Cell Biochem Biophys DOI 10.1007/s12013-014-9964-x
ORIGINAL PAPER
Ecto-Mesenchymal Stem Cells from Facial Process: Potential for Muscle Regeneration Xin Nie • Yongjun Xing • Manjin Deng Li Gang • Rui Liu • Yongjie Zhang • Xiujie Wen
•
Ó Springer Science+Business Media New York 2014
Abstract Ecto-mesenchymal stem cells (EMSCs) originate from the cranial neural crest and participate in the formation of tooth, salivary, and muscle in early development stage. The transplantation of EMSCs, a potential source of myoblast stem cell, might improve muscle regeneration. The purpose of this study was to explore whether EMSCs have the potential to differentiate and display a myogenic phenotype in vitro the in vitro. Here, we characterized the EMSCs isolated from the facial process, and p75 ? EMSCs were collected by a FACS calibur flow cytometer. In vitro, p75 ? EMSCs induced by DMSO can accumulate and fuse into multinucleated myotubes and further differentiate into the skeletal muscle cells in form of cell sheet. Functional myoblast phenotypes of p75 ? EMSCs were found in vivo model of muscle injury. The remarkable ability of stem cells to regenerate skeletal muscle indicated their potential role in the cell therapy and tissue engineering of the skeletal muscle. Keywords Ecto-mesenchymal stem cells Muscle regeneration Cell sheet Transplantation
Xin Nie and Yongjun Xing have contributed equally on this study. X. Nie Y. Xing M. Deng L. Gang R. Liu X. Wen (&) Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, Chongqing 400042, People’s Republic of China e-mail: [email protected] Y. Zhang Center for Tissue Engineering, Qin Du Stomatological College, Fourth Military Medical University, Xi’an, People’s Republic of China
Introduction Functional loss of skeletal muscle tissue due to trauma, vascular injuries, tumor resection, or degenerative muscle disorders, such as muscular dystrophy, represents a significant clinical problem with few solutions [1]. Stem cellbased therapies for repair and regeneration of muscular tissues may provide alternative therapeutic solutions for a number of muscle diseases [2]. For stem cell transplant, a sufficient number of cells would be the key of success. Satellite cells, located beneath the muscle fiber basal lamina, are considered as the main myogenic progenitors in skeletal muscles. Under physiological conditions, these quiescent cells start to expand upon activation into myoblasts to replenish the loss myocytes. However, due to the scarcity of satellite cells and the difficulties in isolating them, options of using freshly isolated satellite cells as a source for the stem cell therapy have been largely disregarded. To find more effective stem cell-based therapies to treat muscular diseases, researchers explored other sources of stem cells for muscle regeneration and repair [3]. Numerous sources of stem cells or progenitor cells for myogenesis and restoration of muscle function have been reported in previous literatures including bone marrow-derived stem cells, adipose stem cells, muscle-derived stem cells, neural crest cells, and embryonic vessel-derived stem cells [4–6]. Although the stem cells from those sources provide some benefit, the incorporation into skeletal muscle to form a functional organ was far from satisfactory. Notably, some types of stem cell from mesenchymal tissue share a common lineage of being derived from neural crest cells, and possess generic ecto-mesenchymal properties, including expression of marker genes and differentiation into mesenchymal cell lineages [7–9]. Developmental
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study about distribution of pluripotent neural crest cells in the embryo reveals that migration of ecto-mesenchymal cells from the cranial neural crest to the facial process gives rise to progeny of different phenotypes including neurogenic, myogenic, chondrogenic, osteogenic, and odontogenic cells, which develop into many of the oral and maxillary tissues with the exception of the enamel organ [10]. The fate of the ecto-mesenchyme appears to be determined mainly by intrinsic genetic programming, but also under the influence of extracellular signals in the local environment. Our previous studies have shown that EMSCs originated from neural crest stem cells have the properties of pluripotent stem cells, and the lineage of those stem cells may be directed by specific molecules [11]. Biochemical and biomechanical signals may trigger the proliferation and differentiation of EMSCs during early development and in regeneration after injury or disease. To date, there are few reports of successful generation or reparation of skeletal muscle using EMSCs. The aim of this study was to evaluate the possibility of p75 ? EMSCs acting as a role of myogenic lineages stem cell and to explore the feasibility of reconstruction of skeletal muscle tissue by cell sheet for the treatment of muscle defect.
Methods Experimental Animals Embryonic BALB/C mice of 11.5 days and adult BALB/C mice of 2–3 months were provided by the Third Military Medical University Animal Laboratory. The experimental protocol was reviewed and approved by the Ethics Committee and is in accordance with the guidelines for the ethical treatment of animals established by the International Council for Laboratory Animal Science (ICLAS). Cell Isolation and Cell Culture Embryonic facial processes from E11.5 BALB/C mice were dissected, minced into fine pieces, digested with 1 % trypsin/ l mM EDTA solution (Sigma, USA), filtered through 75 lm mesh, centrifuged and cultured in Dulbecco Modified Eagles Medium/Ham’s F12 (DMEM/F12) (Gibco, USA), and supplemented with 10 % fetal bovine serum (FBS, Gibco, USA). Cells were sub-cultured under identical conditions and then used in experiments after the third passage. Selection and Identification of p75 ? EMSCs p75 ? EMSCs cells were selected by FACS. The above EMSCs were harvested and washed twice in sterile
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phosphate-buffered saline (PBS). With anti-rat p75-FITC (1:20, Abcam Inc., Cambridge, MA) added, the cells were incubated with 1 % BSA for 60 min at 37 °C in a 5 % CO2 humidified incubator. After washed with PBS, p75 ? stem cells were collected using a FACS calibur flow cytometer (BD Bioscience, San Jose, CA, USA). Flow cytometric analysis was performed to detect the expression of the cell surface markers in p75 ? EMSCs as previously described [12]. Briefly, approximately 5 9 105 cells were harvested and incubated overnight with primary antibodies: anti-rat p75 (1:100), mouse anti-rat CD29, CD44, CD90, CD105, CD146 (1:100, Santa Cruz, CA, USA), and mouse anti-rat Stro-1 (IgM) (1:100, Abcam Inc., Cambridge, MA) according to the manufacturer’s protocol. The corresponding secondary antibodies including antimouse IgG-FITC (1:100) and anti-mouse IgM-FITC (1:100) were added. Cells were analyzed with a FACS calibur flow cytometer. Immunocytochemical identification of EMSC with cell surface makers of p75 ? and Stro-1 was