of dental mesenchyme-marker genes (Msx1, Pax9 and Lhx7) increased in DMSO-EC cells, compared with those in EC cells. Furthermore, the structurally correct ...
Pluripotent stem cells developed into regenerated tooth by organ germ method in combination with tooth germ-derived epithelium Ritsuko Morita1, 2, Kazuhisa Nakao1, 2, Miho Ogawa1, 2, Yasumitsu Saji1, 2, Kentaro Ishida1, 2 & Takashi Tsuji1, 2 1
Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, JAPAN 2 Tissue Engineering Research Center, Tokyo University of Science, Noda, Chiba, 278-8510, JAPAN to all cell types in the body. Recently, it was reported that whereby gene transfer of four transcription factors, somatic fibroblasts could be transfomed into pluripotent stem cell [4-5]. Therefore, pluripotent stem cells are increasingly expected
1. ABSTRACT The regenerative therapy ultimately aims to develop bioengineered organs that can replace lost or damaged organs following disease, injury or aging. Recently, we showed that the artificial organ germ, which generates a structurally correct tooth, could be reconstituted by in vitro cell manipulation (Nature Methods 4, 227-230, 2007). In this study, we revealed that neural crest-like cells, which were differentiated from pluripotent stem cells such as embryonal carcinoma (EC) cells, could develop the regenerated tooth by organ germ method with tooth germ epithelium. EC cells were stimulated with dimethyl sulfoxide (DMSO) and differentiated cells were isolated by cell sorting as DMSO-EC cells. We found that the expressions of pluripotent stem cell marker genes (Oct3/4 and Nanog) could not be detected in DMSO-EC cells. In contrast, the expressions of neural crest-marker genes (Pax3 and Slug) and of dental mesenchyme-marker genes (Msx1, Pax9 and Lhx7) increased in DMSO-EC cells, compared with those in EC cells. Furthermore, the structurally correct tooth can be generated by combining DMSO-EC cells and tooth germ epithelium, both in vitro and in vivo. Our current results indicated the possibility that pluripotent stem cells are applicable as a candidate of cell sources to develop of a bioengineered organ germ for the organ replacement in the future regenerative therapy.
as a useful cell source for regenerative medicine. Teeth develop from tooth germs induced by reciprocal interactions between oral epithelium and its underlying ectomesenchyme which is populated by migratory cranial neural crest (CNC) cells. It is known that snail homolog 2 (Slug) and paired box gene 3 (Pax3) are necessary for migration of neural crest cells [6]. Spatial delimination of mesenchymal marker genes, such as msh-like 1 (Msx1), paired box gene 9 (Pax9), patched homolog 1 (Ptc1), participate in compartmentalization of odontogenic mesenchyme in postmigratory CNC cells [7-8]. Msx1 and Pax9 also have essential roles in tooth development, because tooth development is arrested by knock out of these genes [9-11]. Finally, dental mesenchyme differentiates into odontoblast, which will form dentin, dental pulp, and periodontal tissue. Here, we reported that pluripotent stem cells, such as embryonal carcinoma (EC) cells, could differentiate into dental mesenchyme via CNC cells. EC-derived mesenchymal cells could generate a structurally correct tooth by organ germ method in vitro and in vivo.
2. INTRODUCTION
3. MATERIALS AND METHODS
For regenerative medicine, it has been expected to create fully functioning bioengineered organs that can replace lost or damaged organs following disease, injury or aging [1-2]. We previously established a bioengineered organ germ method, which reconstitute the artificial organ germ by in vitro cell manipulation. The artificial tooth germ could develop in a tooth cavity with natural structure, showing penetration of blood vessels and nerve fibers. These findings increase the potential for the use of bioengineered organ replacement in future regenerative therapies [3]. As next issue of organ regeneration, it is necessary to find the available cell source capable to form the transplantable organ. Pluripotent stem cells can give rise
3.1 Animals C57BL/6 mice (Japan SLC, Inc., Tokyo, Japan) were used. Mouse care and handling conformed to the NIH guidelines for animal research. All experimental protocols were approved by the Tokyo University of Science Animal Care and Use Committee. 3.2 Differentiation and purification of DMSO-EC cells in culture Mouse EC cells derived from 129 were purchased from RIKEN Bioresource Center (Tokyo, Japan). EC cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Wako, Osaka, JAPAN) containing 10% Fetal
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bovine serum (FBS; Hyclone, UT, USA), 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma, St. Louis, MO) and 100 nM β2-Mercaptoethanol (Gibco, NY, USA). For differentiation, EC cells (1.0×105 cells/ml) were cultured in DMEM, containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and Dimethyl sulfoxide (DMSO; Sigma) for 24 hours. Then, the cells were continuously cultured in medium without DMSO for 5-7 days. Differentiated fraction were sorted as DMSO-EC cells using Epics ALTRA (Beckman Coulter, Inc., CA, USA). DMSO-EC-derived clones were obtained by limiting dilution.
