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Generation of different fates from multipotent muscle stem cells

Michiko R. Wada1,*, Masayo Inagawa-Ogashiwa1,*, Shirabe Shimizu2, Shigeru Yasumoto3 and Naohiro Hashimoto1,{dagger}

1 Stem Cell Research Unit, Mitsubishi Kagaku Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194-8511, Japan
2 Department of Plastic Surgery and
3 Laboratory of Molecular Cell Biology and Oncology, Kanagawa Cancer Center Research Institute, Yokohama, Kanagawa 241-0815, Japan
* These authors contributed equally to this work



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  		Fig. 1. Multipotentiality of MMCs derived from muscle satellite cells. (A-D) Expression of myogenic lineage markers in a clone (GB1T) of primary cultured MMCs was determined by immunofluorescence analysis with antibodies to MyoD (A), to Pax7 (B), to desmin (C) or to nestin (D), as well as Cy3-conjugated secondary antibodies. (E) Undifferentiated MMCs resembled fibroblasts when cultured in pmGM. (F) MMCs differentiated into myotubes expressing sarcomeric myosin heavy chain (immunostained with a horseradish peroxidase reaction product) after culture for 4 days in pmDM. (G) MMCs differentiated into immature osteoblasts expressing ALP (activity detected by staining with Fast Blue RR) when cultured in pmDM supplemented with BMP2 (250-500 ng ml–1) for 4 days. (H) MMCs differentiated into adipocytes containing many lipid droplets in their cytoplasm (as revealed by staining with oil red O; nuclei were detected by staining with DAPI) when cultured for 6 days in DMEM supplemented with 10% FBS and 100 µM {gamma}-linolenic acid. Images in A-D,H were obtained by epifluorescence microscopy, that in E was obtained by phase-contrast microscopy, and those in F,G were obtained by bright-field microscopy. Scale bars: 50 µm.

 


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  		Fig. 2. Expression of Runx2 in MMCs. (A) RT-PCR analysis of the expression of MyoD (lane 2), Runx2 (lane 3) and PPAR{gamma} (lane 4) expression in undifferentiated MMCs cultured in pmGM. The abundance of Gapdh mRNA was examined as a positive control (lane 1). DNA size markers in lane M represent 200, 300 and 400 bp. (B) RT-PCR analysis of the expression of Runx2, osteocalcin (Osc) and muscle creatine kinase (MCK) in undifferentiated growing (Gr), myogenically differentiated (Myo) and osteogenically differentiated (Ost) MMCs. The abundance of Gapdh mRNA was again analyzed as a positive control. Three independent samples were analyzed for each experimental condition. (C) Immunofluorescence analysis of Runx2 expression in undifferentiated MMCs. (D) Nuclei in the same field as that shown in C were visualized by DAPI staining. (E) Immunoblot analysis of Runx2 expression in total lysates (20 µg of protein) of undifferentiated MMCs (lane 1) and C2C12 cells (lane 2). Scale bar: 100 µm.

 


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  		Fig. 3. Critical period for BMP2-induced osteogenesis. (A,B) Sensitivity of MMCs to BMP2 under myogenic differentiation conditions. MMCs were cultured in the absence (white bars) or presence (black bars) of BMP2 for various intervals up to 48 hours (A). Numbers on the left represent the period of exposure to BMP2. At the end of the 48-hour culture, the cells were stained for ALP activity (B); images were obtained by bright-field microscopy. The level of ALP activity in each culture is shown as +, +/– or – below the images. (C) Induction of myogenesis-specific proteins in MMCs under myogenic differentiation conditions. Cells were cultured in pmDM for the indicated times, after which total lysates (20 µg of protein) were subjected to immunoblot analysis with antibodies to MHC and to myogenin.

 


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  		Fig. 4. Suppression of BMP2-induced osteogenesis by myogenin. MMCs were transfected with the myogenin expression plasmid pISEmgn or the control vector pISE. (A) Expression of myogenin was induced by ponasterone A simultaneously with the onset of stimulation with BMP2 for 30 hours. The expression of Runx2 was determined by immunofluorescence analysis in 132 or 177 GFP-expressing cells transfected with pISE or pISEmgn, respectively (A). MMCs transfected with pISE (B) or pISEmgn (C) were probed with antibodies to GFP and to Runx2, and immune complexes were detected by FITC or Cy3 fluorescence, respectively. Runx2 (red) was present in nuclei, whereas GFP (green) was localized to both nuclei and cytoplasm. Intrinsic fluorescence of GFP was severely decreased after the immunostaining procedure. Scale bar: 20 µm.

 


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  		Fig. 5. Expression of MyoD and ALP in MMCs during myogenic and osteogenic differentiation. MMCs were cultured in pmDM for 6 days to induce myogenic differentiation (A) or in the presence of BMP2 for 2 (B) or 4 (C) days to induce osteogenic differentiation. The cells were then subjected to staining for ALP activity (blue) and immunostaining for MyoD (brown). Arrows in B indicate cells expressing both MyoD and ALP. Scale bars: 20 µm.

 


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  		Fig. 6. Bone matrix formation by MMCs. MMCs were cultured for 6 days in pmDM supplemented with either BMP2 alone (A) or both BMP2 and 10 mM ß-glycerophosphate (B-H). Transparent nodules were formed by the cells cultured with BMP2 alone (A). By contrast, many opaque nodules were detected in the presence of ß-glycerophosphate (B). Nodules formed by multiclonal (C) and monoclonal (D) MMCs stained with Alizarin Red S. Incubation of fixed cells with 200 mM EGTA for 5 minutes resulted in the removal of the opaque material; the same field of a culture before and after EGTA treatment is shown in E and F, respectively. (G,H) Osteocalcin was detected in the opaque matrix by immunofluorescence analysis with antibodies to osteocalcin and FITC-conjugated secondary antibodies. (H) The same field is shown in G. Scale bars: 100 µm. Images in A and B were obtained by phase-contrast microscopy, whereas those in C-G were obtained by bright-field microscopy.

 


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  		Fig. 7. Multipotentiality of HMCs. (A,B) Undifferentiated growing HMCs were subjected to immunofluorescence analysis with antibodies to MyoD (A) or to Pax7 (B) and with Cy3-conjugated secondary antibodies. Both MyoD and Pax7 were present in the nuclei of all HMCs. (C,D) HMCs were cultured in pmDM for 6 days in the absence (C) or presence (D) of BMP2, and were then stained with Alizarin Red S for the detection of calcium deposition and examined by phase-contrast microscopy. Prominent myotubes developed in the culture shown in C. By contrast, myogenic differentiation was inhibited and calcium deposition was induced in the culture shown in D. Scale bars: 100 µm in A-D. (E) RT-PCR analysis of Runx2 expression in undifferentiated HMCs. Results are shown for two independent samples.

 


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  		Fig. 8. Models for the generation of different fates from multipotent stem cells. The divergence model (A), trans-determination model (B), and stock options model (C) are shown. Triangles, squares and circles represent tissue stem cells, progenitor cells and differentiated cells, respectively. X and Y represent lineage-specific determination genes.

 

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© The Company of Biologists Ltd 2002