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TISSUE-SPECIFIC STEM CELLS

Clonal Multipotency of Skeletal Muscle-Derived Stem Cells Between Mesodermal and Ectodermal Lineage

^aMuscle Physiology & Cell Biology Unit,
^bDepartment of Regenerative Medicine, Division of Basic Clinical Science,
^cTeaching & Research Support Center,
^dDepartment of Orthopedics, Division of Surgery, and
^eDepartment of Urology, Division of Surgery, Tokai University School of Medicine, Isehara, Kanagawa, Japan;
^fStem Cell Research, Research Center Japan, Nihon Schering KK, Chuo-ku, Kobe, Japan

Key Words. Tissue-specific stem cells • Myogenic lineage • Neural lineage • Vasculogenic lineage

Correspondence: Tetsuro Tamaki, Ph.D., Muscle Physiology and Cell Biology Unit, Department of Regenerative Medicine, Division of Basic Clinical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1143, Japan. Telephone: +81-463-93-1121 (ext. 2524); Fax: +81-463-95-0961; e-mail: tamaki{at}is.icc.u-tokai.ac.jp

Received November 16, 2006; accepted for publication May 23, 2007.
First published online in STEM CELLS EXPRESS   June 21, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

 
The differentiation potential of skeletal muscle-derived stem cells (MDSCs) after in vitro culture and in vivo transplantation has been extensively studied. However, the clonal multipotency of MDSCs has yet to be fully determined. Here, we show that single skeletal muscle-derived CD34^–/CD45^– (skeletal muscle-derived double negative [Sk-DN]) cells exhibit clonal multipotency that can give rise to myogenic, vasculogenic, and neural cell lineages after in vivo single cell-derived single sphere implantation and in vitro clonal single cell culture. Muscles from green fluorescent protein (GFP) transgenic mice were enzymatically dissociated and sorted based on CD34 and CD45. Sk-DN cells were clone-sorted into a 96-well plate and were cultured in collagen-based medium with basic fibroblast growth factor and epidermal growth factor for 14 days. Individual colony-forming units (CFUs) were then transplanted directly into severely damaged muscle together with 1 x 10⁵ competitive carrier Sk-DN cells obtained from wild-type mice muscle expanded for 5 days under the same culture conditions using 35-mm culture dishes. Four weeks after transplantation, implanted GFP⁺ cells demonstrated differentiation into endothelial, vascular smooth muscle, skeletal muscle, and neural cell (Schwann cell) lineages. This multipotency was also confirmed by expression of mRNA markers for myogenic (MyoD, myf5), neural (Musashi-1, Nestin, neural cell adhesion molecule-1, peripheral myelin protein-22, Nucleostemin), and vascular (-smooth muscle actin, smoothelin, vascular endothelial-cadherin, tyrosine kinase-endothelial) stem cells by clonal (single-cell derived) single-sphere reverse transcription-polymerase chain reaction. Approximately 70% of clonal CFUs exhibited expression of all three cell lineages. These findings support the notion that Sk-DN cells are a useful tool for damaged muscle-related tissue reconstitution by synchronized vasculogenesis, myogenesis, and neurogenesis.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Muscle-derived stem cells (MDSCs) have been isolated using a variety of methods, such as a series of preplatings in culture [1–3], repeated culture using the freeze-thaw technique [4–6], and flow cytometry-based sorting using cell surface markers [7–9] or Hoechst dye [10–13]. Because of these variations in isolation methods, it is difficult to determine the origin and localization of MDSCs, although their potential to give rise to myogenic [1, 2, 8–11, 13, 14], hematopoietic [7, 12, 14], osteogenic [1], endothelial [2, 8, 9, 12], and neural [2, 9] cell lineages after transplantation has been demonstrated. Skeletal muscle is the largest organ in the body, comprising approximately 40%–50% of total body mass, and it presumably allows donor cells to be obtained relatively easily and safely. Therefore, skeletal muscle is potentially a practical source for autologous stem cell therapy. However, the clonal characteristics of MDSCs have yet to be fully determined, and elucidation of the biological mechanisms for practical cell populations is considered the most important issue for further clinical applications.

