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Myogenic specification of side population cells in skeletal muscle - PubMed

  • ️Tue Jan 01 2002

Myogenic specification of side population cells in skeletal muscle

Atsushi Asakura et al. J Cell Biol. 2002.

Abstract

Skeletal muscle contains myogenic progenitors called satellite cells and muscle-derived stem cells that have been suggested to be pluripotent. We further investigated the differentiation potential of muscle-derived stem cells and satellite cells to elucidate relationships between these two populations of cells. FACS(R) analysis of muscle side population (SP) cells, a fraction of muscle-derived stem cells, revealed expression of hematopoietic stem cell marker Sca-1 but did not reveal expression of any satellite cell markers. Muscle SP cells were greatly enriched for cells competent to form hematopoietic colonies. Moreover, muscle SP cells with hematopoietic potential were CD45 positive. However, muscle SP cells did not differentiate into myocytes in vitro. By contrast, satellite cells gave rise to myocytes but did not express Sca-1 or CD45 and never formed hematopoietic colonies. Importantly, muscle SP cells exhibited the potential to give rise to both myocytes and satellite cells after intramuscular transplantation. In addition, muscle SP cells underwent myogenic specification after co-culture with myoblasts. Co-culture with myoblasts or forced expression of MyoD also induced muscle differentiation of muscle SP cells prepared from mice lacking Pax7 gene, an essential gene for satellite cell development. Therefore, these data document that satellite cells and muscle-derived stem cells represent distinct populations and demonstrate that muscle-derived stem cells have the potential to give rise to myogenic cells via a myocyte-mediated inductive interaction.

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Figures

Figure 1.
Figure 1.

Adult muscle contains hematopoietic progenitors. (A, B, and D–F) Muscle-derived cells formed myeloid hematopoietic colonies in Methocult M3434. Both large tight colonies >1 mm (A) and small colonies (B) were formed in cultures of muscle-derived cells. (C) Muscle-derived cells also formed pre-B cell colonies in Methocult M3630. May-Grunwald's Giemsa staining (D) and immunohistochemistry against Mac1 (E) and Gr1 (F). Arrows and arrowheads indicate Mac-1/Gr-1–positive monocytes containing some vacuoles and granulocytes with polymorphic nuclei, respectively. (G) B220-positive pre-B cells were contained in colonies derived from muscle cells in Methocult M3630. Bars: (A and B) 200 μm; (C) 100 μm; and (D–G) 25 μm.

Figure 2.
Figure 2.

Satellite cells do not exhibit hematopoietic potential. Isolated single muscle fibers prepared from Myf5-nlacZ transgenic mice were cultured in Methocult M3434. (A) Satellite cells expressing Myf5-nlacZ (arrowheads) were detected on freshly isolated muscle fibers (day 0). (B) Migrating satellite cells derived from muscle fibers were detected after 4 d of culture (arrowheads). (C) By day 10, the migrating cells formed colonies containing multinucleated myotubes that expressed Myf5-nlacZ (arrows). (D) By 14 days, differentiated myotubes (arrow) were detected adjacent the original muscle fiber (indicated by F). (E) Single muscle fibers (total fibers; n = 180 for three independent experiments) or 1 × 104 cells prepared from whole muscle, bone marrow, or peripheral blood were cultured in Methocult M3434. By day 14, hematopoietic colonies were counted. The error bars represent the SEM from three independent experiments. Bar: (A–C) 50 μm; and (D) 100 μm.

Figure 3.
Figure 3.

Muscle SP cells express Sca-1 and CD45 but do not express Myf5-nlacZ or MD6.0-lacZ. (A) FACS® fractionation of SP cells from bone marrow and muscle was used for the isolation of adult stem cells by Hoechst dye exclusion. Both muscle and bone marrow SP cells stained with Hoechst dye were sensitive to verapamil. (B) x- and y-axes indicate intensity of CD45-FITC and Sca-1-PE, respectively. The percentage of cells in each quadrant is indicated in the panel. (C) Unsorted (WP) or sorted cells prepared from Myf5-nlacZ or MD6.0-lacZ transgenic mouse muscle were stained with X-gal. The error bars represent the SEM from three independent experiments. At least a total of 1,000 cells were counted in each fraction.