Dental epithelium was then injected into a gel drop and combined with the DMSO-EC cell aggregate to enable direct cell contact by a fine tungsten needle. 3.5 Organ cultures Recombinant explants were cultured for for 14 days as described [3]. 3.6 SRC assays Recombinant explants were transplanted into a subrenal capsule (SRC) in mice for 14 days as described [3]. 3.7 Tissue preparation and immunohistochemistry The tissues were removed and immersed in 4% paraformaldehyde (nacalai tesque, inc., Kyoto, Japan) in PBS(-). After fixation, the tissues were decalcified in 4.5% EDTA (pH 7.4) for a day at 4°C, and embedded in paraffin. The sections were stained with hematoxylin-eosin (HE staining), observed using an Axio Imager A1 (Carl Zeiss, Jena, Germany) with an AxioCAM MRc5 (Zeiss) and processed with AxioVision software (Zeiss).
3.3 Gene expression analysis Total RNA was extracted from cultured cells with TRIzol Reagent (Invitrogen Co., CA, USA). First strand cDNA, primed with pd (N)6 random primers, was synthesized using ReverTra Ace (Toyobo, Osaka, JAPAN). Quantitative real-time polymerase chain reaction (PCR) was performed using SYBR Premix Ex Taq (Takara Bio, Shiga, Japan) according to the manufacture’s protocol. The gene expression levels were measured using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, CA, USA). mRNA copy numbers were calculated from serially diluted standard curves generated from a cDNA template. Data was analyzed using ABI Prism 7000 SDS Software, and all expression levels were normalized to the β-Actin control. The sequence for the primers used in this study were as follows: Oct3/4, 5’-ATTGAGAACCGTGTGAGGTGGA-3’ (sense), 5’-GCGCCGGTTACAGAACCATAC-3’ (anti sense); Nanog, 5’-CAAGGGTCTGCTACTGAGATGCT-3’ (sense), 5’-ATCAGGGCTGCCTTGAAGAG-3’ (anti sense); Slug, 5’-GACCCTGGCTGCTTCAAGGA-3’ (sense), 5’-GACCCTGGCTGCTTCAAGGA-3’ (anti sense); Pax3, 5’-CAAGCTGGAGCCAATCAACTG-3’ (sense), 5’-GCGGTGGGAGGGAATCC-3’ (anti sense); Msx1, 5’-CCAGCCCTATAGAAAGCAAGGA-3’ (sense), 5’-CCCCTCAGAGCAATGCTTTG-3’ (anti sense); Pax9, 5’-GGCCAGGCACCGAATG-3’ (sense), 5’-GCCATGCTGGATGCTGAGA-3’ (anti sense); Lhx7, 5’- AAGTGGAGAACGGTAATGGGATTAG-3’ (sense), 5’-GCTTTGGATGATTGACGTCTTG-3’ (anti sense).