We previously identified two stem cell populations that are able to differentiate into myogenic-vasculogenic cells in the interstitial spaces of skeletal muscle [8, 13]. Cells in the CD34⁺/CD45^– fraction (Sk-34 cells) formed colonies and had the potential to differentiate into mesodermal cells, such as endothelial cells, myogenic cells, and adipocytes in vitro and in vivo [8]. Sk-34 cells were also demonstrated to give rise to ectodermal lineage cells (Schwann cells) after transplantation into severely damaged muscle, with significant functional recovery through the synchronized reconstitution of the vascular, muscular, and peripheral nervous systems [9].

These studies suggested that tissue-specific vasculogenic and neurogenic cells reside in skeletal muscle in addition to myogenic cells, and collaborative lineage development resulted both in mass and functional recovery in severely injured muscles. In addition, the multipotency and plasticity of Sk-34 cells suggest the importance of crosstalk among the constituents of the skeletal muscle, blood vessel, and nerve systems. Failure of functional improvement by blood vessel formation alone was observed with transplanted cultured Sk-34 cells due to the absence of the above collaborative mechanisms through undesired commitment during cell culture. However, positive results were obtained with bulk cell transplantation of Sk-34 cells, but whether this was due to cellular multipotency and/or mixed progenitor cell populations in each lineage is unknown. Therefore, determination of clonal multipotency of MDSCs is important for practical cell expansion culture. Whether "myo-vasculogenic cells" can also give rise to neural cell lineages or whether different cell lineages are present is of particular interest, because differentiation of neural stem cells into endothelial [15] and myogenic [16] cell lineages has been reported.

We have identified a further cell population in the CD34^–/CD45^– (skeletal muscle-derived double negative [Sk-DN]) fraction as putative immature stem cells that can typically form sphere-like colonies with high (>10%) colony-forming activity in a collagen-based cell culture system with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) [13]. Using this basic ability, we designed the present study to define the clonal multipotency of Sk-DN cells after clonal cell sorting by fluorescence-activated cell sorting (FACS). To characterize the clonal plasticity of this cell population, we transplanted a single cell-derived colony into a severe muscle damaged model that allows the determination of injury and transplantation sites through precise histological detection of regenerated organic components [9]. We further divided Sk-DN cells into small and large fractions and performed reverse transcription-polymerase chain reaction (RT-PCR) analysis on a single cell-derived colony in order to detect the expression of putative mRNA markers for early skeletal myogenic, neural, and vascular cells during a 14-day culture period.

	MATERIALS AND METHODS

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Animals
Green fluorescent protein transgenic mice (GFP-Tg mice; C57BL/6 TgN[act EGFP]Osb Y01, provided by Dr. M. Okabe, Osaka University, Osaka, Japan) were used as donor mice for cell transplantation studies [17], and wild-type C57BL/6 mice (CLEA, Tokyo, http://www.clea-japan.com) and female nude rats (F344/NJcl-mu/rnu; CLEA) were used as recipients. Both GFP-Tg and wild-type mice were used for in vitro studies. All experimental procedures were conducted in accordance with the Japanese Physiological Society Guidelines for the Care and Use of Laboratory Animals as approved by the Tokai University School of Medicine Committee on Animal Care and Use.

Cell Purification
Interstitial cells from the thigh and lower leg muscles (tibialis anterior [TA], extensor digitorum longus, soleus, plantaris, gastrocnemius, and quadriceps femoris) of 3–8-week-old GFP-Tg and wild-type mice were obtained following a method for isolating intact, living individual muscle fibers associated with satellite cells [18]. Briefly, whole muscles were treated with 0.1% collagenase type IA (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) in Dulbecco's modified Eagle's medium containing 5%–10% fetal calf serum (FCS) with gentle agitation for 2 hours at 37°C. Extracted cells were filtered through 70-µm, 40-µm, and 20-µm nylon strainers to remove muscle fibers and other debris and were then washed and resuspended in Iscove's modified Dulbecco's medium containing 10% FCS, yielding enzymatically extracted cells. These cells were stained with biotin-conjugated anti-mouse CD34 (RAM34) and streptavidin-allophycocyanin and phycoerythrin-conjugated anti-mouse CD45 (30-F11). CD34⁺/CD45^– and CD34^–/CD45^– cells were then collected. Live cells were counted after cells positive for propidium iodide were excluded as dead cells. All antibodies were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml). Cell analysis and sorting were carried out on a dual-laser FACSVantage (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Details of the isolation methods were described previously [8, 13].

