Fig. 4 a, 1). By contrast, when MMCs were pretreated with high-serum myoblast growth medium (GM), fusion was observed. For instance, when MMCs and myoblasts were each cultured in GM for 3 d and then co-cultured in DM, GFP+ myotubes containing myogenin-expressing nuclei were detected as early as 4 d (Fig. 4, a [2] and b–b”). Notably, when MMCs were cultured in GM for 3 d and then plated together with 3-d-old myotubes in DM, GFP+ myotubes containing myogenin-expressing nuclei were visible as early as 12 h after co-culture (Fig. 4 a, 3), and >90% of GFP+ myotubes contained three or more nuclei (not depicted). Together, these results suggest that exposure to GM is a prerequisite for MMCs to fuse. In addition, these “activated” MMCs can fuse with well-differentiated multinucleated myotubes. In none of these conditions in eight independent experiments were unfused GFP+ mononucleated bone marrow cells that expressed myogenin ever detected (Fig. 4, b–b”, arrowheads). The frequency of these fusion events is low (∼0.013%) but comparable with that previously reported by others for adherent bone marrow mesenchymal cells that are stromal derivatives (Shi et al., 2004; Lee et al., 2005). Thus, nonadherent HSC derivatives are as capable of fusing with muscle as adherent mesenchymal cells.


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)

MMCs can fuse spontaneously in vitro with differentiated myotubes. (a) Schematic of the co-culture experiments. (b and c) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with wild-type primary myoblasts and differentiation induced (DM) for 4 d, according to scheme a (2). Phase-contrast (b) and laser-scanning confocal images of immunofluorescence for myogenin only (b') or for GFP and myogenin (b”) are shown. Arrows indicate an example of GFP+myogenin+ multinucleated myotube. Arrowheads indicate GFP+ MMCs that do not express myogenin. (c) MMCs were isolated by FACS from fresh bone marrow from R26R Cre-reporter transgenic mice and co-cultured with Cre-expressing myoblasts, according to scheme a (2). A β-gal+ multinucleated myotube provides evidence of fusion between MMCs and Cre myotubes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171272&req=5

fig4: MMCs can fuse spontaneously in vitro with differentiated myotubes. (a) Schematic of the co-culture experiments. (b and c) MMCs were isolated by FACS from fresh bone marrow from GFP transgenic mice, maintained in myoblast GM for 3 d, and co-cultured with wild-type primary myoblasts and differentiation induced (DM) for 4 d, according to scheme a (2). Phase-contrast (b) and laser-scanning confocal images of immunofluorescence for myogenin only (b') or for GFP and myogenin (b”) are shown. Arrows indicate an example of GFP+myogenin+ multinucleated myotube. Arrowheads indicate GFP+ MMCs that do not express myogenin. (c) MMCs were isolated by FACS from fresh bone marrow from R26R Cre-reporter transgenic mice and co-cultured with Cre-expressing myoblasts, according to scheme a (2). A β-gal+ multinucleated myotube provides evidence of fusion between MMCs and Cre myotubes.
Mentions: To elucidate the mechanism underlying the IGF-I–enhanced BMDC contribution to skeletal muscle in vivo, BMDCs were co-cultured with myogenic cells in vitro. Because we and others previously showed that the bone marrow derivatives of HSCs capable of fusing with myofibers in vivo are the MMCs (Camargo et al., 2003; Doyonnas et al., 2004; Ojima et al., 2004), experiments were designed to assess whether MMCs in co-culture with muscle cells were able to fuse with myotubes. Various experimental conditions were tested (Fig. 4 a). When freshly isolated MMCs were immediately co-cultured with myoblasts in low-serum myoblast differentiation medium (DM), fusion events were never observed, even after 6 d (Fig. 4 a, 1). By contrast, when MMCs were pretreated with high-serum myoblast growth medium (GM), fusion was observed. For instance, when MMCs and myoblasts were each cultured in GM for 3 d and then co-cultured in DM, GFP+ myotubes containing myogenin-expressing nuclei were detected as early as 4 d (Fig. 4, a [2] and b–b”). Notably, when MMCs were cultured in GM for 3 d and then plated together with 3-d-old myotubes in DM, GFP+ myotubes containing myogenin-expressing nuclei were visible as early as 12 h after co-culture (Fig. 4 a, 3), and >90% of GFP+ myotubes contained three or more nuclei (not depicted). Together, these results suggest that exposure to GM is a prerequisite for MMCs to fuse. In addition, these “activated” MMCs can fuse with well-differentiated multinucleated myotubes. In none of these conditions in eight independent experiments were unfused GFP+ mononucleated bone marrow cells that expressed myogenin ever detected (Fig. 4, b–b”, arrowheads). The frequency of these fusion events is low (∼0.013%) but comparable with that previously reported by others for adherent bone marrow mesenchymal cells that are stromal derivatives (Shi et al., 2004; Lee et al., 2005). Thus, nonadherent HSC derivatives are as capable of fusing with muscle as adherent mesenchymal cells.

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