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First published online 5 October 2004
doi: 10.1242/jcs.01419

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Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway

Department of Biological Structure, School of Medicine, University of Washington, Seattle, WA 98195, USA

View larger version (100K): [in a new window]   Fig. 1. Development of cultures emanating from individual mouse myofibers. (A-E') Myofibers labeled with DAPI (blue) and anti-Pax7 (red); fibers were fixed at time 0 (A and A'), 19 hours (B and B'), 24 hours (C and C'), 48 hours (D and D') and 72 hours (E and E'). (F-F'') Parallel images of 1-week-old myofiber culture labeled with DAPI, anti-Pax7 and anti-MyoD, respectively; image of each stain was merged with the phase-contrast image; arrow points to a cell negative for both Pax7 and MyoD. (G-H') Parallel DAPI-stained and phase-contrast images of two different areas within the same 4-week-old culture, depicting adipocytes (G and G') and myotubes (H and H'); nuclei in the adipogenic area are larger then the myonuclei. Bar, 20 µm (A-F''); 90 µm (G-H').

View larger version (85K): [in a new window]   Fig. 2. Formation of adipocytes in mouse myofiber cultures. (A-D) Representative images of myofiber-derived cultures co-stained with DAPI (blue) and oil-red-O (red). (A) Multilocular cells (indicated with arrows) in a non-myogenic area within a 10-day-old culture. (B) Multi-, pauci- and unilocular cells (indicated by the upper right, middle and lower left arrows, respectively) in a non-myogenic area of a 3-week-old culture. (C,D) Myoblasts and myotubes exhibiting minimal staining with oil-red-O in 10- and 21-day-old cultures; myoblasts are indicated with arrows in panel C and a myotube is indicated with an arrowhead in panel D; a multilocular cell present in panel D is indicated with an arrow. Bar, 20 µm. (E) Graph depicting the number of fiber cultures containing adipogenic cells accumulating during a 6-week period.

View larger version (33K): [in a new window]   Fig. 4. Quantification and classification of 2-week-old clones derived from individual EDL myofibers. (A) Distribution of 242 EDL myofibers according to the number of clones they gave rise to. Clones were prepared from both wild-type and GFP mice (n=17). (B) The incidence of myogenic, non-myogenic and mixed clones per myofiber depicted in panel A. Fibers are grouped according to the number of clones they produced. Each bar represents a specific combination of myogenic, non-myogenic and mixed clones, and the incidence of this combination is depicted at the top of the bar. For example, in the inset of 5 clones/fiber, the first bar is a combination of four myogenic and one non-myogenic clone, and this combination occurred in three fibers.

View larger version (85K): [in a new window]   Fig. 3. Cell morphology and expression of adipogenic-associated markers in non-myogenic (left column) and myogenic (right column) clones. (A,B) Fluorescent images of 7-day-old clones derived from one GFP-myofiber; the clone depicted in panel A shows multiple cell morphologies and the clone depicted in panel B shows myotubes. (C,D) Representative merged images of 2-week-old cultures visualized with DAPI (blue) and oil-red-O (red); adipogenic cells are detected only in the non-myogenic clone. (E,F) Images of 2-week-old cultures labeled with anti-PPAR (red) and DAPI (blue) merged with the phase-contrast image; intense nuclear staining of PPAR was evident only in the adipogenic cells (E); a very low level of PPAR was detected in some of the myonuclei (F). (G-H') Double immunostaining with c/EBP (green) and MyoD (red), image of each stain was merged with the phase-contrast image; the non-myogenic clone was additionally stained with oil-red-O; both clones exhibit similar levels of nuclear expression of c/EBP (G and H) but only the myogenic clone expresses MyoD (G',H'). Note that the two mononucleated cells in panel H' that seem to be negative, in fact express low levels of MyoD. However, this weaker staining is lost when the MyoD immunostaining image and the phase-contrast image are merged. The variations in the level of MyoD probably reflect different phases of the cell cycle as previously reported (Kitzmann et al., 1998). Of particular interest is the pair of cells (top of micrograph in H') that seem to be just separating from each other following cell division with only one cell expressing a high level of MyoD. Bar, 20 µm.

View larger version (58K): [in a new window]   Fig. 5. Expression of cytoskeletal proteins in parallel non-myogenic (left column) and myogenic clones (right column). (A-B') Double immunostaining for SMA (green) and sarcomeric myosin (red); both non-myogenic cells and myotubes express SMA (A,B), however only myotubes also express sarcomeric myosin (A',B'). (C,D) Images of immunolabeled cultures showing nestin expression in both clonal types. (E,F) Images of immunolabeled cultures showing desmin expression only in the myogenic clone. (G,H) Merged images of clones double immunostained for SMA (green) and PPAR (red); both SMA and PPAR were expressed in the non-myogenic clone, whereas only SMA was expressed in myotubes. Bar, 20 µm.

View larger version (60K): [in a new window]   Fig. 6. Images of 4-day-old GFP clones sequentially labeled with anti-Pax7 followed by anti-MyoD. (A-B') Clone 1 is positive for both Pax7 and MyoD. (C-D') Clone 2 is positive for Pax7 but negative for MyoD. The use of GFP-labeled cells allowed detailed characterization of the cell body, demonstrating morphological distinctions between the two clonal phenotypes. Bar, 20 µm.

View larger version (43K): [in a new window]   Fig. 7. A proposed model for the mesenchymal plasticity of skeletal muscle satellite cells. As shown, satellite cells in an appropriate myogenic environment enter myogenic differentiation, yielding myoblasts that form myotubes. De-homed satellite cells enter the MAD route, yielding non-myogenic cells such as adipocytes and osteoblasts. Inappropriate cues within the muscle may direct satellite cells into the MAD program at the expense of myogenesis. Mesenchymal stem cells residing in the bone marrow are in a MAD-equivalent state and when de-homed can give rise to variety of cell types including satellite cells when reaching skeletal muscle. Note that although the diversion of satellite cells into the MAD program and its culmination with mature adipocytes is based on data provided in the present study, the diversion of satellite cells into osteogenesis via the MAD program is suggested based on studies reporting on the development of osteoblasts/chondroblasts in myogenic cultures treated with bone morphogenic proteins (e.g. Asakura et al., 2001; Katagiri et al., 1994). Likewise, the incorporation of bone marrow-derived cells into the myofiber unit as depicted in the model is based on published studies (Dreyfus et al., 2004; Gussoni et al., 1999; LaBarge and Blau, 2002). Such studies have not established that satellite cells are typically originated from bone marrow-derived cells but have shown that bone marrow-derived cells can infrequently enter the satellite cell niche and acquire myogenic properties.

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