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EMBRYONIC STEM CELLS

Paraxial Mesodermal Progenitors Derived from Mouse Embryonic Stem Cells Contribute to Muscle Regeneration via Differentiation into Muscle Satellite Cells

^aDepartment of Immunology, Nagoya University Graduate School of Medicine, Nagoya, Japan;
^bDepartment of Functioning Activation, National Institute for Longevity Sciences, Aichi, Japan

Key Words. Embryonic stem cell • Mesoderm • Muscle satellite cell • Muscle regeneration • Platelet-derived growth factor receptor-

Correspondence: Correspondence: Ken-Ichi Isobe, M.D., Ph.D., Department of Immunology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan. Telephone: 81-52-744-2135; Fax: 81-52-744-2972; e-mail: kisobe{at}med.nagoya-u.ac.jp

Received on February 20, 2008; accepted for publication on April 21, 2008.

First published online in STEM CELLS EXPRESS  May 1, 2008.

	ABSTRACT

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

 
Pluripotent embryonic stem (ES) cells hold great potential for cell-based therapies. Although several recent studies have reported the potential of ES cell-derived progenitors for skeletal muscle regeneration, how the cells contribute to reconstitution of the damaged myofibers has remained elusive. Here, we demonstrated the process of injured muscle regeneration by the engraftment of ES cell-derived mesodermal progenitors. Mesodermal progenitor cells were induced by a conventional differentiation system and isolated by flow cytometer of platelet-derived growth factor receptor- (PDGFR-), a marker of paraxial mesoderm, and vascular endothelial growth factor receptor-2 (VEGFR-2), a marker of lateral mesoderm. The PDGFR-⁺ population that represented the paraxial mesodermal character demonstrated significant engraftment when transplanted into the injured muscle of immunodeficient mouse. Moreover, the PDGFR-⁺ population could differentiate into the muscle satellite cells that were the stem cells of adult muscle and characterized by the expression of Pax7 and CD34. These ES cell-derived satellite cells could form functional mature myofibers in vitro and generate myofibers fused with the damaged host myofibers in vivo. On the other hand, the PDGFR-^–VEGFR-2⁺ population that showed lateral mesodermal character exhibited restricted potential to differentiate into the satellite cells in injured muscle. Our results show the potential of ES cell-derived paraxial mesodermal progenitor cells to generate functional muscle stem cells in vivo without inducing or suppressing gene manipulation. This knowledge could be used to form the foundation of the development of stem cell therapies to repair diseased and damaged muscles.

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

	INTRODUCTION

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

 
Murine embryonic stem (ES) cells have proven to be a suitable model system for the dissection of mammalian-specific differentiation pathways [1–3]. Pluripotent mouse ES cells can be selectively differentiated into all three germ layers that contain mesendoderm, definitive endoderm, visceral endoderm, mesoderm, and neuroectoderm [4–6]. These studies could open the door to new therapies that use specifically differentiated ES cells as treatments for human incurable diseases. To pursue these therapies, it is necessary to determine how to specifically differentiate ES cells in vitro before transplanting to animals because of their ability to generate a teratoma [7]. However, very few experiments have succeeded in the in vivo transplantation of specifically differentiated ES cells for skeletal muscle regeneration [8, 9].

In embryogenesis, all skeletal muscle, except for muscles of the head, is derived from somites, transient condensations of paraxial mesoderm located adjacent to the neural tube and notochord [10]. Previous studies of in vitro ES cell differentiation showed the differentiation pathway of paraxial and lateral mesodermal cells as a divergence of platelet-derived growth factor receptor- (PDGFR-)⁺ and vascular endothelial growth factor receptor-2 (VEGFR-2)⁺ cells, respectively [6, 11]. The validity of these markers for specifying the two mesodermal subsets was based on a previous study of embryos [12]. An in vitro fate analysis of VEGFR-2⁺ cells derived from ES cells demonstrated their potential to differentiate into endothelial cells, hematopoietic cells, and -smooth muscle Actin-positive mesenchymal cells [6, 13, 14]. These analyses indicate that VEGFR-2⁺ cells correspond to lateral mesodermal cells. On the other hand, in vitro fate analysis of the PDGFR-⁺ population showed its potential to generate chondrocytes, osteocytes, and skeletal muscle cells [11, 15], indicating that PDGFR-⁺ cells represent paraxial mesodermal cells.

Injured skeletal muscle has the remarkable ability to initiate a rapid and extensive repair process. Following necrosis of the damaged tissue and activation of an inflammatory response, myogenic cells proliferate, differentiate, and fuse, leading to new myofiber formation. Activation of adult muscle satellite cells is a key element in this process [16]. A homogeneous population of mouse satellite cells has been isolated by flow cytometry from adult skeletal muscles [17]. Such cells, grafted into the muscles of Mdx-null mice, contribute to both myofiber repair and reconstruction of the muscle satellite cell compartment [18]. On the other hand, ES cell-derived myogenic progenitors had shown relatively lower potential to proliferate and regenerate the myofiber compared with adult myoblast [8, 9]. However, recent study of mouse ES cells overcame this difficulty by inducing expression of Pax-3 [19], a key transcription factor of muscle development in somitogenesis [20]. In this excellent study, the PDGFR-⁺, VEGFR-2^– mesodermal population derived from Pax3-induced ES cells exhibited fine potential for therapeutic engraftment in a mouse model of Duchenne's muscular dystrophy [19]. Although ES cell-derived myogenic progenitor cells showed the ability to regenerate muscle, how the cells contributed to reconstitution of the damaged myofibers has remained elusive.