Figure 4.
Figure 4.

Muscle SP cells efficiently form hematopoietic colonies in vitro. (A) 1 × 104 unsorted (WP) or sorted MP or SP cells prepared from muscle and bone marrow were cultured in Methocult M3434 (left). 1 × 103 nucleated sorted muscle SP subfractions (CD45−:Sca-1−, CD45−:Sca-1+, CD45+:Sca-1−, and CD45+:Sca-1+) were cultured in Methocult M3434 (right). By day 14, hematopoietic colonies were counted. (B) 1 × 104 unsorted (WP) or sorted MP or SP cells prepared from muscle and bone marrow were cultured in Methocult M3434 for 14 days (left) and in myoblast growth medium by 10 days (right). Muscle colonies were counted after cultures. (C). In myoblast growth medium, the muscle MP fraction contained Myf5-nlacZ–expressing myoblasts (arrows), whereas the muscle SP fraction did not contain any lacZ- expressing cells. The error bars represent the SEM from three independent experiments. Bar: 50 μm.

Figure 5.
Figure 5.

Sca-1–positive cells are located outside of muscle fibers and are associated with vasculature. (A) Satellite cells on freshly isolated muscle fibers expressed CD34 (arrow) but not Sca-1 or CD45. The satellite cell nuclei and myonuclei were visualized by counterstaining with DAPI. These fibers were one example out of 20 muscle fibers stained with antibodies. (B) Left panels show double immunostaining with anti–Sca-1 and laminin, and anti–Sca-1 and PECAM antibodies for cross sections of tibialis anterior (TA) muscle. Right panels show light field with DAPI nucleus staining for each dark field at the left panel. Sca–1–positive cells (red) indicated by arrows were detected between muscle fibers. Laminin was expressed in basal lamina of muscle fibers (green). Sca-1–positive cells (red) were also detected in endothelium (arrowheads) and outer layer (arrows) of blood vessels indicated by black circles in the right panel. PECAM-positive endothelial cells (green) were detected in blood vessels. Bars: (A) 50 μm; and (B) 25 μm.

Figure 6.
Figure 6.

Muscle SP cells undergo myogenic specification and satellite cell differentiation in vivo after intramuscular injection. Myf5-nlacZ muscle SP cells were injected into regenerating tibialis anterior (TA) muscle of scid/bg mice. (A) By day 14, whole-mount staining of the TA muscle showed clusters of lacZ-positive nuclei. (B and C) By day 14, cross-sections showed that muscle SP cell–derived myocytes that expressed lacZ in the nucleus were integrated into desmin (B) or MHC-positive (C) regenerating muscle fibers (arrows). By 1 month, TA muscles were used for histochemical (D–K) and culture (L) experiments to detect muscle SP cell–derived satellite cells. (D–F) The lacZ- expressing satellite cell was detected between the basal lamina (laminin+; red) and sarcolemma (dystrophin+; green) of muscle fiber (arrows). The lacZ-expressing satellite cells were positive for Pax7 (G–I) in the nucleus and desmin (J and K) in the cytoplasm (arrows). (E, F, I, and K) DAPI staining indicates nuclei (blue). Arrowheads indicate myonuclei. Dissociated cells prepared from the TA muscle contained a lacZ-positive myoblast cluster (L). Bar: (A) 50 μm; (B and C) 10 μm; (D–F) 7 μm; (G–K) 5 μm; and (F) 40 μm.

Figure 7.
Figure 7.

