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
Author for correspondence (e-mail: nao{at}libra.ls.m-kagaku.co.jp)
Accepted 27 March 2002
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SUMMARY |
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Key words: Stem cells, Mouse, Fate generation
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INTRODUCTION |
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Skeletal muscle stem cells, also known as muscle satellite cells, are located adjacent to the plasma membrane of myofibers beneath the basement membrane. During muscle regeneration, satellite cells proliferate and then fuse together to form myotubes. Histopathological analysis has shown that muscle satellite cells differentiate into myotubes and myofibers exclusively (Saito et al., 1994), and there has been no evidence that these cells are able to differentiate into nonmuscle cells in vivo. However, both primary cultured mouse myoblasts and the immortalized mouse myoblastic cell line C2C12 differentiate into osteoblasts and adipocytes as well as myotubes under appropriate culture conditions (Chalaux et al., 1998
; Fujii et al., 1999
; Katagiri et al., 1994
; Nishimura et al., 1998
; Teboul et al., 1995
; Yamamoto et al., 1997
). Although these observations suggest that muscle satellite cells preserve multipotentiality, the source of the muscle-derived cells (so-called myoblasts) analyzed in these studies is unknown. Recently, multipotentiality of muscle satellite cells is also suggested by the analysis of multiclonal myoblasts derived from multiple satellite cells (Asakura et al., 2001
). However, it has been unclear whether different fates are generated from a single satellite cell.
Specific cells within skeletal muscle exhibit apparent stem cell-like plasticity. Side population (SP) cells separated from dissociated muscle cells by fluorescence-activated cell sorting differentiate into muscle and hematopoietic cells (Gussoni et al., 1999; Jackson et al., 1999
). However, SP cells constitute a distinct population from muscle satellite cells (Seale et al., 2000
). Stem-like cells have also been isolated from primary cultured skeletal muscle cells and shown to exhibit multipotentiality (Lee et al., 2000a
; Qu et al., 1998
; Torrente et al., 2001
). The origin of these cells is unknown, however, and their relation to muscle satellite cells is unclear.
Histopathological and molecular biological studies (Bischoff, 1986; Garry et al., 2000
; Megeney et al., 1996
; Saito et al., 1994
; Seale et al., 2000
) indicate that muscle satellite cells are largely responsible for postnatal muscle growth, regeneration and repair, even if SP cells or other stem-like muscle-derived cells also contribute to muscle regeneration. Clarification of the mechanisms of muscle regeneration will therefore require determination of the differentiation potential of muscle satellite cells and its regulation.
We have now characterized a clone of unmanipulated myogenic cells derived from a single mouse muscle satellite cell and revealed its multipotentiality in vitro. Furthermore, multipotent progenitor cells derived from muscle satellite cells were shown to co-express multiple determination genes under growth conditions. On the basis of these observations, we propose a stock options model for the lineage commitment of muscle satellite cells.
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MATERIALS AND METHODS |
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Normal human abdominal muscle tissue was obtained by biopsy from a 44-year-old woman with informed consent at the Kanagawa Cancer Center Research Institute. Human myogenic cells were isolated from a small piece of muscle according to the low cell density culture method.
For induction of myogenic differentiation, myogenic cells were cultured in primary cultured myocyte differentiation medium (pmDM), consisting of the chemically defined medium TIS (Hashimoto et al., 1995; Hashimoto et al., 1994
) supplemented with 2% FBS. Myogenic cells were cultured in pmDM supplemented with recombinant human BMP2 (250-500 ng ml1) (Strathman Biotech, Hamburg, Germany) to induce osteogenic differentiation. ALP activity in cells fixed with 4% paraformaldehyde was detected by incubation of the fixed cells for 20 minutes in a solution containing 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 20 mM MgCl2, 0.01% naphthol AS-MX, and Fast Blue RR (0.5 mg ml1). To induce adipogenic differentiation, we cultured myogenic cells in DMEM supplemented with 10% FBS and 100 µM
-linolenic acid (Sigma, St. Louis, MO) for up to 10 days. Formalin-fixed cells were stained with 0.3% oil red O (Sigma) in 60% isopropanol for 30 minutes at room temperature, and were photographed under epifluorescence conditions with a WIG filter. Nuclei of cells were visualized by staining with 2,4-diamidino-2-phenylindole dihydrochloride n-hydrate (DAPI) (0.5 µg/ml, Sigma).
For induction of bone matrix formation in vitro, Mouse myogenic cells were cultured in pmDM supplemented with BMP-2 and 10 mM ß-glycerophosphate for up to 8 days. Paraformaldehyde-fixed cells were stained with the calcium-staining dye Alizarin Red S (0.01%, Sigma) for 30 minutes.