In this study, we demonstrated that ES cell-derived mesodermal progenitor cells can contribute to the regeneration of injured muscle via satellite cell differentiation. Initially, the PDGFR-⁺ population derived from ES cells differentiated mainly into the muscle satellite cells in injured muscle of mouse by direct intramuscular transplantation. Secondarily, such ES cell-derived satellite cells regenerated myofibers by fusing with host damaged myofibers. Our results also demonstrated the potential of ES cell-derived paraxial mesodermal progenitor cells to generate functional muscle stem cells in vivo without inducing or suppressing gene manipulation. This knowledge could be the foundation for the development of stem cell therapies to repair diseased and damaged muscles.

	MATERIALS AND METHODS

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

 
Cell Culture and In Vitro ES Cell Differentiation
ES cells expressing the LacZ gene (CCE/nLacZ) were a kind gift of Dr. S. Nishikawa [13]. CCE/nLacZ ES cells were maintained as described previously [6].

Induction of ES cell differentiation was performed as described previously [6]. Briefly, ES cells were seeded into a 10-cm dish coated with type IV collagen (Nitta Gelatin, Inc., Osaka, Japan, http://www.nitta-gelatin.com) at a density of 8 x 10⁴ cells per dish in -minimal essential medium (-MEM) (Gibco, Gaithersburg, MD, http://www.gibcobrl.com) supplemented with 10% fetal calf serum (FCS) (Roche Diagnostics, Basel, Switzerland, http://www.roche.com) and 50 µM 2-mercaptoethanol (2-ME). Four days later, the cells were harvested and collected for further study.

Antibodies, Cell Staining, Fluorescence-Activated Cell Sorting Analyses, and Cell Sorting
Rat monoclonal antibodies (MoAbs) APA5 (anti-PDGFR-) [21] and AVAS12 (anti-VEGFR-2) [12] were kind gifts of Dr. S. Nishikawa [6]. Phycoerythrin-conjugated streptavidin (BD Pharmingen, San Diego, http://www.bdbiosciences.com/index_us.shtml) was used to detect biotinylated-APA5 antibody. AVAS12 MoAbs were directly conjugated by a standard method using allophycocyanin.

Cultured cells were harvested and collected in 0.05% trypsin-EDTA (Gibco, Grand Island, NY, http://www.invitrogen.com). Single-cell suspensions were stained as previously described [6] and analyzed or sorted by FACSCalibur or FACSVantage-HG (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com).

Transplantation of ESC-Derived Mesodermal Progenitors into Injured Mouse Muscle
We carried out mouse experiments according to protocols approved by the Animal Care and Use Committee of Nagoya University Graduate School of Medicine. The PDGFR⁺ (Fig. 1Ai), PDGFR^–VEGFR2⁺ (Fig. 1Aii), and PDGFR^–VEGFR2^– (Fig. 1Aiii) populations were purified and collected by fluorescence-activated cell sorting (FACS) (> 5 x 10⁵ cells). Cells were resuspended at a density of 2.5 x 10⁴ cells per microliter in MEM supplemented with 10% FCS and 50 µM 2-ME. A quadriceps femoris muscle of a KSN nude mouse (Japan SLC, Inc., Hamamatsu, Japan, http://www.jsk.co.jp/) was injured by direct cramping with the anesthesia diethyl ether. Twenty microliters of collected cell suspension was directly injected into the injured quadriceps of each mouse.

View larger version (22K): [in this window] [in a new window]   Figure 1. Mesoderm differentiation during in vitro embryonic stem (ES) cell differentiation. CCE/nLacZ ES cells were cultured on type IV collagen-coated dishes with differentiation medium in the absence of leukemia inhibitory factor. (A): Four days postinduction, differentiated ES cells were harvested, and the expression of VEGFR-2 and PDGFR- was analyzed by fluorescence-activated cell sorting. Three distinct populations were isolated and purified by FACSVantage-HG: (Ai), PDGFR-+; (Aii), PDGFR-–VEGFR-2+; and (Aiii), PDGFR-–VEGFR-2– populations. (B): RNA was isolated from each population to investigate the expression of lineage specific markers by reverse transcription-polymerase chain reaction. Expressions of PDGFR- and VEGFR-2 were indicators of isolation efficiency. T, pan-mesoderm marker; Eomes, early mesodermal marker; Msgn, Mesp-2 and Tbx-6, paraxial mesodermal markers; Pax-3 and Pax-7, dermomyotome markers; Myf-5 and Myo-D, myogenic cell markers; GATA-2 and Tal-1, lateral mesodermal markers; Oct-3/4 and Nanog, undifferentiated ES cell markers. The paraxial mesodermal markers were observed mainly in the PDGFR-+ population; on the other hand, the lateral mesodermal markers were detected only in the PDGFR-–VEGFR-2+ population. Expression of Oct-3/4 and Nanog in the PDGFR-–VEGFR-2– population indicated that the residual undifferentiated ES cells were contained in this population. Almost the same results were obtained by four separate experiments. Abbreviations: PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor.

Whole-Mount β-Galactosidase Staining
For whole-mount detection of β-galactosidase activity, transplanted tissue was extracted and fixed with 2% formaldehyde, 0.2% glutaraldehyde, and 0.02% Nonidet-P-40 in phosphate-buffered saline (PBS) for 2 hours on ice. The fixed sample was washed twice in PBS for 1 hour at 4°C. β-Galactosidase activity was detected by incubating the sample in 3.1 mM potassium ferricyanide, 3.1 mM potassium ferrocyanide, and 1 mM MgCl₂ in PBS with 0.4 mg/ml X-gal (Gibco) overnight at 37°C. The stained sample was washed twice in PBS at 4°C for 30 minutes, frozen in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA, http://www.sakura.com), and cut into 5-µm sections with a cryostat. The sectioned sample was counterstained with hematoxylin-eosin or by immunohistochemical staining.

