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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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IGF-I promotes survival of MMCs. (a) MMCs were obtained by FACS from freshly isolated bone marrow and 6 × 104 cells plated in myoblast GM for 3 d, with or without 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as a percentage of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. *, P < 0.02. (b) Cell cycle profile using propidium iodide for analysis of DNA content in MMCs with or without IGF-I after 3 d of culture in the media indicated. Percentages of the cells in each specific cell cycle phase are indicated on the graphs.
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fig6: IGF-I promotes survival of MMCs. (a) MMCs were obtained by FACS from freshly isolated bone marrow and 6 × 104 cells plated in myoblast GM for 3 d, with or without 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as a percentage of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. *, P < 0.02. (b) Cell cycle profile using propidium iodide for analysis of DNA content in MMCs with or without IGF-I after 3 d of culture in the media indicated. Percentages of the cells in each specific cell cycle phase are indicated on the graphs.

Mentions: To test the potential role of IGF-I on the fusion of MMCs with myotubes, we had to distinguish its effects on fusion from those on survival. Accordingly, experiments were first designed to determine whether IGF-I affects the survival and proliferation of MMCs. Freshly isolated MMCs were isolated from GFP+ bone marrow by FACS. MMCs were cultured for 3 d in GM with or without IGF-I (100 ng/ml). Cells were then harvested and counted, and cell cycle analysis was performed using propidium iodide and FACS. As shown in Fig. 6, IGF-I led to an increase in the number of cells within this Lin−ckit+Sca1− bone marrow fraction (P < 0.02). By contrast, IGF-I did not lead to a change in the number of Lin+ cells (unpublished data). This potent effect on cell number resulted from an increase in cell survival because no effect of IGF-I on proliferation was observed, as indicated by the cell cycle profile, which was not altered relative to untreated controls. Indeed, 14% of the cells were in S–G2/M phase in each case (Fig. 6 b). As a control, when MMCs were cultured in bone marrow medium in the presence of cytokines (IL3, IL6, and stem cell factor), a medium in which they were highly stimulated to proliferate, 35% of the cells were found in the S–G2/M phase of the cell cycle (Fig. 6 b). Thus, IGF-I increased survival but not proliferation of MMCs under the experimental conditions used here.


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)

IGF-I promotes survival of MMCs. (a) MMCs were obtained by FACS from freshly isolated bone marrow and 6 × 104 cells plated in myoblast GM for 3 d, with or without 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as a percentage of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. *, P < 0.02. (b) Cell cycle profile using propidium iodide for analysis of DNA content in MMCs with or without IGF-I after 3 d of culture in the media indicated. Percentages of the cells in each specific cell cycle phase are indicated on the graphs.
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Related In: Results  -  Collection

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fig6: IGF-I promotes survival of MMCs. (a) MMCs were obtained by FACS from freshly isolated bone marrow and 6 × 104 cells plated in myoblast GM for 3 d, with or without 100 ng/ml IGF-I. Cell counts were performed on days 0 and 3. Final cell numbers are expressed as a percentage of the initial number of cells plated on day 0 (± SEM). P value was determined with a t test. *, P < 0.02. (b) Cell cycle profile using propidium iodide for analysis of DNA content in MMCs with or without IGF-I after 3 d of culture in the media indicated. Percentages of the cells in each specific cell cycle phase are indicated on the graphs.
Mentions: To test the potential role of IGF-I on the fusion of MMCs with myotubes, we had to distinguish its effects on fusion from those on survival. Accordingly, experiments were first designed to determine whether IGF-I affects the survival and proliferation of MMCs. Freshly isolated MMCs were isolated from GFP+ bone marrow by FACS. MMCs were cultured for 3 d in GM with or without IGF-I (100 ng/ml). Cells were then harvested and counted, and cell cycle analysis was performed using propidium iodide and FACS. As shown in Fig. 6, IGF-I led to an increase in the number of cells within this Lin−ckit+Sca1− bone marrow fraction (P < 0.02). By contrast, IGF-I did not lead to a change in the number of Lin+ cells (unpublished data). This potent effect on cell number resulted from an increase in cell survival because no effect of IGF-I on proliferation was observed, as indicated by the cell cycle profile, which was not altered relative to untreated controls. Indeed, 14% of the cells were in S–G2/M phase in each case (Fig. 6 b). As a control, when MMCs were cultured in bone marrow medium in the presence of cytokines (IL3, IL6, and stem cell factor), a medium in which they were highly stimulated to proliferate, 35% of the cells were found in the S–G2/M phase of the cell cycle (Fig. 6 b). Thus, IGF-I increased survival but not proliferation of MMCs under the experimental conditions used here.

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