4. RESULTS AND DISCUSSION We first stimulated mEC cells by DMSO to induce differentiation toward neural crest cells. After 5-7 days of stimulation, we harvested the cells and purified differentiated fraction (DMSO-EC cells) by cell sorting (Fig. 1a). We then replated them in tissue culture flasks and expanded in DMEM containing 10% FBS. DMSO-EC cells have fibroblastic morphology, which are different from that of EC cells (Fig. 1b-d). To characterize DMSO-EC cells, we used real-time PCR to analyze gene expressions for pluripotent stem cells (Oct3/4 and Nanog); for neural crest cells (Slug and Pax3); for dental mesenchymal cells (Msx1, Pax9 and Lhx7). The undifferentiated EC cells expressed pluripotent stem cell marker genes, but did not express genes for neural crest cells and dental mesenchymal cells (Fig. 2). After the differentiation, the expression of pluripotent stem cell marker genes decreased, and the expression of genes for neural crest cells and dental mesenchymal cells increased in DMSO-EC cells, compared with those in EC cells (Fig. 2). These profiles of gene expression were similar in the cloned cells which were obtained by limiting dilution. In order to assess the potential of DMSO-EC cells to differentiate toward tooth germ mesenchyme, we next reconstituted the artificial tooth germ by combining DMSO-EC cells and dental epithelial tissue by a bioengineered organ germ method (Fig. 3a). Within a day
3.4 Organ germ method Bioengineered tooth germ was reconstituted as previously described [3]. Incisor epithelial tissues were separated from the mandibles of E14.5 mice. To prepare bioengineered tooth germ, the high-density DMSO-EC cells were injected (0.2-0.3 μl) using 0.1-10 μl pipette tip (Molecular Bio Products, San Diego, CA) into a 30 μl gel drop of Cellmatrix type I-A (Nitta gelatin, Osaka, Japan).
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a Culture for 5-7 days Cultured cells after differentiation
EC cells
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as formation of embryoid bodies and stimulating by many cytokines and drugs [12-18]. We thought that it is adequate to differentiate EC cells into neural crest cells, because tooth germ mesenchyme is originated from neural crest cells. So far, there were some reports that neural crest cells were induced from ES cells and EC cells using retinoic acids (RA) or some cytokines [16-18]. In the present study, we obtained the neural crest like cells, which have odontogenic potential, from EC cells using DMSO. The gene expression profiles of DMSO-EC cells indicated that DMSO-EC cells seem to have properties of both neural crest cells and tooth germ mesenchyme. We think that our organ germ method will enable us to study the cellular differentiation process in tooth development. To reconstitute the organs in vitro, it has been thought to need not only the development of cell manipuration technology, but also the exploration of available cell source [1-2]. It has reported that adult bone-marrow-derived adherent cells reconstituted with dental epithelial tissue of 10 days embryo could form tooth in subrenal capsule [19]. And Tomooka et al. reported a tooth can be generated by combining oral epithelial cells and tooth germ mesenchymal tissue by organ germ method [20]. ES cells can differentiate into many cell types in vitro, and transplantation of these cells led to recovery in an animal models of disease [12-14]. However, there have been no reports showing that the organogenesis from pluripotent stem cells was achieved, although the use of pluripotent stem cells in cell transplantation therapy is widely reported. It may be due to no useful method that enable to reconstruct the organs. Our organ germ method can lead to the strong epithelial-mesenchymal interactions by the high-cell density and correct cell compartmentalization between epithelial and mesenchymal cells, and thus enabled us to assess whether the cells have the odontogenic potencial to form tooth [3, 20]. We think that this study could provide
Cell Sorting of differentiated cells Sorted cells
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Figure 1 Differentiation of mouse embryonal carcinoma (EC) cells into neural crest cells. (a) Purification of differentiated cells from EC cells. Undifferentiated EC cells cultured with DMSO for a day to induce neural crest cells. differentiated population was purified by flowcytometry sorting. (b-d) Phase-contrast images of EC cells (b), cultured cells after differentiation (c) and sorted cells (d). Scale bars, 200 μm.