Clonal Cell Culture and Transplantation
Sk-DN cells from a GFP-Tg mouse were clone-sorted (one cell per well) into a 96-well plate and were cultured in collagen-based semisolid medium (CollagenCult H4742; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) with 10 ng/ml bFGF and 20 ng/ml EGF for 14 days in order to obtain single cell-derived colony-forming units (CFUs) as described in detail elsewhere [13]. Individual CFUs were suspended in 0.5% collagenase/phosphate-buffered saline (PBS) for 5 minutes at 37°C and were washed with serum-free medium. Single CFUs were then transplanted directly into severely damaged muscle together with 1 x 10⁵ competitive carrier cells (Sk-DN cells from wild-type mice expanded for 5 days under the same culture conditions using 35-mm culture dishes). This is because the clone would presumably contribute to muscle regeneration but not be solely responsible for total muscle regeneration of severe muscle damage. Surgery and cell transplantation were performed under halothane anesthesia (Fluothane; Takeda, Osaka, Japan, http://www.takeda.com) based on previously published methods [9]. Briefly, the left TA muscle of a wild-type C57BL/6 mouse was exposed by skin incision, and its fascia was minimally cut. Muscle fibers with nerve and blood vessels were then manually removed (torn off) from the region surrounding the motor point of the TA muscle using forceps. Four weeks after transplantation, treated muscles were prepared for fluorescence immunohistochemical analysis after macroscopic detection of GFP-positive structures (see descriptions below). Clonal transplantation was performed in two trials using four recipient mice per trial x2. Donor clones (four clones per trial x2) were randomly selected from 30 clone-sorted cells (30 individual cells in 96-well plate) obtained from the muscles of three GFP-Tg mice after 14 days of culture. Carrier cells were also obtained from the muscles of three wild-type mice per trial and after 5 days of culture.

Clonal RT-PCR
Sk-DN cells (from the muscles of three mice) were further divided into small- and large-cell populations based on forward scatter axis (see particular gate in Fig. 5A), and 30 cells from each fraction were clonally sorted into a 96-well plate. Two weeks after culture, approximately 12–14 clonal CFUs were obtained, and 10 of these were randomly selected from each fraction and prepared for clonal RT-PCR. The same trial was repeated twice, and 20 clonal CFUs were finally obtained from each fraction. The setting of particular gates for small- and large-cell fractions was fixed throughout all experiments. Clonal RT-PCR was performed based on highly optimized global RT-PCR procedure described previously [19, 20]. Single CFUs of small and large Sk-DN cells were suspended in microtubes and were lyzed with 9 µl of cold lysis-first strand synthesis solution containing first-strand buffer (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 1% NP-40, 1 mM dithiothreitol, 0.01 mM deoxynucleoside-5'-triphosphates (dNTPs), 3.4 nM dT30-containing primer (AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT), and RNase inhibitors (Ambion, Austin, TX, http://www.ambion.com; Eppendorf AG, Hamburg, Germany, http://www.eppendorf.de). Samples were quickly frozen with liquid nitrogen and were stored at –80°C until use. For analysis, samples were heated at 65°C for 5 minutes and placed on ice. Each lysate was equally divided into two PCR tubes, to which was added 100 U of SuperScript III reverse transcriptase (Invitrogen) or 0.5 µl of nuclease-free water (negative control). The first cDNA strand was synthesized by incubation for 60 minutes at 45°C. The reaction was stopped by heating at 65°C for 10 minutes. After cooling on ice, 1.5 µl of 1 U RNase H solution (Invitrogen), 0.5 µl of 75 mM MgCl₂, and 0.5 µl of nuclease-free water were added to the test sample.