carried out as follows: cells at the third passage were harvested and incubated in 1 % BSA-PBS containing rabbit anti-rat p75 (1:20, Abcam Inc., Cambridge, MA) and Stro-1 antibody (1:100, Abcam Inc., Cambridge, MA) for 30 min on ice. After washing with PBS, then incubated with corresponding FITC-conjugated secondary antibodies for another 30 min, cells were counter-stained with DAPI (nuclear marker) and observed under the confocal laser scanning microscope. Myoblast Induction of p75 ? EMSCs p75 ? EMSCs cells were plated at a density of 1 9 105 cells/well in 6-well plates and allowed to grow until confluence. DMSO (Sigma, USA) was added at a final concentration of 1 %. The medium containing DMSO was renewed every 2 days for a total of 8 days of treatment. Confirmation of the Phenotype by Western Blot and Electron Microscope Cells after DMSO induction were rinsed twice with PBS and lysed for western blot. Protein concentration was estimated by the BCA assay (Pierce, USA). Equal amounts of protein extracts were fractionated by 10 % sodium dodecyl sulfate–polyacrylamide gels, and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad, USA). The membranes were incubated with mouse monoclonal antibodies against myogenin (1:1,000; Sigma, USA) and myosin heavy chain (1:5,000; Sigma, USA). For ultra microstructural analysis, cells after 1- and 2-week culture were collected, centrifuged, and fixed in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)
Cell Biochem Biophys
overnight at 4 °C. These cells were then rinsed in 0.1 M cacodylate buffer, postfixed in 1 % OsO4 for 1 h at 4 °C, dehydrated in a graded ethanol series, and embedded in Epon to obtain ultrathin sections on grids. Grids were stained in 4 % uranyl acetate in 50 % methanol for 25 min, followed by Reynold’s lead citrate for 5 min, and examined with a transmission electron microscope (JEM-2000, JPN). Fabrication of Cell Sheet for Tissue-Engineered Muscle Tissue-engineered differenced EMSCs sheets were fabricated ex vivo by culturing harvested cells for 2 weeks on surfaces with collagen membranes. In brief, scaffolds were prepared by 3 % collagen solutions freeze-dried to form at 4 °C for 8 h, and 20 °C for 4 h, and then sterilized with gamma irradiation. For cell seeding, the collagen membranes were rehydrated with 4 ml of standard growth medium for 30 min. 1 9 106 cells were washed twice with PBS and trypsinized. The prepared cell suspension was dripped on the surface of collagen membranes and incubated overnight. The culture dishes were rinsed twice with PBS to remove the unattached cells. After 1 week of culture with DMSO, cell sheet was taken out, rinsed, and stained with hematoxylin and eosin. Some cell sheets were further analyzed by scanning electron microscopy (SEM) as follows: specimens were fixed in 1 % buffered glutaraldehyde and 0.1 % buffered formaldehyde for 1 and 24 h, respectively, then dehydrated with a graded ethanol series, critical point dried, sputter coated with platinum, and observed by SEM (S520, Hitachi, Japan). Transplantation of Cells Sheet in the Mouse Model of Muscle Defect BALB/C mice of 2–3 month old were anesthetized by intraperitoneal injection of 30 mg/kg amylobarbitone sodium. The model of muscle injury was made by removal of part of the quadriceps in leg. Cells sheets harvested after 1 week of induced myogenesis were fresh rolled into spindle, transplanted into defect site. The site of each transplant was marked with suture. Two weeks after the transplantation, all mice were euthanized with excessive amylobarbitone sodium. Transplanted constructs were then carefully separated from surrounding fibrous capsule for histological and RT-PCR analysis. Myogenic Differentiation In vivo Assessed by Histology and RT-PCR Transplanted tissues were fixed in 4 % paraformaldehyde, paraffin embedded, then sectioned and stained with hematoxylin and eosin.
To further confirm myogenic differentiation of transplanted tissue, RT-PCR assay was also performed for MyoD expression as previously described [13]. In brief, total RNA was extracted from all the specimens. About 1 lg of total RNA was reversed transcribed by reverse transcriptase, and PCR amplification of target message RNA was performed. PCR amplification was performed for 30 cycles in a thermal cycler, with initial denaturation at 94 °C for 30 s, subsequent annealing at 60 °C for 60 s, and extension at 72 °C for 90 s. The PCR products were visualized on a 1.5 % agarose gel containing 5 mg/ml ethidium bromide.
Results Isolation and Cell Characteristics of p75 ? EMSCs The initial culture of cells before selection was inhomogeneous containing a majority of fibroblast-like cells and some spindle-shaped cells similar to what was described in cultures of EMSC. After selection, p75 ? EMSCs presented a homogeneous fibroblast-like morphology (Fig. 1a, b). The cell surface makers of p75 and Stro-1 were observed under the confocal laser scanning microscope. High expression of proteins was detected, indicating that those cells in embryo mesenchymal tissue were originated from cranial neural crest (Fig. 1c, d). In live cell sorting, p75 ? EMSCs and Stro-1 EMSCs accounted for 31.37 and 89.2 % respectively; the strong expressions of CD29, CD44, CD90, and CD105, which were associated with stem cell origin, suggested that these cells are the earliest progenitors and the most rapidly replicating cells (Fig. 2). Myoblast Induction of p75 ? EMSCs EMSCs are known to have a potential to differentiate spontaneously into smooth muscle cell phenotype. After confluence, cells became columnar and enlarged, tending to align themselves in straight parallel lines (Fig. 3a). After 7 day of treatment with DMSO, profound changes in morphology features more flatted the cell at high density. Some of the cells stopped dividing at later stage, and myotube fusion was observed through continuous culture (Fig. 3b). Myogenin and myosin protein constitutively expressing in high levels were confirmed by western blot (Fig. 3c). Ultramicrostructural observation showed that cells after DMSO induction differentiated into skeletal myoblasts that fused to form myotubes. The changes of the differentiated cells were almost similar with the normal skeletal muscle cell. The nucleoli of some cells were enlarged and pale. The myofilament and sarcomere of the
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Fig. 1 Isolation and cell characteristics of p75 ? EMSCs: The characteristics of the EMSC before and after sorted and collected on a FACS calibur flow cytometry (a, b). Immunocytochemical identity of EMSC with cell surface makers of p75 ? and Stro-1(c, d)
Fig. 2 The expression of the cell surface markers Stro-1, p75, CD29, CD44, CD90, and CD105 in EMSCs with flow cytometric analysis
majority were well oriented, and vacuolarization was also seen in some mitochondria (Fig. 3d). Fabrication of Cell Sheet for Tissue-Engineered Muscle Collagen membranes were used in our experiment as extracellular matrix material to support cell sheet (Fig. 4a).