Immunohistochemistry
For histochemical analysis, a sectioned sample was fixed with 2% paraformaldehyde in PBS for 5 minutes on ice and washed twice in PBS on ice. Antibodies were diluted in 0.2% bovine serum albumin, 1% normal goat serum, and 0.2% Triton X-100 in PBS as follows: rabbit anti-β-galactosidase (Chemicon, Temecula, CA, http://www.chemicon.com) at 1:2,000, mouse anti-Dystrophin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) at 1:200, and mouse anti-CD34 biotin conjugated (Biolegend, San Diego, http://www.biolegend.com) at 1:1,000. Alexa 488-conjugated anti-rabbit IgG (Invitrogen) was used as secondary antibody for rabbit anti-β-galactosidase. Biotin-conjugated anti-mouse IgG (Sigma-Aldrich) was used as the secondary antibody for mouse anti-Dystrophin. To detect the biotin-conjugated antibodies, phycoerythrin-conjugated streptavidin (BD Pharmingen) was used in 0.2% Triton X-100 in PBS at 1:250. Pax7 expression was detected with mouse anti-Pax7 (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and the Zenon Alexa Fluor 647 Mouse IgG₁ Labeling Kit (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) according to the manufacturers' instructions. Green fluorescent protein (GFP), Alexa 488, and phycoerythrin fluorescent signals were analyzed with a µRadiance Confocal Microscopy System (Bio-Rad, Hercules, CA, http://www.bio-rad.com). The Alexa 647 fluorescent signal was analyzed by a fluorescence in situ histochemistry system (Hamamatsu Photonics, Hamamatsu, Japan, http://jp.hamamatsu.com).

Cultures of Tissue-Dissociated Mouse Satellite Cells
Tissue-dissociated mouse satellite cells were isolated as described previously [22]. Briefly, satellite cells were isolated from the quadriceps femoris muscles of the KSN nude mouse in which ES cell-derived mesodermal progenitor cells were engrafted. Muscles, cleaned from the surrounding connective tissue, were minced into small fragments and incubated in 0.2% collagenase type I (Sigma-Aldrich) at 37.5°C for 2.5 hours. The collagenase-treated preparations were subsequently triturated, and the resulting suspensions were filtered through a 35-nm nylon mesh. Cells were harvested by centrifugation (300g, 5 minutes), resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 5% horse serum (Gibco), filtered again through the nylon mesh, and collected again by centrifugation. The final cell pellet was resuspended in growth medium and cultured in 35-mm plates precoated with 2% gelatin at 10⁴ to 5 x 10⁴ cells per plate. The growth medium, which was changed every 3 days, consisted of DMEM containing 25% FCS (Roche) and 10% horse serum (Gibco). The cells isolated from the quadriceps femoris muscles were cultured for 12 days, and the following histochemical analyses were performed.

Immunohistochemistry of Culture Cells
For detection of β-galactosidase activity in culture cells, cells were fixed with 2% formaldehyde, 0.2% glutaraldehyde, and 0.02% Nonidet-P-40 in PBS for 10 minutes on ice. The fixed cells were washed twice in PBS for 10 minutes at 4°C. β-galactosidase activity was detected by same procedure as whole-mount staining. After overnight incubation for β-galactosidase activity, cells were washed twice in PBS at 4°C for 10 minutes.

For detection of skeletal muscle Actin expression in culture cells, the following immunohistochemical analysis was done after β-galactosidase detection. The cells were fixed again in 100% methanol on ice for 15 minutes and blocked by 2% skim milk, 0.2% Triton X-100 in PBS for 1 hour at room temperature. Rabbit anti-skeletal muscle Actin antibody (Abcam, Cambridge, MA, http://www.abcam.com) was diluted in blocking solution at 1:100, and cells were incubated in the first antibody solution overnight at 4°C. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Chemicon) was used as secondary antibody, and HRP signals were detected by diaminobenzidine (Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english) and H₂O₂ as described previously [21]. Cells were photographed using a DP-20 microscopic digital camera system (Olympus, Tokyo, http://www.olympus-global.com).

Tolerance Induction of GFP Mouse
Tolerance to CCE/nLacZ ES cells was induced by subcutaneous transplantation of 1.5 x 10⁶ CCE/nLacZ ES cells at day 4 of differentiation into GFP-expressing mice (C57BL/6-Tg(UBC-GFP)30Scha/J mice (Jackson Laboratory, Bar Harbor, ME, http://www.jax.org]) 2 days after birth. On the basis of tumor generation, a tolerance-induced mouse was selected 6 weeks after transplantation.

	RESULTS

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

 
In Vitro Characterization of Mesodermal Progenitor Population in Differentiated ES Cell Culture
In a previous study, mesodermal progenitor cells emerged from an in vitro ES cell differentiation culture [6]. In this culture system, the PDGFR-⁺VEGFR-2^– population represented paraxial mesodermal characteristics, the PDGFR-^–VEGFR-2⁺ population showed lateral mesodermal characteristics, and the PDGFR-⁺VEGFR-2⁺ population exhibited an immature mesodermal character [11]. The PDGFR-⁺VEGFR-2⁺ population had the character of both paraxial and lateral mesodermal progenitor cells, and it was able to differentiate into the paraxial mesodermal progenies, such as myocytes, osteocytes, and chondrocytes, as well as the PDGFR-⁺VEGFR-2^– paraxial mesodermal lineage could do [11]. Therefore, in this study, we handled the PDGFR-⁺VEGFR-2^– and the PDGFR-⁺VEGFR-2⁺ population as a whole population of paraxial mesodermal progenitors and named it the PDGFR-⁺ population. The expression patterns of these mesodermal markers were confirmed in this culture system with CCE/nLacZ ES cells (Fig. 1A). Differentiation of CCE/nLacZ ES cells was induced according to the method previously described [6]. The proportions of the PDGFR-⁺ population and the PDGFR-^–VEGFR-2⁺ population were assessed by FACS on day 4. Under these culture conditions, the PDGFR-⁺ cells were 35%–40% of the population, and the PDGFR-^–VEGFR-2⁺ cells existed at 5%–10% of the population (Fig. 1Ai and 1Aii). The PDGFR-^–VEGFR-2^– cells appeared at 50%–60% of the population (Fig. 1Aiii).