RNA was degraded by incubation for 15 minutes at 37°C and RNase H was inactivated for 10 minutes at 65°C. Samples were immediately cooled on ice, and 6.5 µl of 2x poly(dA) tailing solution containing 2x terminal deoxynucleotidyl transferase buffer, 3 mM CoCl₂, 1.5 mM 2'-deoxyadenosine 5'-triphosphate, and 15 U of terminal deoxynucleotidyl transferase (Promega, Madison, WI, http://www.promega.com) were added to 6.5 µl of the first-strand cDNA solution. Poly(dA) tailing was performed for 15 minutes at 37°C and was stopped by heating at 65°C for 10 minutes. Poly(dA) tailed cDNA was preamplified using a sequence nonspecific two-step PCR protocol with dT30-containing primer. Preamplification was performed in a 20-µl volume containing ExTaq buffer, 1 U of ExTaq-polymerase (Takara Bio, Shiga, Japan, http://www.takara-bio.com), 8.3 µM dT30-containing primer, 0.65 mM dNTPs, and 4 µl of poly(dA) tailed cDNA.

The second strand was synthesized by incubating for 1 minute at 94°C, 2 minutes at 50°C, and 2 minutes at 72°C followed by 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes. Duplicate reaction tubes were employed in order to compensate for experimental errors. The reaction products from the two tubes were mixed, and this mixture was used as a template for the second preamplification. The second PCR was performed in a 20-µl volume containing ExTaq buffer, 1 U of ExTaq-polymerase, 2 µM dT30-containing primer, 0.2 mM dNTPs, and 2 µl of the first PCR product. The reaction comprised 35 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 2 minutes.

The second PCR product was subjected to 2% agarose gel electrophoresis and was confirmed as a smear having a peak of 500–700 base pairs. Specific PCR (30 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 2 minutes at 72°C) was performed in a 15-µl volume containing ExTaq buffer, 0.8 U of ExTaq-polymerase, 0.7 µM specific sense and antisense primers, 0.2 mM dNTPs, and 0.2 µl of the second PCR product. Preparations without reverse transcriptase served as negative controls in cDNA synthesis, preamplification, and specific PCR. Amplification of genomic DNA was not detected in specific PCR preparations lacking reverse transcriptase. The specific primers, skeletal and smooth myogenic, vascular, and possible neural stem cells, are summarized in Table 1.

Fluorescence In Situ Hybridization Analysis
In order to confirm the intrinsic differentiation potential of clonal Sk-DN cells, fluorescence in situ hybridization (FISH) analysis was performed for mouse to rat heterografts using GFP-mice and nude rats. In this case, a single sphere obtained from GFP mouse muscle was directly implanted into the nude rat TA injury model (not severe, but slight injury) with no carrier cells. For this purpose, genomic DNA was extracted from nude rat and C57BL/6 mouse liver, and genomic DNA probes labeled with dinitrophenyl-dUTP and digoxigenin-dUTP by the nick translation method were visualized using anti-digoxygenin-Cy3 (red reactions for rat chromosomes) and anti-dinitrophenyl-Cy5 (yellow reactions for mouse chromosomes). Nuclei were counterstained with 4,6-diamidino-2-phenylindole.

Purification of Sk-DN Cells After Bone Marrow Transplantation
In order to examine whether the Sk-DN cells were derived from bone marrow and/or blood circulation, whole bone marrow cells from a GFP mouse (1 x 10⁶, i.v.) were transplanted into five lethally irradiated wild-type mice. After 6 months, blood samples were obtained and analyzed for GFP chimerism. Sk-DN cell populations were purified as normal from their muscles using CD34 and CD45 (see Cell Purification), and the presence of GFP-positive cells in four fractions was examined by FACS analysis.