Immunofluorescence, immunocytochemical and immunoblot analyses
For immunofluorescence or immunocytochemical analysis, paraformaldehyde-fixed cells were incubated for 12 to 36 hours at 4°C with a mouse monoclonal antibody to Runx2 (Zhang et al., 2000), a mouse monoclonal antibody to MyoD (Novocastra, Newcastle, UK), rabbit antibodies to rat myogenin (Hashimoto et al., 1994
), a mouse monoclonal antibody to MHC (Bader et al., 1982
), a mouse monoclonal antibody to Pax7 (Ericson et al., 1996
) (DSHB, Iwoa City, IA), goat antibodies to mouse osteocalcin (Biomedical Tech, Stoughton, MA), rabbit antibodies to nestin (Arimatsu et al., 1999
), rabbit antibodies to desmin (Progen, Heiderberg, Germany) or rabbit antibodies to GFP (Medical and Biological Laboratory, Nagoya, Japan) in the presence of 0.1% saponin (Sigma). Biotinylated or Cy3-labeled antibodies to mouse or rabbit immunoglobulin G as well as fluorescein isothiocyanate (FITC)-labeled antibodies to goat immunoglobulin G (Jackson ImmunoResearch Laboratory, Bar Harbor, ME) were used as secondary antibodies. The biotinylated antibodies were detected with streptavidin-conjugated horseradish peroxidase or FITC. The antibody to Runx2 was detected by biotinylated antibodies to mouse immunoglobulin G and a TSA Direct kit (New England Nuclear, Boston, MA). Cell nuclei were stained with DAPI.
Immunoblot analysis and corresponding sample preparation were performed as described (Hashimoto and Ogashiwa, 1997; Hashimoto et al., 1995
). Immune complexes were detected by colorimetry with a BCIP/NBT detection kit (Nakarai, Kyoto, Japan) or with the use of chemiluminescence reagents (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Reverse transcription and polymerase chain reaction (RT-PCR)
Total RNA was extracted from cultured myogenic cells with TRIzol (Life Technologies, Rockville, MD), treated with RNase-free DNase (RQ-1; Promega, Madison, WI), and then reverse transcribed with the use of a Ready-To-Go Your Prime cDNA synthesis kit (Amersham Pharmacia Biotech) and random hexamers as primers. Targeted genes were amplified by PCR with the following primers (sense and antisense, respectively) and conditions: 5'-AGGACACGACTGCTTTCTTC-3' and 5'-GCACCGCAGTAGAGAAGTGT-3' (25 cycles of 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 30 seconds) for the mouse Myod1 (encoding MyoD) gene; 5'-TGAGATTTGTGGGCCGGAGC-3' and 5'-GGGACACCTACTCTCATACTGG-3' (25 cycles of 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 30 seconds) for the mouse Runx2 gene; 5'-TTGCTGAACGTGAAGCCCATCGAGG-3' and 5'-GTCCTTGTAGATCTCCTGGAGCAG-3' (30 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 60 s) for the mouse Pparg (encoding PPAR) gene; 5'-TGACGGAGCAGGAACAGCAG-3' and 5'-GACGAAGGCGAGTGAGAATC-3' (25 cycles of 94°C for 30 seconds, 57°C for 30 seconds and 72°C for 30 seconds) for the mouse muscle creatine kinase gene; 5'-TTCATGTCCAAGCAGGAGGGCAA-3' and 5'-ACCGTAGATGCGTTTGTAGGCGGT-3' (27 cycles of 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 30 s) for the mouse osteocalcin gene; 5'-GCATGGACTGTGGTCATGAG-3' and 5'-CCATCACCATCTTCCAGGAG-3' (22 cycles of 94°C for 30 seconds, 55°C for 30 seconds and 72°C for 60 seconds) for the mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene; 5'-GTCTTACCCCTCCTACCTGA-3' and 5'-TGCCTGGCTCTTCTTACTGA-3' (30 cycles of 94°C for 30 seconds, 53°C for 30 seconds and 72°C for 30 seconds) for the human RUNX2 gene; and 5'-GCATGGACTGTGGTCATGAG-3' and 5'-CCATCACCATCTTCCAGGAG-3' (30 cycles of 94°C for 30 seconds, 58°C for 30 seconds and 72°C for 60 seconds) for the human GAPDH gene.