The three populations were characterized on the basis of genes specifically expressed in nascent immature mesoderm, paraxial mesoderm, lateral mesoderm, and undifferentiated cells (Fig. 1B). Each population was purified by FACSVantage-HG, and RNA was extracted for RT-PCR. The expression of PDGFR- and VEGFR-2 was examined to evaluate the efficiency of the FACS sorting, and it was confirmed that contamination from the other fractions was negligible (Fig. 1B).

We measured the expression of T and Eomes, which are known to be expressed primarily in mesoderm that is newly exfoliated from the primitive streak [23, 24]. The expression of T was detected equally in all populations because the T gene is expressed from the early blastocyst stage [25] and widely in mesodermal cells [24]. Although expression of Eomes was detected in the PDGFR-⁺ population and the PDGFR-^–VEGFR-2⁺ population, the higher expression was found in the PDGFR-⁺ population (Fig. 1B). These results show that the PDGFR-⁺ cells and the PDGFR-^–VEGFR-2⁺ cells actually contain mesodermal precursors correlated with the embryonic primitive streak-stage mesoderm.

The paraxial mesoderm and early somite markers Mesogenin, Mesp-2, and Tbx-6 were highly expressed in the PDGFR-⁺ population. Their expressions in the other populations were very low. However, the myotome markers, such as Pax-3 and Pax-7, and the myogenic markers Myf-5 and Myo-D were never expressed in all populations (Fig. 1B). These results indicate that paraxial mesodermal differentiation has progressed to early somitic stage but not to myotome or myogenic stage at day 4 differentiation ES cell culture. GATA-2 and Tal-1 were detected only in the PDGFR-^–VEGFR-2⁺ population (Fig. 1B). According to these results, the PDGFR-⁺ population contains the cells from the early paraxial mesodermal stage to the somitic stage. The PDGFR-^–VEGFR-2⁺ population represents the lateral mesodermal progenitors specifically. Oct-3/4 and Nanog, markers for undifferentiated ES cells, were dominantly detected in the PDGFR-^–VEGFR-2^– population (Fig. 1B). Only the PDGFR-^–VEGFR-2^– cells contained residual undifferentiated ES cells at day 4 of differentiation. We also detected a faint expression of Oct-3/4 in the PDGFR-⁺ population and considered that the PDGFR-⁺ population represented the primitive streak mesoderm that expressed Oct-3/4 at a lower level in early embryogenesis [26].

Localization of ES Cell-Derived Mesodermal Progenitor Cells in Injured Muscle
We next attempted to determine whether the mesodermal progenitors have a myogenic potential in vivo. The PDGFR-⁺, PDGFR-^–VEGFR-2⁺, and PDGFR-^–VEGFR-2^– populations were purified by FACSVantage-HG and transplanted into the injured quadriceps femoris muscle of KSN nude mice. Transplanted tissues were isolated at day 28 and stained with hematoxylin-eosin after whole-mount β-galactosidase staining. ES cell-derived cells had a light blue nuclear staining (Fig. 2A, 2B; Fig. 2C, top row, center and right panels). Each fraction of differentiated ES cells displayed the ability to survive in injured muscle. Tumorigenesis was observed only in the tissues transplanted with the PDGFR-^–VEGFR-2^– population (Fig. 2C, top row, left panel). This tumor expressed β-galactosidase clearly (Fig. 2C, top row, center and right panels) and was teratoma that consisted of various tissues derived from all three germ layers, including gut-like epithelial tissues (endoderm), keratin-containing epidermal tissues (ectoderm), and cartilage (mesoderm) (Fig. 2C, middle row). These tissues were derived from undifferentiated ES cells confirmed by the β-galactosidase expression in fluorescent immunohistochemical analysis (Fig. 2C, bottom row). Teratoma formation was not detected in the muscle transplanted with two types of mesodermal progenitor cells, the PDGFR-⁺ and PDGFR-^–VEGFR-2⁺ populations.

View larger version (61K): [in this window] [in a new window]   Figure 2. Localization of embryonic stem (ES) cell-derived cells in injured muscle of immunodeficient mouse. The ES cell-derived populations were purified by fluorescence-activated cell sorting and transplanted into the injured quadriceps femoris muscle of a KSN nude mouse. The engrafted tissues were isolated 28 days after transplantation and stained by hematoxylin-eosin after whole-mount β-galactosidase staining. ES cell-derived cells showed a light blue nuclear staining. (A): The PDGFR-+ cells were primarily located adjacent to the myofibers (arrowheads) of KSN nude mice (n = 9). Some nuclei localized to the center of the myofiber (arrow). (B): The PDGFR-–VEGFR-2+ cells were also identified in adjacent to the myofibers (arrowheads) and in the center of the myofiber (arrow) of KSN nude mice (n = 3). (C): The PDGFR-–VEGFR-2– cells induced tumorigenesis in the injured muscle of mice (top row, left; dotted circle; n = 3). This tumor clearly had β-galactosidase expression (top row, center and right). This tumor was a teratoma that consisted of various tissues, including gut-like epithelial tissue (middle row, left), epidermal tissues (middle row, center), and cartilage (middle row, right). The β-galactosidase expressions in these tissues were confirmed by immunostaining (bottom row, green signals). Scale bars = 20 µm ([A, B], middle and bottom rows of [C]) and 100 µm (top row of [C]). Abbreviations: PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor.