	RESULTS

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In Vivo Differentiation Potential of Clonal Sk-DN Spheres
Although only 38 x 10⁴ primary Sk-DN cells were obtained by fresh isolation, this number can be expanded by 10–15 times with 5–6 days of cell culture. Sk-DN cells typically form sphere-like colonies derived from a single cell on collagen-based clonal cell culture (Fig. 1), which greatly simplifies clonal analysis. The number of cells composing individual spheres varied from approximately 50–200 cells per sphere. To evaluate the clonal plasticity of Sk-DN cells in vivo, we transplanted a single GFP⁺ sphere into a severe muscle damage model [9] with 1 x 10⁵ 6-day cultured competitive carrier Sk-DN cells obtained from wild-type mice. At 4 weeks after transplantation, GFP⁺ muscle fiber bundles were apparent in the injured skeletal muscle under a dissection microscope (dotted frame in Fig. 2A). Several histological sections were obtained from portions of transplanted muscle, indicated by lines 1 and 2 in Figure 2A. Apparent GFP⁺ muscle fibers having GFP⁺ central nuclei (arrows) were anti--skeletal muscle actin positive (Fig. 2B), thus supporting the dissection microscopic observations. Similarly, muscle fiber bundles were clearly seen in the section (Fig. 2C). This bundle also was associated with GFP⁺ capillaries and/or blood vessels positive for CD31 around the GFP⁺ muscle fibers (Fig. 2C, arrows). Relatively large blood vessels positive for anti--smooth muscle actin were also observed in the histological section from line 2 (Fig. 2D, arrows). Donor cell-derived (GFP⁺) circle and/or arc-shapes having MAP-2⁺ reactions inside were evident adjacent to the GFP⁺ muscle fibers (Fig. 2E, arrows). These were thought to be the myelin sheaths surrounding MAP-2⁺ axons formed by donor cells (GFP⁺). In fact, Schwann cells adjacent to a small myelin sheath (probably early stage of myelin formation) were positive for anti-GFP (cells having black dots in Fig. 3A, 3B). Muscles, in five of eight single sphere-transplanted mice, yielded similar results. Typically, coformation of muscle fibers and blood vessels was frequently observed (>90% of obtained histological sections), but myelin formation was detected in <20% of sections. This may be due to characteristic tissue distribution; muscle fibers and blood vessels are distributed throughout whole muscle, but nerve fibers are distributed in only limited regions. Most GFP⁺ cells remained near the site of injection. These results indicate that sphere-forming units derived from single cells (clone) in the Sk-DN cell fraction can gave rise to skeletal myogenic, vascular, and neural cell lineages in vivo. In order to confirm the intrinsic differentiation potential of clonal Sk-DN cells, we performed FISH analysis for mouse to rat transplantation (Fig. 4). Mouse genomic DNA (yellow) was detected in myofiber nuclei (Fig. 4A, 4B), corresponding to the location of GFP⁺ fiber in adjacent sections (Fig. 4C). There were no mixed reactions for mouse (yellow) and rat (red) genome color, thus suggesting fusion-independent differentiation.

View larger version (17K): [in this window] [in a new window]   Figure 1. Sphere-like colony formation. Representative sphere-like colonies were formed by a single skeletal muscle-derived double negative cell during 5-day culture. Scale bar = 10 µm. Abbreviation: d, day.

View larger version (43K): [in this window] [in a new window]   Figure 2. Implanted tissues after transplantation of single sphere derived from single skeletal muscle-derived double negative cell. (A): In vivo view of single sphere-transplanted muscle. Implanted green tissue is seen in the dotted frame. Lines 1 and 2 indicate the histological sections shown in the following panels (B–E). Scale bar = 200 µm. (B): Histological section obtained at line 1 in (A) clearly shows GFP+ muscle fibers positive for SkMA. Centrally located nuclei were also GFP+ (arrows). Scale bar = 10 µm. (C): Histological section obtained at line 2 shows a group of GFP+ muscle fibers associated with GFP+ blood vessels positive for CD31 (red reactions, white arrows). Scale bar = 10 µm. (D): Histological section obtained at line 2 also shows GFP+ blood vessels positive for SMA (red reactions, white arrows). Scale bar = 10 µm. (E): MAP-2+ nerve axons (red reactions, white arrows) surrounded by GFP+ walls (probably myelin sheath) were evident in the histological section obtained at line 1. Scale bar = 10 µm. Blue staining in (B–E), DAPI. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; MAP2, microtubule associated protein-2; SkMA, anti--skeletal muscle actin; SMA, anti--smooth muscle actin.

View larger version (41K): [in this window] [in a new window]   Figure 3. Immunoelectron microscopic detection of donor cell-derived Schwann cells. (A, B): Schwann cells with little myelin (arrows), probably due to their early stage, are GFP+, as the black dots of 3,3-diaminobenzidine products are evident in their nuclei and cytoplasm. GFP+ myofibers were also evident in left side of panels (A) and (B). In panel (B), a GFP– capillary is located between a GFP+ myofiber and Schwann cell as a negative control. Scale bar = 2 µm. Abbreviations: Cap, capillary; GFP, green fluorescent protein; Mf, myofibers; N, nuclei; Scw, Schwann cell.