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During the organ culture, the collagen membrane was completely absorbed, but the adhesion molecules on the cell surface and cell–cell interactions remain intact (Fig. 4b). No enzymatic treatments, such as trypsin digestion, were necessary to harvest the cell sheet. It can easily detach from the culture dish. SEM analysis demonstrated cells aggregate on the surface of the collagen material,
Cell Biochem Biophys
Fig. 3 Morphological changes of p75 ? EMSCs toward myoblast during differentiation: Typical morphologic changes appeared after confluence (a). Myotube fusion observed in some cells through
continuous culture (b). Dramatical induction of myogenin and myosin assessed by western blot analysis (c). Ultramicrostructure of myofilament and sarcomere were obvious in the differentiated cells (d)
allowing them to contact with each other, grow, and become confluence. Complete formation of cell sheet occurred within 1 week (Fig. 4c). Cells after 2 weeks of culture became columnar and enlarged, tending to align themselves in straight parallel lines (Fig. 4d).
challenge for orthopedic surgeons. Skeletal muscle stem cell therapy is based on the regenerative properties of the myoblasts and their potential for proliferation and differentiation. Early studies have demonstrated that skeletal muscle regeneration and reparation by mesenchymal stem cells for degenerative muscle diseases or skeletal muscle injuries [14]. We recently discovered another type of MSCs in the embryonic facial process termed ecto-mesenchy stem cells [11, 15]. EMSCs originated from neural crest stem cells possess a multipotent and self-renewing capacity [16]. The key issue of using EMSCs transplantation is to identify cells that can differentiate into myoblasts and participate in the regeneration of muscle. Since EMSCs are not well known, it is important to characterize and investigate the possible differentiation of EMSCs in terms of myogenesis before applying in muscle injury repair. In our study, EMSCs sorted by p75 can differentiate into skeletal muscle cells either cultured with DMSO in vitro or transplanted into a model of muscle defect. In particular, spontaneous fusion of p75 ? EMSCs and subsequent formation of myotube-like structure was observed when cultured in cell sheet technique at high cell density. In a muscular defect model, p75 ? EMSCs were able to
Myogenic Differentiation In vivo A gross view of graft showed that the histological features of the regenerative tissue were similar to those of the native muscle, indicating the transplanted cell sheet facilitated the regeneration of skeletal muscle fibers (Fig. 5a). The HE staining demonstrated that newly formed muscle tissue around the defect site 2 weeks after transplantation, forming several mosaic multinuclear muscle fibers (Fig. 5b). This finding was further confirmed by PCR result. The PCR signal of MyoD from transplanted tissue was detected (Fig. 5c).
Discussion Injury of skeletal muscle is common in traffic accidents and warfare. Failure of reparation poses as a common clinical
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Fig. 4 Fabrication of cell sheet for tissue-engineered muscle: Morphological characteristics of collagen membrane (a). Formation of the cell sheet after continuous culture (HE staining) (b). Complete
formation of cell sheet occurred within 1-week (SEM) (c). Cells became columnar and enlarged, tending to align themselves in straight parallel lines after 2-week culture (SEM) (d)
Fig. 5 Myogenic differentiation of p75 ? EMSCs at the model of muscle defect. Gross observations of graft after 2-week transplantation (a). Histological analysis of tissue-engineered muscle with HE
staining (b). The PCR signal of MyoD observed from transplanted tissue. Exemplary data from 3 mice (c)
facilitate the regeneration of skeletal muscle fibers following direct transplantation into the affected muscle. The isolation methods and the techniques used to evaluate whether the isolated cells are indeed the stem cells are keys for investigators. The histological evidence suggests that p75 ? EMSCs exist in the early stage of embryo development and contributes to the development of many
tissues, including the tooth, muscle, salivary and nerve. A population of self-renewing EMSCs was first isolated by our group [11, 17]. The easy availability from low maintenance requirements of cell culture, and their capacity to differentiate into odontoblasts, osteoblasts, adipocytes, and chondrocytes has been demonstrated in previous studies [18–20]. Recently, other groups reported that different
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types of EMSCs can be isolated from developing and developed tissues [21–23]. In vitro, the stem cell characteristic and p75 marker were further confirmed by our study; moreover, p75 ? EMSCs display the characteristics of myogenesis and the activation of the gene expression encoding the muscle special marker myogenin and myosin. Other in vitro study of skeletal muscle also showed a similar morphologic and functional appearance in the myogenic regulation of stem cell [13]. This finding was further confirmed by immunohistochemical. Proper spatial and temporal expression of molecule markers such as myogenin, myoD, and myosin protein during the differentiation of myoblast is critical for successful myogenesis. These muscle tissue specific proteins are required for the initial formation of skeletal muscle [24, 25]. The expression of these proteins, which is apparent in p75 ? EMSCs treated with DMSO (an efficient myogenical differentiating solvent for embryo stem cell), is considered to be a phenotypic marker for myoblasts [26]. Previous study demonstrates that the presence of myogenin and myosin was not detected in mesenchymal stem cell, which agrees with our observation that there is no detectable expression in the initial migratory neural crest-like cell [27]. However, the expression of the muscle specific proteins increase as the cells were treated with DMSO, spread into a flattened pattern, and skeletal muscle fibers appear. The stem cell therapy as a treatment option for skeletal muscle disorders, particularly muscular defect, has become an imminent possibility as progress has been made in elucidating the developmental capabilities of both muscular progenitor cells and mesenchymal stem cells [28, 29]. A variety of approaches to graft tissue engineering have shown to be promising, including seeding cells onto natural and synthetic scaffolds or by culturing cell sheets without exogenous scaffolds [30]. The major restriction of scaffoldbased designs is the flexibility and tendency to shrink of muscle. If transplantation of EMSCs without scaffold could enhance repair at the injury site, then scaffold-free cellbased treatments could be employed, representing an ideal approach to the treatment of muscle injury. Therefore, we have developed a cell transplantation method in which p75 ? EMSCs are cultured and lifted as a cell sheet structure. No special equipment, such as temperature-based culture dishes and thermoresponsive films, is necessary as EMSCs are cultured only on materials constructed with extracellular matrix, and lifted by a scraper as a single cell sheet structure. This suggests that using a direct cell seeding approach, it is possible to develop completely biologic cellderived tissue, and it may also possible to modulate matrix synthesis by optimizing cell culture conditions. At present, there are few effective therapeutic approaches for individuals with muscle injury. The mechanism of
stem cell gives rise to the fully differentiated muscle tissue, and the process of muscle regeneration still remains to be clarified. In this study, we suggest that p75 ? EMSCs may provide an interesting cell model for regenerative medicine. We further characterized the stem cell properties of p75 ? EMSCs using morphometric, immunocytofluorescent, and ultramicrostructural analysis. The data in vitro and in vivo suggested that tissue-engineered cell sheet fused into multinucleated myotubes and incorporate into the defect site. Further functional study of transplanted skeletal muscle will be necessary to show the possible clinical application. Acknowledgments This work was supported by the Grants from National Natural Science Foundation of China (Grant No. 31070863, 81271097) and from the Natural Science Foundation Project of Chongqing, China (Grants No. CSTC2010BB5161 and CSTC2011BA5013).