The Expression of Myogenic Markers in ES Cell-Derived Mesodermal Progenitor Cells in Injured Muscle
We next examined whether the ES cell-derived mesodermal progenitor cells had a myogenic character in transplanted injured muscles. The PDGFR-⁺ population and the PDGFR-^–VEGFR-2⁺ population were purified by FACS and transplanted into the injured quadriceps of KSN nude mice. The tissues were isolated 28 days after transplantation and were stained by immunofluorescent antibodies with whole-mount β-galactosidase staining (Fig. 3).

View larger version (48K): [in this window] [in a new window]   Figure 3. Multicolor immunofluorescent staining of myogenic marker proteins in cell transplanted muscle. Injured muscle tissues were isolated 28 days after transplantation. The tissues were stained by whole-mount β-galactosidase staining and sectioned prior to immunofluorescent staining (n = 3 mice per experiment). (A): A major muscle satellite cell marker, Pax7 (red), was expressed in LacZ+ (green) cells located adjacent to myofibers (white arrows). Both types of mesodermal progenitor-derived engrafted cells that, located adjacent to myofibers, expressed Pax7. (B): Another satellite cell marker, CD34 (red), was also expressed in LacZ+ mesodermal progenitor-derived cells (black) adjacent to myofibers (white arrows). (C): Dystrophin, a marker of the myofiber surface, also colocalized with LacZ+ cells derived from both types of mesodermal populations (white arrowheads). Scale bars = 20 µm. Abbreviations: PDGFR, platelet-derived growth factor receptor; VEGFR, vascular endothelial growth factor receptor.

In Vitro Myogenic Potentials of the Adult Satellite Cells Derived from ES Cell Differentiation Culture
We next analyzed the function of the satellite cells derived from ES cell differentiation culture. We transplanted the PDGFR-⁺ cells in quadriceps femoris muscle of KSN nude mouse as mentioned in Materials and Methods. Then we isolated the satellite cells, cultured them for 12 days, and analyzed them by β-galactosidase staining (Fig. 4). Many LacZ⁺ cells were observed in the culture, and some of them exhibited a fiber formation like that of other host satellite cell-derived myofibers (Fig. 4A, arrowheads). To analyze whether these fibers with LacZ⁺ nuclei are myofibers or not, we examined the expression of skeletal muscle Actin in the culture cells (Fig. 4B). We detected the brown myofibers that had LacZ⁺ blue nuclear staining (Fig. 4B, black arrows). This result indicated that the satellite cells derived from the PDGFR-⁺ population had the same potential to grow into a mature myofiber as the host satellite cells did. However, the frequency was only 1% (5 LacZ⁺ myotubes in approximately 450 host-derived myotubes in a well). In addition, some LacZ⁺ cells displayed a fibroblastic form that had a large cytoplasm and did not express the skeletal muscle Actin (Fig. 4B, red arrows). We considered that the PDGFR-⁺ population in day 4 ES cell differentiation culture consisted of heterogeneous mesodermal progenitor cells that could differentiate into various types of the cells, and this was one cause for the lower frequency of engraftment of ES cell-derived mesodermal progenitor cells.

View larger version (116K): [in this window] [in a new window]   Figure 4. In vitro differentiation of satellite cells isolated from the muscle in which the embryonic stem (ES) cell-derived platelet-derived growth factor receptor-+ mesodermal progenitor cells were transplanted. The satellite cells were isolated from the ES-derived-cell engrafted muscle of KSN nude mice (n = 3), 28 days after transplantation. The satellite cells were cultured for 12 days, and the origin of the cells was analyzed by β-galactosidase staining. (A): Some LacZ+ multinuclei cells formed a fiber shape (arrowhead) in many host-derived myofibers that did not express β-galactosidase. (B): Expression of skeletal -actin in cultured satellite cells (brown). All LacZ+ multinuclei cells that formed fiber shapes showed skeletal muscle Actin expression (black arrow). However, some LacZ+ mononuclear cells that did not express skeletal muscle Actin were observed (red arrow). Scale bars = 50 µm.

Time-Course Behavior of ES Cell-Derived PDGFR-⁺ Transplanted Cells in Injured Muscle

View larger version (64K): [in this window] [in a new window]   Figure 5. Time-course behavior of transplanted platelet-derived growth factor receptor (PDGFR)-+ mesodermal cells. (A): LacZ-embryonic stem (ES) cell-derived PDGFR-+ cells were transplanted into crushed muscle of KSN nude mice (n = 4, each term). Tissues were isolated at 7 and 14 days after transplantation and stained by hematoxylin-eosin after whole-mount β-galactosidase staining. ES cell-derived cells displayed a light blue nuclear staining. At 7 days after transplantation (left panels), ES cell-derived mesodermal cells were detected in postinflammatory connective tissue (upper panel), whereas host myogenic cells were regenerated with multinuclei formation (lower panel). At 14 days after transplantation (right panels), ES cell-derived mesodermal cells were located both in postinflammatory connective tissue (arrow) and adjacent to myofibers (arrowheads). (B): Satellite cell marker expression of ES cell-derived cells at 14 days after transplantation. LacZ+ cells (green) adjacent to a myofiber expressed both CD34 (upper panels, red) and Pax7 (lower panels, red). Scale bars = 50 µm (A) and 20 µm (B).