View larger version (19K): [in this window] [in a new window]   Figure 4. Fluorescence in situ hybridization analysis after mouse to rat single-sphere transplantation. (A): Donor mouse cell-derived nuclei (yellow, arrows) were evident among host rat nuclei (red). (B): Nuclei were confirmed by 4,6-diamidino-2-phenylindole staining. (C): Location of mouse-derived nuclei (yellow) corresponds to the location of green fluorescent protein-positive myofibers (dotted circle) in adjacent section (21 µm apart). Scale bar = 10 µm.

View larger version (29K): [in this window] [in a new window]   Figure 5. Flow cytometric characterization and clonal expression of specific mRNAs. (A): Distribution and determination of small and large cells among enzymatically extracted cells. (B): Basic sorting pattern of skeletal muscle-derived double negative (Sk-DN) and Sk-34 cells. (C): Percentage of small and large cells between Sk-DN and Sk-34 cell populations. (D): Expression of specific mRNAs in single Sk-DN cell-derived colonies after 14 days of culture. Specific primers for early skeletal myogenic (MyoD, myf5), neural (Musashi-1, Nestin, NCAM, PMP22, Nucleostemin), smooth myogenic (-smooth muscle actin, smoothelin), and vascular (VE-cadherin, TEK) marker mRNAs, as well as for Pax3 and Pax7, are summarized in Table 3. Colony shapes and expression patterns of mRNAs varied in each clone; however, some were found to express all cell lineage marker genes, thus demonstrating the multipotency of cells in the Sk-DN fraction. Note that typical sphere colony formation was not observed in large-cell clones, whereas sphere formation can be seen in small-cell clones. HPRT was used as a housekeeping gene. Scale bars = 100 µm. Abbreviations: alpha-SMA, -smooth muscle actin; APC, allophycocyanin; bp, base pairs; FSC, forward scatter; HPRT, hypoxanthine phosphoribosyltransferase; NCAM1, neural cell adhesion molecule-1; PE, phycoerythrin; PMP, peripheral myelin protein; Sk-34, skeletal muscle-derived CD34 positive cells; SSC, side scatter; TEK, tyrosine kinase-endothelial; VE, vascular endothelial.

The percentage expression of mRNAs in the clonal CFUs from small and large cells is shown in Table 2. Typically, 100% (20/20) of small clonal CFUs expressed skeletal (MyoD, Myf5) and smooth (-smooth muscle actin, smoothelin) myogenic and possible neural (Musashi-1, Nestin, neural cell adhesion molecule-1, peripheral myelin protein-22, and Nucleostemin) stem cell markers, and approximately 65%–75% (1315/20) of clones expressed endothelial (vascular endothelial [VE]-cadherin, tyrosine kinase-endothelial [TEK]) cell markers. Among clones from large cells, 100% expressed neural, smooth muscle, and endothelial (TEK) markers, whereas fewer than 25% (15/20) of clones expressed skeletal myogenic markers. In addition, 55% (11/20) and 5% (1/20) of small-cell clones expressed Pax7 and Pax3, whereas large-cell clones did not express either Pax3 or Pax7. Thus, higher multipotency was observed in the small-cell fraction.

View larger version (15K): [in this window] [in a new window]   Figure 6. Detection of GFP+ Sk-DN cells from wild-type mouse muscle 6 months after GFP+ bone marrow transplantation. (A): Distribution of GFP+ cells (approximately 5%) in total extracted cells from muscle. (B): Analysis of GFP+ cells using CD34 and CD45. There were no cells in the Sk-DN fraction, thus indicating that Sk-DN cells are not derived from bone marrow cells. Abbreviations: APC, allophycocyanin; GFP, green fluorescent protein; PE, phycoerythrin; Sk-DN, skeletal muscle-derived double negative.

	DISCUSSION

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The origins of locally preserved adult stem cells have been extensively reviewed by Young et al. [25, 26]. Based on their classification, cells that are able to differentiate into ectodermal and mesodermal lineages including vascular-related cells are epiblastic-like stem cells. Thus, the present Sk-DN cells can be categorized as epiblastic-like stem cells. Epiblasts are situated just under the inner-cell mass in mammalian embryonic development and are highly immature cells. Therefore, we speculate that Sk-DN cells are residual cells from embryonic developmental stages and are preserved in the interstitial spaces of skeletal muscle, even after birth, as a cellular reserve, as described by Young et al. [25, 26]. The fact that Sk-34 and Sk-DN cells are not derived from bone marrow [9] also supports this notion (Fig. 6). The notion that residual cells from embryonic developmental stages were preserved in the interstitium can also be applied to other tissue-specific stem cells residing in the adipose tissue [27] and dermis [28, 29].