Oct 3/4
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of organ culture, we observed formation of a tooth germ with cell-to-cell compaction. At 3 days of organ culture, epithelium started to elongate toward the aggregated DMSO-EC cells (Fig. 3b). After 10 days, the reconstituted tooth germ developed a tooth which has natural tooth components such as odontoblasts, dentin, ameloblasts, enamel and dental pulp (Fig. 3c). We also found that this occurred with a frequency of 51.3±8.8% and DMSO-EC cell-derived clones have odontogenic potencial as equal as DMSO-EC cells (Data not shown). Then, we transplanted this reconstituted tooth germ into subrenal capsules (SRC) in mice. Over 2-week period, we found that this explant could generate a structurally correct tooth, which has natural tooth components and blood vessels. But, alveolar bone and periodontal ligaments were not observed around a generated tooth (Fig. 3d). And, we did not observed teratoma formation in any of the organ cultures and the transplantation (data not shown). This study indicated that DMSO-EC cells which derived from pluripotent stem cells is neural crest like cells by their gene expression profiles. And we showed that DMSO-EC cells can generate into a tooth by organ germ method. Our findings encourage the future development of organ replacement by pluripotent stem cells in regenerative therapy. To differentiate pluripotent stem cells to target cells, researchers therefore have examined various methods such
DM EC SO -EC Cl on e# Clo 1 ne #2
Differentiation with DMSO for a day
Figure 2 Characterization of DMSO-EC cells. The expression of pluripotent stem cells (Oct3/4 and Nanog), for neural crest cells (Slug and Pax3) and for mesenchyme of tooth germ (Msx1, Pax9 and Lhx7) were analyzed by real-time PCR in EC cells and DMSO-EC cells. mRNA expression levels were normalized relative to beta-actin.
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a E14.5 Tooth germ
Epithelium Tissue a
High-density reconstituted tooth germ
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Transplantation into SRC assay for 14 days Organ culture for 14 days
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Figure 3 Analysis of tooth generation by bioengineered tooth germ derived from tooth germ epitherium and DMSO-EC cells. (a) Schematic diagram of the bioengineering system used for the generation of a reconstituted tooth germ. The epithelial tissues were isolated from incisor tooth germ of ED14.5 mice. The bioengineered tooth germ was then reconstituted using incisor epitherium and DMSO-EC cells. The explants were either transplanted under a subrenal capsule in mouse or were continuously cultured. (b) Phase-contrast images of bioengineered incisor tooth germ after 1 d (top) and 3 d (bottom) of organ culture. E, epithelium; M, DMSO-EC cells. Scale bar, 200 μm. (c) Histological analysis of the reconstituted tooth germ for 14 d of organ culture. am, ameloblasts; PD, pre-dentin; D, dentin; E, enamel; od, odontoblasts; p, pulp cells. Scale bars, 200 μm. (d) Histological analysis of the reconstituted tooth germ under a subrenal capsule for 14 days after transplantation. am, ameloblasts; bv, blood vessels; PD, pre-dentin; D, dentin; E, enamel; od, odontoblasts; p, pulp cells. Scale bars, 100 μm.
the first evidence of an organ reconstruction from pluripotent stem cells. Although we showed here that tooth organogenesis is possible using the mesenchymal cells derived from pluripotent stem cells, further studies will be needed to induce both mesenchymal and epitherial cells. These studies will provide important insights into the development of stem cell biology and the organ replacement strategy for regenerative medicine.
[3] Nakao K, Morita R et al. The development of a bio engineered organ germ method. Nature Methods 4: 227-230, 2007. [4] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126: 663-676, 2006 [5] Okita K, Ichisaka T et al. Generation of germline-competent induced pluripotent stem cells. Nature 448: 313-317, 2007. [6] Jones N, Trainor P. Role of Morphogens in Neural Crest Cell Determination. J Neurobiol 64, 388-404, 2005. [7] Cobourne MT, Miletich I et al. Restriction of sonic hedgehog signalling during early tooth development. Development 131: 2875-85, 2004. [8] Aberg T, Wang XP et al. Runx2 mediates FGF signaling from epithelium to mesenchyme during tooth morphogenesis. Dev Biol 270: 76-93, 2004. [9] Satokata I, Maas R. Msx1 deficient mice exhibit cleft palate and abnormalities of craniofacial and tooth development. Nat Genet 6:348-56, 1994. [10] Bei M, Maas R. FGFs and BMP4 induce both Msx1-independent and Msx1-dependent signaling pathways in early tooth development. Development 125: 4325-4333, 1998. [11] Peters H, Neubuser A et al. Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit
5. ACKNOWLEDGEMENT This work was supported in part by an “Academic Frontier” Project for Private Universities (to Y.T. and T.T.): a matching fund subsidy from MEXT Japan (2003-2007). This work was also supported in part by a Grant-in Aid for Scientific Research in Priority Areas (No. 18048039) to T.T. from MEXT Japan. 6. REFERENCE [1] Brockes JP, Kumar A. Appendage Regeneration in Adult Vertebrates and Implications for Regenerative Medicine. Science 310: 1919-1923, 2005. [2] Sharpe PT, Young CS. Test-tube teeth. Sci Am 293: 34-41, 2005.