References 1. Vilquin, J. T., Catelain, C., & Vauchez, K. (2011). Cell therapy for muscular dystrophies: Advances and challenges. Current Opinion in Organ Transplantation, 16(6), 640–649. 2. Bhagavati, S. (2008). Stem cell based therapy for skeletal muscle diseases. Current Stem Cell Research & Therapy, 3(3), 219–228. 3. Collins, C. A., Olsen, I., Zammit, P. S., et al. (2005). Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell, 122, 289–301. 4. Peault, B., Rudnicki, M., Torrente, Y., et al. (2007). Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Molecular Therapy, 15, 867–877. 5. De, B. C., Dell’Accio, F., Vandenabeele, F., et al. (2003). Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane. Journal of Cell Biology, 160, 909–918. 6. Chan, J., Waddington, S. N., O’Donoghue, K., et al. (2007). Widespread distribution and muscle differentiation of human fetal mesenchymal stem cells after intrauterine transplantation in dystrophic mdx mouse. Stem Cells, 25, 875–884. 7. Zhang, J., Duan, X., Zhang, H., et al. (2006). Isolation of neural crest-derived stem cells from rat embryonic mandibular processes. Biology of the Cell, 98(10), 567–575. 8. Degistirici, O., Jaquiery, C., Scho¨nebeck, B., Siemonsmeier, J., Go¨tz, W., Martin, I., et al. (2008). Defining properties of neural crest-derived progenitor cells from the apex of human developing tooth. Tissue Engineering Part A, 14(2), 317–330. 9. Davies, L. C., Locke, M., Webb, R. D., et al. (2010). A multipotent neural crest derived progenitor cell population is resident within the oral mucosa lamina propria. Stem Cells Development, 19(6), 819–830. 10. Bhattacherjee, V., Mukhopadhyay, P., Singh, S., Johnson, C., Philipose, J. T., Warner, C. P., et al. (2007). Neural crest and mesoderm lineage-dependent gene expression in orofacial development. Differentiation, 75(5), 463–477. 11. Deng, M. J., Jin, Y., Shi, J. N., et al. (2004). Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch. Tissue Engineering, 10, 1597–1606. 12. Wen, X., Nie, X., Zhang, L., Liu, L., & Deng, M. (2011). Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro. Biochemistry Biophysics Research Communication, 409(3), 583–589.
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Cell Biochem Biophys 13. Qin, R. F., Mao, T. Q., Gu, X. M., Hu, K. J., Liu, Y. P., Chen, J. W., et al. (2007). Regulation of skeletal muscle differentiation in fibroblasts by exogenous MyoD gene in vitro and in vivo. Molecular and Cellular Biochemistry, 302(1–2), 233–239. 14. Conboy, I. M., & Rando, T. A. (2005). Aging, stem cells and tissue regeneration: Lessons from muscle. Cell Cycle, 4, 407–410. 15. Nie, X., Zhang, Y. J., Tian, W. D., Jiang, M., Dong, R., Chen, J. W., et al. (2007). Improvement of peripheral nerve regeneration by a tissue-engineered nerve filled with ectomesenchymal stem cells. International Journal of Oral and Maxillofacial Surgery, 36(1), 32–38. 16. Hu, S., Shen, X., Zhang, R., et al. (2008). Development of rat antigen-presenting cells from pluripotent ecto-mesenchymal stem cells in vitro and in vivo. Molecular Immunology, 45(14), 3818–3826. 17. Deng, M., Shi, J., Smith, A. J., & Jin, Y. (2005). Effects of transforming growth factor beta1 (TGF beta-1) and dentin noncollagenous proteins (DNCP) on human embryonic ectomesenchymal cells in a three-dimensional culture system. Archives of Oral Biology, 50(11), 937–945. 18. Chai, Y., & Maxson, R. E, Jr. (2006). Recent advances in craniofacial morphogenesis. Developmental Dynamics, 235(9), 2353–2375. 19. Zhang, Y., Wang, S., Song, Y., Han, J., Chai, Y., & Chen, Y. (2003). Timing of odontogenic neural crest cell migration and tooth-forming capability in mice. Developmental Dynamics, 226(4), 713–718. 20. Jiang, H. B., Tian, W. D., Liu, L. K., & Xu, Y. (2008). In vitro odontoblast-like cell differentiation of cranial neural crest cells induced by fibroblast growth factor 8 and dentin non-collagen proteins. Cell Biology International, 32(6), 671–678. 21. Paino, F., Ricci, G., De Rosa, A., et al. (2010). Ecto-mesenchymal stem cells from dental pulp are committed to differentiate into active melanocytes. Eur Cell Mater, 20, 295–305.
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22. Yan, Z., Lin, Y., Jiao, X., et al. (2006). Characterization of ectomesenchymal cells isolated from the first branchial arch during multilineage differentiation. Cells Tissues Organs, 183(3), 123–132. 23. Lin, Y., Yan, Z., Liu, L., et al. (2006). Proliferation and pluripotency potential of ectomesenchymal cells derived from first branchial arch. Cell Proliferation, 39(2), 79–92. 24. Gabillard, J. C., Sabin, N., & Paboeuf, G. (2010). In vitro characterization of proliferation and differentiation of trout satellite cells. Cell and Tissue Research, 342(3), 471–477. 25. Morosetti, R., Mirabella, M., Gliubizzi, C., et al. (2006). MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle. Proceedings of the National Academy of Sciences of the United States of America, 103, 16995–17000. 26. Nishimura, M., Mikura, M., Hirasaka, K., Okumura, Y., Nikawa, T., Kawano, Y., et al. (2008). Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells. Journal of Biochemistry, 144(6), 717–724. 27. Jeffery, W. R., Strickler, A. G., & Yamamoto, Y. (2004). Migratory neural crest-like cells form body pigmentation in a urochordate embryo. Nature, 431, 696–699. 28. Montarras, D., Morgan, J., Collins, C., et al. (2005). Direct isolation of satellite cells for skeletal muscle regeneration. Science, 309, 2064–2067. 29. Lecourt, S., Marolleau, J. P., Fromigue, O., et al. (2010). Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro. Experimental Cell Research, 316(15), 2513–2526. 30. Zhao, J., Liu, L., Wei, J., et al. (2012). A novel strategy to engineer small-diameter vascular grafts from marrow-derived mesenchymal stem cells. Artificial Organs, 36(1), 93–101.