View larger version (60K): [in this window] [in a new window]   Figure 6. Transplantation of the platelet-derived growth factor receptor (PDGFR)-+ cells into GFP+ mice to assess how embryonic stem (ES) cell-derived cells form myofibers. (A): Tolerance to CCE/nLacZ ES cells was induced in GFP mice (n = 6) by subcutaneous transplantation of the entire population of day 4 differentiated CCE/nLacZ ES cells 2 days after birth. Six weeks after transplantation, tumorigenesis was observed (left panel). This teratoma was induced by a residual undifferentiated ES cell population (right panel, light blue). (B): The PDGFR+ cells derived from LacZ-ES cells were transplanted into injured quadriceps femoris muscle of tolerance-induced GFP mice (n = 4). Tissues were isolated 28 days after transplantation, stained by whole-mount β-galactosidase staining, and sectioned prior to immunofluorescent staining. LacZ+ cells (red), located in the center of the myofibers, were detected only in GFP+ myofibers (green). No LacZ+ cells that formed GFP– myofibers were detected (n = 3). Scale bars = 200 µm (A) and 25 µm (B). Abbreviations: GFP, green fluorescent protein; H.E., hematoxylin and eosin.

PDGFR-⁺ cells derived from CCE/nLacZ ES cells were transplanted into the injured muscle of tolerant GFP-expressing mice. Tissues were isolated 28 days after transplantation, stained by whole-mount β-galactosidase staining, and sectioned prior to immunofluorescent staining. LacZ⁺ cells located in the center of myofibers were detected only in GFP⁺ myofibers (Fig. 6B, arrows). We concluded that ES cell-derived mesodermal progenitor cells could contribute to muscle regeneration by fusion with host damaged myofiber.

	DISCUSSION

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

 
Many studies have exploited the potential of cell transplantation therapies to promote muscle regeneration. Various cell types isolated from adult muscle have exhibited their regenerative potential, including side-population cells [34], muscle-derived stem cells [35], mesoangioblast obtained from postnatal skeletal muscle vessels [36], and satellite cells isolated by flow cytometry [17, 18]. All of these fractions have been reported to produce engraftment when transplanted into dystrophic mice. Although such adult myogenic stem cells may be useful for cell therapy, an ES cell-based therapy would have advantages, including, first, differentiation from pluripotent stage by developmental manner, allowing the transplantation of a more primitive cell with higher replicative potential, which would enable more durable engraftment; and second, the probability that patient-specific induced pluripotent stem cells could be induced from adult somatic cells [37]. Recently, several reports have shown that the ES cell-derived myogenic progenitor cells have the potential to engraft to the dystrophic muscle [8, 9, 19]. Among these reports, Darabi et al. succeeded in the significant engraftment of Pax3-induced ES cell-derived paraxial mesodermal progenitors in dystrophic muscle and improvement of the function of impaired muscle [19]. However, the process of the engraftment of ES cell-derived progenitor cells to impaired muscle is still unclear. Here, we demonstrated the process of regeneration that ES cell-derived mesodermal progenitors initially differentiated into satellite cells and secondary generated the myofiber by fusion with host damaged muscle. These satellite cells, which are derived from donor ES cells, exhibited the same ability to form functional myofibers as host satellite cells in vitro primary culture study. Satellite cells are the key population in adult muscle regeneration [16], and this satellite cellular repopulation would have an advantage therapeutically for repeated regeneration by engrafted ES cell-derived cells in the recipient muscle that has a genetic disorder.

Satellite cellular repopulation of ES cell-derived mesodermal PDGFR-⁺ progenitors may be due to their character, because our progenitor cells were in a much earlier stage than those of other ES cell-based studies [8, 9, 19]. We showed that the PDGFR-⁺ progenitor cells did express early-stage markers, such as Mesogenin, Mesp-2, and Tbx-6, which were expressed primarily in the paraxial mesoderm, presomitic mesoderm, and somites [38–40], but did not express Pax-3 and Pax-7, which were expressed in the dermomyotome, a derivative from somite [41]. Besides, myogenic regulatory genes, such as Myf-5 [42] and Myo-D [43], were also negative in the PDGFR-⁺ population, whereas other ES cell-derived myoblasts in previous studies had Myf-5 and Myo-D expression. Our results show the potential to differentiate into functional satellite cells in vivo in the early paraxial mesodermal population, which has not committed to myogenic cells yet. The early paraxial mesodermal progenitors would be superior in therapeutic transplantation because of their replicative potential with satellite cell differentiation. However, their multipotency would cause the heterogeneity of ES cell-derived cells in the engrafted muscle, and the low-level engraftment would be due to this heterogeneity. Another marker selection that isolates specific myogenic fraction or a modulation of the environment by adding appropriate growth factors to enrich the myogenic population would be necessary for overcoming this heterogeneity of the early mesodermal progenitors.