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craniofacial and limb abnormalities. Genes Dev 12: 2735-2747, 1998. [12] Kim JH, Auerbach JM. Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease. Nature 418: 50-56, 2002. [13] Soto-Gutiérrez A, Kobayashi N et al. Reversal of mouse hepatic failure using an implanted liver-assist device containing ES cell-derived hepatocytes. Nat Biotechnol 24: 1412-1419, 2006. [14] Gouon-Evans V, Boussemart L et al. BMP-4 is required for hepatic specification of mouse embryonic stem cell-derived definitive endoderm. Nat Biotechnol 24: 1402-1411, 2006. [15] McBurney MW, Jones-villeneuve EM. Control of muscle and neuronal differentiation in a cultured embryonal carcinoma cell line. Nature 299:165-167, 1982. [16] Okada Y, Shimazaki T et al. Retinoic-acid-concentration-dependent acquisition of neural cell identity during in vitro differentiation of mouse embryonic stem cells. Dev Biol 275: 124-42, 2004. [17] Motohashi T, Aoki H et al. Multipotent cell fate of neural crest-like cells derived from embryonic stem cells. Stem cells 25: 402-410, 2007. [18] Mizuseki K, Sakamoto T et al. Generation of neural crest-derived peripheral neurons and floor plate cells from mouse and primate embryonic stem cells. Proc Natl Acad Sci USA 100:5828-5833, 2003. [19] Ohazama A, Modino SA et al. Stem-cell based tissue engineering of murine teeth. J Dent Res 83: 518-522, 2004. [20] Komine A, Suenaga M et al. Tooth regeneration from newly established cell line from a molar tooth germ epitherium. Biochem Biophys Res Commun 355: 758-763, 2007.
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Pluripotent stem cells developed into regenerated tooth by organ germ method in combination with tooth germ-derived epithelium 1
Ritsuko Morita1,2, Kazuhisa Nakao1,2, Miho Ogawa1,2, Yasumitsu Saji1,2, Kentaro Ishida1,2& Takashi Tsuji1,2 Faculty of Industrial Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510, JAPAN 2 Tissue Engineering Research Center, Tokyo University of Science, Noda, Chiba, 278-8510, JAPAN
The regenerative therapy ultimately aims to develop bioengineered organs that can replace lost or damaged organs following disease, injury or aging. Recently, we showed that the artificial organ germ, which generates a structurally correct tooth, could be reconstituted by in vitro cell manipulation (Nature Methods 4, 227-230, 2007). In this study, we revealed that neural crest-like cells, which were differentiated from pluripotent stem cells such as embryonal carcinoma (EC) cells, could develop the regenerated tooth by organ germ method with tooth germ epithelium. EC cells were stimulated with dimethyl sulfoxide (DMSO) and differentiated cells were isolated by cell sorting as DMSO-EC cells. We found that the expressions of pluripotent stem cell marker genes (Oct3/4 and Nanog) could not be detected in DMSO-EC cells. In contrast, the expressions of neural crest-marker genes (Pax3 and Slug) and of dental mesenchyme-marker genes (Msx1, Pax9 and Lhx7) increased in DMSO-EC cells, compared with those in EC cells. Furthermore, the structurally correct tooth can be generated by combining DMSO-EC cells and tooth germ epithelium, both in vitro and in vivo. Our current results indicated the possibility that pluripotent stem cells are applicable as a candidate of cell sources to develop of a bioengineered organ germ for the organ replacement in the future regenerative therapy.
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