ORIGINAL PAPER
Ecto-Mesenchymal Stem Cells from Facial Process: Potential for Muscle Regeneration Xin Nie • Yongjun Xing • Manjin Deng Li Gang • Rui Liu • Yongjie Zhang • Xiujie Wen
•
Ó Springer Science+Business Media New York 2014
Abstract Ecto-mesenchymal stem cells (EMSCs) originate from the cranial neural crest and participate in the formation of tooth, salivary, and muscle in early development stage. The transplantation of EMSCs, a potential source of myoblast stem cell, might improve muscle regeneration. The purpose of this study was to explore whether EMSCs have the potential to differentiate and display a myogenic phenotype in vitro the in vitro. Here, we characterized the EMSCs isolated from the facial process, and p75 ? EMSCs were collected by a FACS calibur flow cytometer. In vitro, p75 ? EMSCs induced by DMSO can accumulate and fuse into multinucleated myotubes and further differentiate into the skeletal muscle cells in form of cell sheet. Functional myoblast phenotypes of p75 ? EMSCs were found in vivo model of muscle injury. The remarkable ability of stem cells to regenerate skeletal muscle indicated their potential role in the cell therapy and tissue engineering of the skeletal muscle. Keywords Ecto-mesenchymal stem cells Muscle regeneration Cell sheet Transplantation
Xin Nie and Yongjun Xing have contributed equally on this study. X. Nie Y. Xing M. Deng L. Gang R. Liu X. Wen (&) Department of Stomatology, Daping Hospital & Research Institute of Surgery, Third Military Medical University, Chongqing 400042, People’s Republic of China e-mail: [email protected] Y. Zhang Center for Tissue Engineering, Qin Du Stomatological College, Fourth Military Medical University, Xi’an, People’s Republic of China
Introduction Functional loss of skeletal muscle tissue due to trauma, vascular injuries, tumor resection, or degenerative muscle disorders, such as muscular dystrophy, represents a significant clinical problem with few solutions [1]. Stem cellbased therapies for repair and regeneration of muscular tissues may provide alternative therapeutic solutions for a number of muscle diseases [2]. For stem cell transplant, a sufficient number of cells would be the key of success. Satellite cells, located beneath the muscle fiber basal lamina, are considered as the main myogenic progenitors in skeletal muscles. Under physiological conditions, these quiescent cells start to expand upon activation into myoblasts to replenish the loss myocytes. However, due to the scarcity of satellite cells and the difficulties in isolating them, options of using freshly isolated satellite cells as a source for the stem cell therapy have been largely disregarded. To find more effective stem cell-based therapies to treat muscular diseases, researchers explored other sources of stem cells for muscle regeneration and repair [3]. Numerous sources of stem cells or progenitor cells for myogenesis and restoration of muscle function have been reported in previous literatures including bone marrow-derived stem cells, adipose stem cells, muscle-derived stem cells, neural crest cells, and embryonic vessel-derived stem cells [4–6]. Although the stem cells from those sources provide some benefit, the incorporation into skeletal muscle to form a functional organ was far from satisfactory. Notably, some types of stem cell from mesenchymal tissue share a common lineage of being derived from neural crest cells, and possess generic ecto-mesenchymal properties, including expression of marker genes and differentiation into mesenchymal cell lineages [7–9]. Developmental
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study about distribution of pluripotent neural crest cells in the embryo reveals that migration of ecto-mesenchymal cells from the cranial neural crest to the facial process gives rise to progeny of different phenotypes including neurogenic, myogenic, chondrogenic, osteogenic, and odontogenic cells, which develop into many of the oral and maxillary tissues with the exception of the enamel organ [10]. The fate of the ecto-mesenchyme appears to be determined mainly by intrinsic genetic programming, but also under the influence of extracellular signals in the local environment. Our previous studies have shown that EMSCs originated from neural crest stem cells have the properties of pluripotent stem cells, and the lineage of those stem cells may be directed by specific molecules [11]. Biochemical and biomechanical signals may trigger the proliferation and differentiation of EMSCs during early development and in regeneration after injury or disease. To date, there are few reports of successful generation or reparation of skeletal muscle using EMSCs. The aim of this study was to evaluate the possibility of p75 ? EMSCs acting as a role of myogenic lineages stem cell and to explore the feasibility of reconstruction of skeletal muscle tissue by cell sheet for the treatment of muscle defect.
Methods Experimental Animals Embryonic BALB/C mice of 11.5 days and adult BALB/C mice of 2–3 months were provided by the Third Military Medical University Animal Laboratory. The experimental protocol was reviewed and approved by the Ethics Committee and is in accordance with the guidelines for the ethical treatment of animals established by the International Council for Laboratory Animal Science (ICLAS). Cell Isolation and Cell Culture Embryonic facial processes from E11.5 BALB/C mice were dissected, minced into fine pieces, digested with 1 % trypsin/ l mM EDTA solution (Sigma, USA), filtered through 75 lm mesh, centrifuged and cultured in Dulbecco Modified Eagles Medium/Ham’s F12 (DMEM/F12) (Gibco, USA), and supplemented with 10 % fetal bovine serum (FBS, Gibco, USA). Cells were sub-cultured under identical conditions and then used in experiments after the third passage. Selection and Identification of p75 ? EMSCs p75 ? EMSCs cells were selected by FACS. The above EMSCs were harvested and washed twice in sterile
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phosphate-buffered saline (PBS). With anti-rat p75-FITC (1:20, Abcam Inc., Cambridge, MA) added, the cells were incubated with 1 % BSA for 60 min at 37 °C in a 5 % CO2 humidified incubator. After washed with PBS, p75 ? stem cells were collected using a FACS calibur flow cytometer (BD Bioscience, San Jose, CA, USA). Flow cytometric analysis was performed to detect the expression of the cell surface markers in p75 ? EMSCs as previously described [12]. Briefly, approximately 5 9 105 cells were harvested and incubated overnight with primary antibodies: anti-rat p75 (1:100), mouse anti-rat CD29, CD44, CD90, CD105, CD146 (1:100, Santa Cruz, CA, USA), and mouse anti-rat Stro-1 (IgM) (1:100, Abcam Inc., Cambridge, MA) according to the manufacturer’s protocol. The corresponding secondary antibodies including antimouse IgG-FITC (1:100) and anti-mouse IgM-FITC (1:100) were added. Cells were analyzed with a FACS calibur flow cytometer. Immunocytochemical identification of EMSC with cell surface makers of p75 ? and Stro-1 was carried out as follows: cells at the third passage were harvested and incubated in 1 % BSA-PBS containing rabbit anti-rat p75 (1:20, Abcam Inc., Cambridge, MA) and Stro-1 antibody (1:100, Abcam Inc., Cambridge, MA) for 30 min on ice. After washing with PBS, then incubated with corresponding FITC-conjugated secondary antibodies for another 30 min, cells were counter-stained with DAPI (nuclear marker) and observed under the confocal laser scanning microscope. Myoblast Induction of p75 ? EMSCs p75 ? EMSCs cells were plated at a density of 1 9 105 cells/well in 6-well plates and allowed to grow until confluence. DMSO (Sigma, USA) was added at a final concentration of 1 %. The medium containing DMSO was renewed every 2 days for a total of 8 days of treatment. Confirmation of the Phenotype by Western Blot and Electron Microscope Cells after DMSO induction were rinsed twice with PBS and lysed for western blot. Protein concentration was estimated by the BCA assay (Pierce, USA). Equal amounts of protein extracts were fractionated by 10 % sodium dodecyl sulfate–polyacrylamide gels, and electrophoretically transferred to a nitrocellulose membrane (Bio-Rad, USA). The membranes were incubated with mouse monoclonal antibodies against myogenin (1:1,000; Sigma, USA) and myosin heavy chain (1:5,000; Sigma, USA). For ultra microstructural analysis, cells after 1- and 2-week culture were collected, centrifuged, and fixed in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)
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overnight at 4 °C. These cells were then rinsed in 0.1 M cacodylate buffer, postfixed in 1 % OsO4 for 1 h at 4 °C, dehydrated in a graded ethanol series, and embedded in Epon to obtain ultrathin sections on grids. Grids were stained in 4 % uranyl acetate in 50 % methanol for 25 min, followed by Reynold’s lead citrate for 5 min, and examined with a transmission electron microscope (JEM-2000, JPN). Fabrication of Cell Sheet for Tissue-Engineered Muscle Tissue-engineered differenced EMSCs sheets were fabricated ex vivo by culturing harvested cells for 2 weeks on surfaces with collagen membranes. In brief, scaffolds were prepared by 3 % collagen solutions freeze-dried to form at 4 °C for 8 h, and 20 °C for 4 h, and then sterilized with gamma irradiation. For cell seeding, the collagen membranes were rehydrated with 4 ml of standard growth medium for 30 min. 1 9 106 cells were washed twice with PBS and trypsinized. The prepared cell suspension was dripped on the surface of collagen membranes and incubated overnight. The culture dishes were rinsed twice with PBS to remove the unattached cells. After 1 week of culture with DMSO, cell sheet was taken out, rinsed, and stained with hematoxylin and eosin. Some cell sheets were further analyzed by scanning electron microscopy (SEM) as follows: specimens were fixed in 1 % buffered glutaraldehyde and 0.1 % buffered formaldehyde for 1 and 24 h, respectively, then dehydrated with a graded ethanol series, critical point dried, sputter coated with platinum, and observed by SEM (S520, Hitachi, Japan). Transplantation of Cells Sheet in the Mouse Model of Muscle Defect BALB/C mice of 2–3 month old were anesthetized by intraperitoneal injection of 30 mg/kg amylobarbitone sodium. The model of muscle injury was made by removal of part of the quadriceps in leg. Cells sheets harvested after 1 week of induced myogenesis were fresh rolled into spindle, transplanted into defect site. The site of each transplant was marked with suture. Two weeks after the transplantation, all mice were euthanized with excessive amylobarbitone sodium. Transplanted constructs were then carefully separated from surrounding fibrous capsule for histological and RT-PCR analysis. Myogenic Differentiation In vivo Assessed by Histology and RT-PCR Transplanted tissues were fixed in 4 % paraformaldehyde, paraffin embedded, then sectioned and stained with hematoxylin and eosin.
To further confirm myogenic differentiation of transplanted tissue, RT-PCR assay was also performed for MyoD expression as previously described [13]. In brief, total RNA was extracted from all the specimens. About 1 lg of total RNA was reversed transcribed by reverse transcriptase, and PCR amplification of target message RNA was performed. PCR amplification was performed for 30 cycles in a thermal cycler, with initial denaturation at 94 °C for 30 s, subsequent annealing at 60 °C for 60 s, and extension at 72 °C for 90 s. The PCR products were visualized on a 1.5 % agarose gel containing 5 mg/ml ethidium bromide.
Results Isolation and Cell Characteristics of p75 ? EMSCs The initial culture of cells before selection was inhomogeneous containing a majority of fibroblast-like cells and some spindle-shaped cells similar to what was described in cultures of EMSC. After selection, p75 ? EMSCs presented a homogeneous fibroblast-like morphology (Fig. 1a, b). The cell surface makers of p75 and Stro-1 were observed under the confocal laser scanning microscope. High expression of proteins was detected, indicating that those cells in embryo mesenchymal tissue were originated from cranial neural crest (Fig. 1c, d). In live cell sorting, p75 ? EMSCs and Stro-1 EMSCs accounted for 31.37 and 89.2 % respectively; the strong expressions of CD29, CD44, CD90, and CD105, which were associated with stem cell origin, suggested that these cells are the earliest progenitors and the most rapidly replicating cells (Fig. 2). Myoblast Induction of p75 ? EMSCs EMSCs are known to have a potential to differentiate spontaneously into smooth muscle cell phenotype. After confluence, cells became columnar and enlarged, tending to align themselves in straight parallel lines (Fig. 3a). After 7 day of treatment with DMSO, profound changes in morphology features more flatted the cell at high density. Some of the cells stopped dividing at later stage, and myotube fusion was observed through continuous culture (Fig. 3b). Myogenin and myosin protein constitutively expressing in high levels were confirmed by western blot (Fig. 3c). Ultramicrostructural observation showed that cells after DMSO induction differentiated into skeletal myoblasts that fused to form myotubes. The changes of the differentiated cells were almost similar with the normal skeletal muscle cell. The nucleoli of some cells were enlarged and pale. The myofilament and sarcomere of the
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Fig. 1 Isolation and cell characteristics of p75 ? EMSCs: The characteristics of the EMSC before and after sorted and collected on a FACS calibur flow cytometry (a, b). Immunocytochemical identity of EMSC with cell surface makers of p75 ? and Stro-1(c, d)
Fig. 2 The expression of the cell surface markers Stro-1, p75, CD29, CD44, CD90, and CD105 in EMSCs with flow cytometric analysis
majority were well oriented, and vacuolarization was also seen in some mitochondria (Fig. 3d). Fabrication of Cell Sheet for Tissue-Engineered Muscle Collagen membranes were used in our experiment as extracellular matrix material to support cell sheet (Fig. 4a).