Myogenic specification of muscle SP cells after co-culture with myoblasts. (A) Cultures of ROSA26 muscle SP cells alone or together with equal numbers of primary myoblasts were performed followed by staining with X-gal to mark all input ROSA26 cells and antidesmin to identify myogenic cells. Muscle SP cells co-cultured with myoblasts gave rise to X-gal/desmin-positive multinucleated myotubes (black arrows). (B) GFP-expressing muscle SP cells prepared from GFP mice were cultured alone or together with equal numbers of primary myoblasts. Muscle SP cells co-cultured with myoblasts formed GFP/MHC-positive myotubes (white arrows). White arrowheads indicate GFP-negative and MHC-positive myoblast–derived myotubes. (C) Myf5-nlacZ muscle SP cells were cultured alone or together with equal numbers of primary myoblasts. Muscle SP cells co-cultured with primary myoblasts underwent Myf5-nlacZ– expressing multinucleated myotubes (black arrows) and mononuclear myocytes (arrowheads). Bars: 25 μm.

Figure 8.
Figure 8.

CD45-positive and -negative muscle SP cells exhibit myogenic potential. Sorted CD45-negative (CD45− mSP) and CD45-positive muscle SP cells (CD45+ mSP) prepared from GFP mice were cultured together with equal numbers of primary myoblasts. Both CD45− mSP and CD45+ mSP gave rise to GFP/MHC-positive myotubes (arrows). Bar: 25 μm.

Figure 9.
Figure 9.

Myogenic differentiation of muscle SP cells after co-culture with myoblasts is Pax7. Muscle SP cells were prepared from Pax7+/+ and Pax7−/− mice, followed by infection with GFP retrovirus. Muscle SP cells were cultured alone or together with equal numbers of primary myoblasts. Both Pax7+/+ and Pax7−/− muscle SP cells formed GFP/MHC-positive myotubes (arrows) after co-culture with myoblasts. Arrowheads indicate GFP-negative and MHC-positive myoblast–derived myotubes. Bar: 25 mm.

Figure 10.
Figure 10.

MyoD-induced myogenic differentiation of muscle SP cells is Pax7 independent. Muscle SP cells prepared from Pax7+/+ (A–F) and Pax7−/− (G–L) mice were infected with control retrovirus vector (A–C and G–I) or with MyoD retrovirus vector (D–F and J–L). Muscle SP cells after infection with control retrovirus did not form any desmin-positive myoblasts (C and I). Infection with MyoD retrovirus induced desmin-positive myoblast differentiation of both muscle SP cells (F and L). MyoD expression was detected in both muscle SP cells after infection with MyoD retrovirus (D and J). Pax7 protein was not detected in MyoD-induced myoblasts derived from Pax7+/+ muscle SP cells (E). Bar: 25 μm.

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References

    1. Asakura, A., G.E. Lyons, and S.J. Tapscott. 1995. The regulation of MyoD gene expression: conserved elements mediate expression in embryonic axial muscle. Dev. Biol. 171:386–398. - PubMed
    1. Asakura, A., M. Komaki, and M. Rudnicki. 2001. Muscle satellite cells are multipotential stem cells that exhibit myogenic, osteogenic, and adipogenic differentiation. Differentiation. 68:245–253. - PubMed
    1. Beauchamp, J.R., J.E. Morgan, C.N. Pagel, and T.A. Partridge. 1999. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J. Cell Biol. 144:1113–1122. - PMC - PubMed
    1. Beauchamp, J.R., L. Heslop, D.S. Yu, S. Tajbakhsh, R.G. Kelly, A. Wernig, M.E. Buckingham, T.A. Partridge, and P.S. Zammit. 2000. Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J. Cell Biol. 151:1221–1234. - PMC - PubMed
    1. De Angelis, L., L. Berghella, M. Coletta, L. Lattanzi, M. Zanchi, M.G. Cusella-De Angelis, C. Ponzetto, and G. Cossu. 1999. Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration. J. Cell Biol. 147:869–878. - PMC - PubMed

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