Cell transfection and inducible expression of myogenin
The ecdysone-inducible expression system (Invitrogen, San Diego, CA) was modified for the present study. The inducible expression plasmid pISE was constructed by introducing an internal ribosome entry site (Hashimoto and Ogashiwa, 1997) and the GFP cDNA into the pIND(SP1) vector. The rat myogenin cDNA (Wright et al., 1989
) was then subcloned into pISE to generate the myogenin expression plasmid pISEmgn. MMCs (5x104 cells in a 35 mm dish) were transfected with 0.5 µg of pISEmgn (or pISE) and 1.5 µg of pVgRXR in the presence of 9 µl of FuGENE6 transfection reagent (Roche Diagnostic, Mannheim, Germany) as described (Hashimoto and Ogashiwa, 1997
; Hashimoto et al., 1995
; Hashimoto et al., 1994
). Expression of both myogenin and GFP was induced simultaneously by ponasterone A in pISEmgn-transfected cells cultured in pmDM supplemented with BMP2 (250 ng ml1).
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RESULTS |
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To determine the multipotentiality of unmanipulated muscle satellite cells, we cultured GB1T under various conditions. Immunofluorescence analysis revealed that GB1T expressed MyoD, Pax7, desmin and nestin (Fig. 1A-D), all proteins that are known to be expressed in muscle lineage cells. GB1T therefore appeared to preserve the myogenic phenotype during in vitro culture. GB1T cells differentiated into myotubes when cultured in the differentiation medium pmDM (Fig. 1F), although they continued to proliferate as undifferentiated cells in the growth medium pmGM (Fig. 1E). Furthermore, GB1T differentiated into immature osteoblasts expressing alkaline phosphatase (ALP), an early marker of osteogenic differentiation, when cultured for 2-4 days in pmDM supplemented with bone morphogenetic protein 2 (BMP2) (Fig. 1G); ALP activity was not detected in GB1T cultured in the absence of BMP2. GB1T also differentiated into adipocytes, containing numerous lipid droplets in the cytoplasm, when cultured in the presence of 100 µM -linolenic acid for 6 days (Fig. 1H). These results indicate that unmanipulated satellite cells derived from adult skeletal muscle preserve multipotentiality.
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Co-expression of multiple lineage determination genes in undifferentiated MMCs
The expression of master genes essential for myogenesis, osteogenesis, and adipogenesis was examined in undifferentiated MMCs by RT-PCR analysis. Undifferentiated MMCs expressed the genes for MyoD, a muscle-determining factor; Runx2, a transcription factor essential for osteogenesis (Komori et al., 1997; Otto et al., 1997
); and PPAR
, a nuclear receptor essential for adipogenesis (Barak et al., 1999
; Kubota et al., 1999
; Rosen et al., 1999
) (Fig. 2A).
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To determine whether a Runx2-expressing subpopulation of MMCs is lost from the culture under myogenic differentiation conditions, we examined the expression of Runx2 protein in undifferentiated MMCs by immunofluorescence analysis. Runx2 was present in the nuclei of all MMCs (Fig. 2C,D). The downregulation of Runx2 expression in MMCs under myogenic differentiation conditions was therefore not due to the selective elimination of a Runx2-expressing subpopulation. Immunoblot analysis revealed that the abundance of Runx2 in undifferentiated MMCs was markedly greater than that in C2C12 cells (Fig. 2E). A low level of Runx2 expression in C2C12 cells was also observed previously (Katagiri et al., 1994; Lee et al., 2000b
; Zhang et al., 2000
).
Critical period for induction of osteogenic differentiation by BMP-2
The downregulation of Runx2 expression during myogenesis suggested that the osteogenic differentiation potential of MMCs may change. To detect any alteration in the response of MMCs in myogenic differentiation culture to BMP2 as well as to determine the reversibility of BMP2-induced osteogenesis, we exposed MMCs to BMP2 for various periods during culture under myogenic differentiation conditions (Fig. 3A). BMP2 induced marked ALP expression in MMCs that were exposed to this factor for 0-12, 12-24 or 12-48 hours of a 48-hour culture period (Fig. 3B). Exposure to BMP2 within a critical period thus induced osteogenic differentiation even after withdrawal of BMP2. The osteogenesis triggered by BMP2 during this critical period thus appears to be irreversible. By contrast, MMCs lost the potential to undergo osteogenic differentiation after culture for 24 hours under myogenic differentiation conditions.
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Inhibition of osteogenic differentiation by myogenin
The loss of osteogenic differentiation potential in MMCs during myogenesis suggested that a myogenesis-specific factor (or factors) is responsible for the downregulation of Runx2 expression. A possible candidate for the suppressor of osteogenesis was myogenin, given that MMCs lost the ability to undergo osteogenic differentiation after the induction of myogenin expression. To determine whether myogenin contributes to the downregulation of Runx2 expression, we transfected MMCs with a bicistronic expression plasmid (pISEmgn) that encodes both myogenin and green fluorescent protein (GFP). Effects of myogenin on the growth properties of MMCs were avoided by inducing the expression of myogenin with ponasterone A under osteogenic differentiation conditions. Immunofluorescence analysis revealed that Runx2 was expressed in 66% of GFP-positive MMCs transfected with the control vector pISE, indicating that Runx2 expression was inhibited slightly by transfection itself (Fig. 4A,B). By contrast, Runx2 was detected in no more than 32% of GFP-expressing MMCs transfected with pISEmgn (Fig. 4A,C). In addition, myogenin was detected in only 50% of GFP-positive MMCs transfected with pISEmgn, probably because myogenin is more labile than is GFP. These results thus indicated that forced expression of myogenin resulted in marked inhibition of Runx2 expression in MMCs even under osteogenic differentiation conditions.