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During the organ culture, the collagen membrane was completely absorbed, but the adhesion molecules on the cell surface and cell–cell interactions remain intact (Fig. 4b). No enzymatic treatments, such as trypsin digestion, were necessary to harvest the cell sheet. It can easily detach from the culture dish. SEM analysis demonstrated cells aggregate on the surface of the collagen material,
Cell Biochem Biophys
Fig. 3 Morphological changes of p75 ? EMSCs toward myoblast during differentiation: Typical morphologic changes appeared after confluence (a). Myotube fusion observed in some cells through
continuous culture (b). Dramatical induction of myogenin and myosin assessed by western blot analysis (c). Ultramicrostructure of myofilament and sarcomere were obvious in the differentiated cells (d)
allowing them to contact with each other, grow, and become confluence. Complete formation of cell sheet occurred within 1 week (Fig. 4c). Cells after 2 weeks of culture became columnar and enlarged, tending to align themselves in straight parallel lines (Fig. 4d).
challenge for orthopedic surgeons. Skeletal muscle stem cell therapy is based on the regenerative properties of the myoblasts and their potential for proliferation and differentiation. Early studies have demonstrated that skeletal muscle regeneration and reparation by mesenchymal stem cells for degenerative muscle diseases or skeletal muscle injuries [14]. We recently discovered another type of MSCs in the embryonic facial process termed ecto-mesenchy stem cells [11, 15]. EMSCs originated from neural crest stem cells possess a multipotent and self-renewing capacity [16]. The key issue of using EMSCs transplantation is to identify cells that can differentiate into myoblasts and participate in the regeneration of muscle. Since EMSCs are not well known, it is important to characterize and investigate the possible differentiation of EMSCs in terms of myogenesis before applying in muscle injury repair. In our study, EMSCs sorted by p75 can differentiate into skeletal muscle cells either cultured with DMSO in vitro or transplanted into a model of muscle defect. In particular, spontaneous fusion of p75 ? EMSCs and subsequent formation of myotube-like structure was observed when cultured in cell sheet technique at high cell density. In a muscular defect model, p75 ? EMSCs were able to
Myogenic Differentiation In vivo A gross view of graft showed that the histological features of the regenerative tissue were similar to those of the native muscle, indicating the transplanted cell sheet facilitated the regeneration of skeletal muscle fibers (Fig. 5a). The HE staining demonstrated that newly formed muscle tissue around the defect site 2 weeks after transplantation, forming several mosaic multinuclear muscle fibers (Fig. 5b). This finding was further confirmed by PCR result. The PCR signal of MyoD from transplanted tissue was detected (Fig. 5c).
Discussion Injury of skeletal muscle is common in traffic accidents and warfare. Failure of reparation poses as a common clinical
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Fig. 4 Fabrication of cell sheet for tissue-engineered muscle: Morphological characteristics of collagen membrane (a). Formation of the cell sheet after continuous culture (HE staining) (b). Complete
formation of cell sheet occurred within 1-week (SEM) (c). Cells became columnar and enlarged, tending to align themselves in straight parallel lines after 2-week culture (SEM) (d)
Fig. 5 Myogenic differentiation of p75 ? EMSCs at the model of muscle defect. Gross observations of graft after 2-week transplantation (a). Histological analysis of tissue-engineered muscle with HE
staining (b). The PCR signal of MyoD observed from transplanted tissue. Exemplary data from 3 mice (c)
facilitate the regeneration of skeletal muscle fibers following direct transplantation into the affected muscle. The isolation methods and the techniques used to evaluate whether the isolated cells are indeed the stem cells are keys for investigators. The histological evidence suggests that p75 ? EMSCs exist in the early stage of embryo development and contributes to the development of many
tissues, including the tooth, muscle, salivary and nerve. A population of self-renewing EMSCs was first isolated by our group [11, 17]. The easy availability from low maintenance requirements of cell culture, and their capacity to differentiate into odontoblasts, osteoblasts, adipocytes, and chondrocytes has been demonstrated in previous studies [18–20]. Recently, other groups reported that different
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types of EMSCs can be isolated from developing and developed tissues [21–23]. In vitro, the stem cell characteristic and p75 marker were further confirmed by our study; moreover, p75 ? EMSCs display the characteristics of myogenesis and the activation of the gene expression encoding the muscle special marker myogenin and myosin. Other in vitro study of skeletal muscle also showed a similar morphologic and functional appearance in the myogenic regulation of stem cell [13]. This finding was further confirmed by immunohistochemical. Proper spatial and temporal expression of molecule markers such as myogenin, myoD, and myosin protein during the differentiation of myoblast is critical for successful myogenesis. These muscle tissue specific proteins are required for the initial formation of skeletal muscle [24, 25]. The expression of these proteins, which is apparent in p75 ? EMSCs treated with DMSO (an efficient myogenical differentiating solvent for embryo stem cell), is considered to be a phenotypic marker for myoblasts [26]. Previous study demonstrates that the presence of myogenin and myosin was not detected in mesenchymal stem cell, which agrees with our observation that there is no detectable expression in the initial migratory neural crest-like cell [27]. However, the expression of the muscle specific proteins increase as the cells were treated with DMSO, spread into a flattened pattern, and skeletal muscle fibers appear. The stem cell therapy as a treatment option for skeletal muscle disorders, particularly muscular defect, has become an imminent possibility as progress has been made in elucidating the developmental capabilities of both muscular progenitor cells and mesenchymal stem cells [28, 29]. A variety of approaches to graft tissue engineering have shown to be promising, including seeding cells onto natural and synthetic scaffolds or by culturing cell sheets without exogenous scaffolds [30]. The major restriction of scaffoldbased designs is the flexibility and tendency to shrink of muscle. If transplantation of EMSCs without scaffold could enhance repair at the injury site, then scaffold-free cellbased treatments could be employed, representing an ideal approach to the treatment of muscle injury. Therefore, we have developed a cell transplantation method in which p75 ? EMSCs are cultured and lifted as a cell sheet structure. No special equipment, such as temperature-based culture dishes and thermoresponsive films, is necessary as EMSCs are cultured only on materials constructed with extracellular matrix, and lifted by a scraper as a single cell sheet structure. This suggests that using a direct cell seeding approach, it is possible to develop completely biologic cellderived tissue, and it may also possible to modulate matrix synthesis by optimizing cell culture conditions. At present, there are few effective therapeutic approaches for individuals with muscle injury. The mechanism of
stem cell gives rise to the fully differentiated muscle tissue, and the process of muscle regeneration still remains to be clarified. In this study, we suggest that p75 ? EMSCs may provide an interesting cell model for regenerative medicine. We further characterized the stem cell properties of p75 ? EMSCs using morphometric, immunocytofluorescent, and ultramicrostructural analysis. The data in vitro and in vivo suggested that tissue-engineered cell sheet fused into multinucleated myotubes and incorporate into the defect site. Further functional study of transplanted skeletal muscle will be necessary to show the possible clinical application. Acknowledgments This work was supported by the Grants from National Natural Science Foundation of China (Grant No. 31070863, 81271097) and from the Natural Science Foundation Project of Chongqing, China (Grants No. CSTC2010BB5161 and CSTC2011BA5013).