The PDGFR-^–VEGFR-2⁺ cells exhibited specific GATA-2 and Tal-1 expression; GATA-2 and Tal-1 are known as hematopoietic and endothelial cell markers. However, the PDGFR-^–VEGFR-2⁺ population, which represented the lateral mesodermal character, also showed the potential to differentiate into satellite cells at a low frequency. The satellite cell differentiation from the PDGFR-^–VEGFR-2⁺ population would be explained by the potential of lineage conversion between paraxial and lateral mesodermal progenitors in early ES cell differentiation, shown in detail in our previous study [11]. Or, more likely, the early vascular endothelial progenitor would have the potential to differentiate into myogenic population. Embryonic skeletal myogenic cells arise from somitic progenitors that are transient condensations of the paraxial mesoderm that forms on either side of the neural tube in bird and mouse embryos [44, 45]. Early avian studies concluded that satellite cells, like embryonic and fetal myoblast lineages, are of somitic origin [46]. However, a recent report has revealed that clonal myogenic precursors are present in the murine embryonic dorsal aorta [47]. Furthermore, the same study showed that myogenic precursor clones can be derived from the limbs of c-Met^–/– and Pax3^–/– mutants, which lack appendicular musculature because of the absence of migratory myoblasts of somatic origin. A second study demonstrated that wild-type mouse embryonic mesoangioblasts, a class of vessel-associated stem cells, morphologically and functionally repair the dystrophic muscles in adult immunocompetent -sarcoglycan (-SG)-null mice by intra-arterial transplantation [36]. These findings suggest that some population of cells in the embryonic vasculature has myogenic capacity, and this capacity may cause the satellite cell differentiation from the ES cell-derived lateral mesodermal progenitors.

Our system for differentiation of mesodermal progenitors without gene manipulation would be suitable for use in therapeutic applications. Gene transfection is one of the advantages of ES cell-based engraftment; however, it will be a hurdle for transplantation to patients. Of course, many improvements are necessary to overcome the low frequency of engraftment and the heterogeneity of the population; our conventional differentiation system would be suitable for clinical use because of its simplified procedure, without gene manipulation. This study shows the process of ES cell-based muscle regeneration: first, ES cell-derived mesodermal progenitor cells differentiate into satellite cells in vivo without forming a teratoma, and second, these cells generate myofibers by fusion with host myofibers. This knowledge could be used as a foundation for the development of stem cell therapies for the repair of diseased and damaged muscles.

	CONCLUSION

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

 
We address an issue of myogenic potential of ES cell-derived mesodermal progenitors in vivo in this study. Several recent studies have reported the potential of the ES cell-derived progenitors for skeletal muscle regeneration. However, the process of the engraftment of ES cell-derived progenitor cells to impaired muscle is still unclear. Here, we demonstrated the process of regeneration: ES cell-derived mesodermal progenitors initially differentiated into satellite cells and then generated the myofibers by fusion with host-damaged muscle. This study presents three important findings: (a) paraxial mesodermal progenitor cells derived from ES cells can differentiate into functional muscle satellite cells in injured muscle. (b) ES cell-derived satellite cells contribute to repair of the injured muscle by fusing with host damaged myofibers. (c) ES cell-derived mesodermal progenitor cells have the potential to generate functional muscle stem cells in vivo without inducing or suppressing gene manipulation.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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

 
We thank Drs. M. Haneda and Y. Ishida for technical support. We also thank M. Tanaka in the Division for Medical Research Engineering, Nagoya University Graduate School of Medicine for maintenance of the FACS instrument and confocal microscopy.

	FOOTNOTES

 
Author contributions: H.S.: conception and design, provision of study material or patients, collection and/or assembly of data, data analysis and interpretation, manuscript writing; Y.O.: conception and design; Y.I. and N.N.: provision of study material or patients, collection and/or assembly of data; K.I.: conception and design, financial support, administrative support, final approval of manuscript.

	REFERENCES

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

 

1. Nishikawa S, Jakt LM, Era T. Embryonic stem-cell culture as a tool for developmental cell biology. Nat Rev Mol Cell Biol 2007;8:502–507.[CrossRef][Medline]

2. Izumi N, Era T, Akimaru H et al. Dissecting the molecular hierarchy for mesendoderm differentiation through a combination of embryonic stem cell culture and RNA interference. STEM CELLS 2007;25:1664–1674.[Abstract/Free Full Text]

3. Takashima Y, Era T, Nakao K et al. Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 2007;129:1377–1388.[CrossRef][Medline]

4. Kawasaki H, Mizuseki K, Nishikawa S et al. Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000;28:31–40.[CrossRef][Medline]

5. Tada S, Era T, Furusawa C et al. Characterization of mesendoderm: A diverging point of the definitive endoderm and mesoderm in embryonic stem cell differentiation culture. Development 2005;132:4363–4374.[Abstract/Free Full Text]

6. Nishikawa SI, Nishikawa S, Hirashima M et al. Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 1998;125:1747–1757.[Abstract]

7. Hardy K, Carthew P, Handyside AH et al. Extragonadal teratocarcinoma derived from embryonal stem cells in chimaeric mice. J Pathol 1990;160:71–76.[CrossRef][Medline]

8. Bhagavati S, Xu W. Generation of skeletal muscle from transplanted embryonic stem cells in dystrophic mice. Biochem Biophys Res Commun 2005;333:644–649.[CrossRef][Medline]

9. Barberi T, Bradbury M, Dincer Z et al. Derivation of engraftable skeletal myoblasts from human embryonic stem cells. Nat Med 2007;13:642–648.[CrossRef][Medline]

10. Christ B, Ordahl CP. Early stages of chick somite development. Anat Embryol 1995;191:381–396.[CrossRef][Medline]

11. Sakurai H, Era T, Jakt LM et al. In vitro modeling of paraxial and lateral mesoderm differentiation reveals early reversibility. STEM CELLS 2006;24:575–586.[Abstract/Free Full Text]

12. Kataoka H, Takakura N, Nishikawa S et al. Expressions of PDGF receptor alpha, c-Kit and Flk1 genes clustering in mouse chromosome 5 define distinct subsets of nascent mesodermal cells. Dev Growth Differ 1997;39:729–740.[CrossRef][Medline]

13. Yamashita J, Itoh H, Hirashima M et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature 2000;408:92–96.[CrossRef][Medline]