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DISCUSSION |
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Ectopic bone formation within skeletal muscle has been described. Osteoprogenitor cells have thus been thought to reside in skeletal muscle, although their source has remained unknown (Bosch et al., 2000). We have now shown that MMCs form bone in vitro in the presence of ß-glycerophosphate. These cells are therefore able to undergo terminal osteogenic differentiation. Our results suggest that the descendants of muscle satellite cells are responsible for ectopic ossification of skeletal muscle, including that caused by genetic deficiency (Feldman et al., 2000
).
Bone marrow stromal cells are considered a source of autologous osteogenic cells for bone repair. However, the collection of these cells entails substantial risk to the individual. Furthermore, freshly isolated bone marrow stromal cells are limited in number and are difficult to culture continuously in order to provide sufficient cells for therapy. Our demonstration of the multipotentiality of HMCs and MMCs suggests that myogenic satellite cells as well as previously described muscle-derived stem-like cells might serve as an alternative source of autologous osteogenic cells for the repair of bone defects.
Undifferentiated myogenic cells were shown to express multiple differentiation-determining genes that are essential for the commitment to distinct cell lineages. The co-expression of these determination genes was apparent in independently isolated MMCs as well as HMCs. The expression of Runx2 was thus not induced artifactually in myogenic cells during culture. Furthermore, undifferentiated MMCs continued to express several myogenic lineage markers. These observations thus indicate that Runx2-expressing MMCs preserve the myogenic phenotype during in vitro culture. Muscle satellite cells were previously thought to be already committed to the myogenic lineage. Our results, however, suggest that the fate of muscle satellite cells is not committed even in adult skeletal muscle. The myogenic commitment of muscle satellite cells during muscle regeneration probably involves both the maintenance of Myod1 expression and the repression of nonmyogenic determination genes including Runx2. The present study suggests that myogenin suppresses osteogenesis by inhibiting Runx2 expression during myogenesis.
In contrast to myogenic differentiation, commitment of muscle satellite cells to the osteogenic lineage was accompanied by the suppression of myogenic determination genes, including those for MyoD and myogenin. The observation that Runx2 is already expressed in undifferentiated myogenic cells before BMP2-induced osteogenesis suggests that osteogenic differentiation of muscle satellite cells might not be trans-determined. Given that they also express genes essential for the commitment to nonmyogenic lineages and that they preserve multipotentiality, it might be more accurate to refer to these multipotent progenitor cells as multiblasts (multiple tissue blasts) rather than as myoblasts.
Myogenesis of C2C12 cells is thought to be inhibited by suppression of the expression of MyoD and myogenin genes by both Runx2 and Smad proteins, which act downstream of the BMP2 receptor (Fujii et al., 1999; Lee et al., 2000b
; Nishimura et al., 1998
). However, we have now shown that the induction of ALP preceded the down-regulation of MyoD expression during osteogenesis of MMCs. The downregulation of MyoD expression is thus not a prerequisite for osteogenic differentiation. MMCs are able to undergo osteogenesis directly; they do not first have to undergo dedifferentiation as has been proposed for the trans-differentiation of iris epithelial cells of adult newts into lens epithelial cells (Yamada and McDevitt, 1984
).
An understanding of the mechanisms of tissue regeneration requires characterization of the mechanisms by which different cell fates are generated from multipotent stem cells. According to the divergence model, a lineage determination gene, such as Myod1 or Runx2, is induced in uncommitted multipotent stem cells and converts them to the corresponding committed monopotent progenitor cells (Fig. 8A). In the trans-determination model, the determination gene expressed initially (X in Fig. 8B) is downregulated and another determination gene (Y in Fig. 8B) is induced in stem cell-derived progenitor cells. In addition to these models, we now propose an alternative that we refer to as the stock options model for the generation of different fates from multipotent stem cells. According to this model, stem cells are induced to express multiple determination genes and are converted to multipotent progenitor cells (multiblasts). Depending on the differentiation-inducing signal, the progenitor cells select an option for the terminal differentiation pathway (Fig. 8C). During muscle regeneration, muscle satellite cells would thus be committed to the myogenic lineage as a result of the process proposed in the stock options model.
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ACKNOWLEDGMENTS |
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