References 1. Vilquin, J. T., Catelain, C., & Vauchez, K. (2011). Cell therapy for muscular dystrophies: Advances and challenges. Current Opinion in Organ Transplantation, 16(6), 640–649. 2. Bhagavati, S. (2008). Stem cell based therapy for skeletal muscle diseases. Current Stem Cell Research & Therapy, 3(3), 219–228. 3. Collins, C. A., Olsen, I., Zammit, P. S., et al. (2005). Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell, 122, 289–301. 4. Peault, B., Rudnicki, M., Torrente, Y., et al. (2007). Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Molecular Therapy, 15, 867–877. 5. De, B. C., Dell’Accio, F., Vandenabeele, F., et al. (2003). Skeletal muscle repair by adult human mesenchymal stem cells from synovial membrane. Journal of Cell Biology, 160, 909–918. 6. Chan, J., Waddington, S. N., O’Donoghue, K., et al. (2007). Widespread distribution and muscle differentiation of human fetal mesenchymal stem cells after intrauterine transplantation in dystrophic mdx mouse. Stem Cells, 25, 875–884. 7. Zhang, J., Duan, X., Zhang, H., et al. (2006). Isolation of neural crest-derived stem cells from rat embryonic mandibular processes. Biology of the Cell, 98(10), 567–575. 8. Degistirici, O., Jaquiery, C., Scho¨nebeck, B., Siemonsmeier, J., Go¨tz, W., Martin, I., et al. (2008). Defining properties of neural crest-derived progenitor cells from the apex of human developing tooth. Tissue Engineering Part A, 14(2), 317–330. 9. Davies, L. C., Locke, M., Webb, R. D., et al. (2010). A multipotent neural crest derived progenitor cell population is resident within the oral mucosa lamina propria. Stem Cells Development, 19(6), 819–830. 10. Bhattacherjee, V., Mukhopadhyay, P., Singh, S., Johnson, C., Philipose, J. T., Warner, C. P., et al. (2007). Neural crest and mesoderm lineage-dependent gene expression in orofacial development. Differentiation, 75(5), 463–477. 11. Deng, M. J., Jin, Y., Shi, J. N., et al. (2004). Multilineage differentiation of ectomesenchymal cells isolated from the first branchial arch. Tissue Engineering, 10, 1597–1606. 12. Wen, X., Nie, X., Zhang, L., Liu, L., & Deng, M. (2011). Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro. Biochemistry Biophysics Research Communication, 409(3), 583–589.
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Cell Biochem Biophys 13. Qin, R. F., Mao, T. Q., Gu, X. M., Hu, K. J., Liu, Y. P., Chen, J. W., et al. (2007). Regulation of skeletal muscle differentiation in fibroblasts by exogenous MyoD gene in vitro and in vivo. Molecular and Cellular Biochemistry, 302(1–2), 233–239. 14. Conboy, I. M., & Rando, T. A. (2005). Aging, stem cells and tissue regeneration: Lessons from muscle. Cell Cycle, 4, 407–410. 15. Nie, X., Zhang, Y. J., Tian, W. D., Jiang, M., Dong, R., Chen, J. W., et al. (2007). Improvement of peripheral nerve regeneration by a tissue-engineered nerve filled with ectomesenchymal stem cells. International Journal of Oral and Maxillofacial Surgery, 36(1), 32–38. 16. Hu, S., Shen, X., Zhang, R., et al. (2008). Development of rat antigen-presenting cells from pluripotent ecto-mesenchymal stem cells in vitro and in vivo. Molecular Immunology, 45(14), 3818–3826. 17. Deng, M., Shi, J., Smith, A. J., & Jin, Y. (2005). Effects of transforming growth factor beta1 (TGF beta-1) and dentin noncollagenous proteins (DNCP) on human embryonic ectomesenchymal cells in a three-dimensional culture system. Archives of Oral Biology, 50(11), 937–945. 18. Chai, Y., & Maxson, R. E, Jr. (2006). Recent advances in craniofacial morphogenesis. Developmental Dynamics, 235(9), 2353–2375. 19. Zhang, Y., Wang, S., Song, Y., Han, J., Chai, Y., & Chen, Y. (2003). Timing of odontogenic neural crest cell migration and tooth-forming capability in mice. Developmental Dynamics, 226(4), 713–718. 20. Jiang, H. B., Tian, W. D., Liu, L. K., & Xu, Y. (2008). In vitro odontoblast-like cell differentiation of cranial neural crest cells induced by fibroblast growth factor 8 and dentin non-collagen proteins. Cell Biology International, 32(6), 671–678. 21. Paino, F., Ricci, G., De Rosa, A., et al. (2010). Ecto-mesenchymal stem cells from dental pulp are committed to differentiate into active melanocytes. Eur Cell Mater, 20, 295–305.
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22. Yan, Z., Lin, Y., Jiao, X., et al. (2006). Characterization of ectomesenchymal cells isolated from the first branchial arch during multilineage differentiation. Cells Tissues Organs, 183(3), 123–132. 23. Lin, Y., Yan, Z., Liu, L., et al. (2006). Proliferation and pluripotency potential of ectomesenchymal cells derived from first branchial arch. Cell Proliferation, 39(2), 79–92. 24. Gabillard, J. C., Sabin, N., & Paboeuf, G. (2010). In vitro characterization of proliferation and differentiation of trout satellite cells. Cell and Tissue Research, 342(3), 471–477. 25. Morosetti, R., Mirabella, M., Gliubizzi, C., et al. (2006). MyoD expression restores defective myogenic differentiation of human mesoangioblasts from inclusion-body myositis muscle. Proceedings of the National Academy of Sciences of the United States of America, 103, 16995–17000. 26. Nishimura, M., Mikura, M., Hirasaka, K., Okumura, Y., Nikawa, T., Kawano, Y., et al. (2008). Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells. Journal of Biochemistry, 144(6), 717–724. 27. Jeffery, W. R., Strickler, A. G., & Yamamoto, Y. (2004). Migratory neural crest-like cells form body pigmentation in a urochordate embryo. Nature, 431, 696–699. 28. Montarras, D., Morgan, J., Collins, C., et al. (2005). Direct isolation of satellite cells for skeletal muscle regeneration. Science, 309, 2064–2067. 29. Lecourt, S., Marolleau, J. P., Fromigue, O., et al. (2010). Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro. Experimental Cell Research, 316(15), 2513–2526. 30. Zhao, J., Liu, L., Wei, J., et al. (2012). A novel strategy to engineer small-diameter vascular grafts from marrow-derived mesenchymal stem cells. Artificial Organs, 36(1), 93–101.