The relationship between the present Sk-DN and other reported MDSCs is of interest. However, it is difficult to directly compare them due to their high variability in the extraction and purification methods of MDSCs. The extraction method can be roughly divided into two techniques: muscles are minced or not minced before enzymatic treatment. We refrain from mincing muscle tissue, but others have consistently minced muscle. The extracted cells can also be purified using two methods: cells are sorted and fractionated by FACS using cell surface markers and/or uptake of Hoechst dye [7–13] or a series of cell cultures (preplating) with and without freeze-thaw technique [1–6]. Some of these reports have used cell characterization by FACS with cell surface markers after culture; however, expression of cell surface markers (and probably other markers) is markedly affected by culture conditions such as term, medium, supplied cytokines, fetal bovine serum concentration, culture plate coating materials, and even applied cell densities. Therefore, it is impossible to directly compare these cell behaviors.

It is therefore likely that muscle side-population (SP) cells and present Sk-DN cells can be compared, and we have previously discussed their relationship [13]. Our extracted cells included <0.1% SP cells and were mainly composed of main-population (MP) cells. In addition, differentiation capacity and sphere-forming activity are not different between SP-Sk-DN and MP-Sk-DN cells, and thus it is possible that present results are 99.9% attributable to MP-Sk-DN cells. In our experiment, SP cell content in the total extracted cells was quite small (0.08%) [13] when compared with other previous reports (0.21%) [12], whereas the distribution of CD34⁺ (58%), CD45⁺ (6%), and CD34^–/45^– (36%) cells in the SP fraction was similar [10, 13].

Other common points with MDSCs include non- and/or less- (or slowly) adherent capacity [1–4, 6, 8, 13, 20, 30], round shape [1, 3, 4, 8, 13, 20, 30], spontaneous contraction [8, 13, 20, 30], and sphere-like colony formation [4, 13, 20, 30] during culture, even under a variety of culture conditions. Previously reported MDSCs showed one or more of the above characteristics, and our fractionated Sk-DN and Sk-34 cells showed all of these characteristics [8, 9, 13]. Therefore, these may be the typical characteristics of primitive MDSCs.

Muscle fibers, blood vessels, and peripheral nerve fibers are the main components of skeletal muscle, and we have reported the reconstitution of severely damaged muscle by synchronized vasculogenesis, myogenesis, and neurogenesis following freshly isolated Sk-34 cell transplantation [9]. The observed reconstitution of these components was reflected by significant mass and functional recovery [9]. In addition, we also confirmed the significant therapeutic potential of Sk-DN cells, which achieved mainly cell fusion-independent differentiation that was strictly confirmed by the combination of three methods, such as using the mouse X- and Y-chromosome FISH, cell transplantation to nonmuscle tissue (beneath the kidney capsule), and FISH using mouse and rat genomic DNA probes (manuscript submitted for publication). Furthermore, Sk-DN cells are situated upstream of Sk-34 cells in the same cell lineage and are potentially capable of self-renewal, as confirmed by the expression of CD34 and Sca-1 in Sk-DN cells during in vitro culture and in vivo transplantation (our unpublished data). Therefore, the significant therapeutic potential of Sk-DN cells and reported Sk-34 cells [9] is largely supported by the clonal multipotency of Sk-DN cells.

In conclusion, clonal analysis of Sk-DN cells in vivo and in vitro clearly demonstrated the multipotency of skeletal muscle-derived single stem cells that can give rise to skeletal myogenic, vascular (endothelial and smooth muscle cells), and neural (Schwann cells) cell lineages. Due to these mesodermal and ectodermal differentiation potentials, Sk-DN cells are probably highly immature cells residing in the interstitium of skeletal muscle as reserve cells to contribute to the postnatal adaptive and/or regenerative capacity of skeletal muscle.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (to T.T.), the New Energy and Industrial Technology Development Organization of Japan (to T.T.), and by Tokai University Research aid (to T.T.).

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