14. Wang L, Li L, Shojaei F et al. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 2004;21:31–41.[CrossRef][Medline]

15. Nakayama N, Duryea D, Manoukian R et al. Macroscopic cartilage formation with embryonic stem-cell-derived mesodermal progenitor cells. J Cell Sci 2003;116:2015–2028.[Abstract/Free Full Text]

16. Chargé SB, Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004;84:209–238.[Abstract/Free Full Text]

17. Sherwood RI, Christensen JL, Conboy IM et al. Isolation of adult mouse myogenic progenitors: Functional heterogeneity of cells within and engrafting skeletal muscle. Cell 2004;119:543–554.[CrossRef][Medline]

18. Montarras D, Morgan J, Collins C et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005;309:2064–2067.[Abstract/Free Full Text]

19. Darabi R, Gehlbach K, Bachoo RM et al. Functional skeletal muscle regeneration from differentiating embryonic stem cells. Nat Med 2008;14:134–143.[CrossRef][Medline]

20. Goulding M, Lumsden A, Paquette AJ. Regulation of Pax-3 expression in the dermomyotome and its role in muscle development. Development 1994;120:957–971.[Abstract]

21. Takakura N, Yoshida H, Kunisada T et al. Involvement of platelet-derived growth factor receptor-alpha in hair canal formation. J Invest Dermatol 1996;107:770–777.[CrossRef][Medline]

22. Yablonka-Reuveni Z, Rudnicki MA, Rivera AJ et al. The transition from proliferation to differentiation is delayed in satellite cells from mice lacking MyoD. Dev Biol 1999;210:440–455.[CrossRef][Medline]

23. Ciruna BG, Rossant J. Expression of the T-box gene Eomesodermin during early mouse development. Mech Dev 1999;81:199–203.[CrossRef][Medline]

24. Wilkinson DG, Bhatt S, Herrmann BG. Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 1990;343:657–659.[CrossRef][Medline]

25. Yoshikawa T, Piao Y, Zhong J et al. High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization. Gene Expr Patterns 2006;6:213–224.[CrossRef][Medline]

26. Rosner MH, Vigano MA, Ozato K et al. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 1990;345:686–692.[CrossRef][Medline]

27. Campion DR. The muscle satellite cell: A review. Int Rev Cytol 1984;87:225–251.[Medline]

28. Hall-Craggs EC, Seyan HS. Histochemical changes in innervated and denervated skeletal muscle fibers following treatment with bupivacaine (marcain). Exp Neurol 1975;46:345–354.[CrossRef][Medline]

29. Seale P, Sabourin LA, Girgis-Gabardo A et al. Pax7 is required for the specification of myogenic satellite cells. Cell 2000;102:777–786.[CrossRef][Medline]

30. Beauchamp JR, Heslop L, Yu DS et al. Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 2000;151:1221–1234.[Abstract/Free Full Text]

31. Chamberlain JS, Pearlman JA, Muzny DM et al. Expression of the murine Duchenne muscular dystrophy gene in muscle and brain. Science 1988;239:1416–1418.[Abstract/Free Full Text]

32. Encut I, Cioloca L, Liciu F et al. A model for inducing tolerance to RS rat tumor in mice. Transplantation 1970;10:339–340.[CrossRef][Medline]

33. Brüner K, Opitz HG, Kolsch E. The thymus as primary site for antigen-specific T suppressor cells in neonatally induced tolerance to bovine serum albumin. Immunobiology 1982;162:221–228.[Medline]

34. Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123–134.[Abstract/Free Full Text]

35. Lee JY, Qu-Petersen Z, Cao B et al. Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing. J Cell Biol 2000;150:1085–1100.[Abstract/Free Full Text]

36. Sampaolesi M, Torrente Y, Innocenzi A et al. Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts. Science 2003;301:487–492.[Abstract/Free Full Text]

37. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676.[CrossRef][Medline]

38. Yoon JK, Moon RT, Wold B. The bHLH class protein pMesogenin1 can specify paraxial mesoderm phenotypes. Dev Biol 2000;222:376–391.[CrossRef][Medline]

39. Saga Y, Hata N, Koseki H et al. Mesp2: A novel mouse gene expressed in the presegmented mesoderm and essential for segmentation initiation. Genes Dev 1997;11:1827–1839.[Abstract/Free Full Text]

40. Chapman DL, Papaioannou VE. Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 1998;391:695–697.[CrossRef][Medline]

41. Jostes B, Walther C, Gruss P. The murine paired box gene, Pax7, is expressed specifically during the development of the nervous and muscular system. Mech Dev 1990;33:27–37.[CrossRef][Medline]

42. Arnold HH, Braun T. The role of Myf-5 in somitogenesis and the development of skeletal muscles in vertebrates. J Cell Sci 1993;104:957–960.[Medline]

43. Sassoon D, Lyons G, Wright WE et al. Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 1989;341:303–307.[CrossRef][Medline]

44. Pourquié O. Vertebrate somitogenesis. Annu Rev Cell Dev Biol 2001;17:311–350.[CrossRef][Medline]

45. Summerbell D, Rigby PW. Transcriptional regulation during somitogenesis. Curr Top Dev Biol 2000;48:301–318.[Medline]

46. Armand O, Boutineau AM, Mauger A et al. Origin of satellite cells in avian skeletal muscles. Arch Anat Microsc Morphol Exp 1983;72:163–181.[Medline]

47. De Angelis L, Berghella L, Coletta M et al. Skeletal myogenic progenitors originating from embryonic dorsal aorta coexpress endothelial and myogenic markers and contribute to postnatal muscle growth and regeneration. J Cell Biol 1999;147:869–878.[Abstract/Free Full Text]

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