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The Similarity Between Human Embryonic Stem Cell-Derived Epithelial Cells and Ameloblast-Lineage Cells

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The Similarity Between Human Embryonic Stem Cell-Derived Epithelial Cells and Ameloblast-Lineage Cells International Journal of Oral Science (2013) 5, 1–6 ß 2013 WCSS. All rights reserved 1674-2818/13 www.nature.com/ijos ORIGINAL ARTICLE The similarity between human embryonic stem cell-derived epithelial cells and ameloblast-lineage cells Li-Wei Zheng1, Logan Linthicum2, Pamela K DenBesten1 and Yan Zhang1 This study aimed to compare epithelial cells derived from human embryonic stem cells (hESCs) to human ameloblast-lineage cells (ALCs), as a way to determine their potential use as a cell source for ameloblast regeneration. Induced by various concentrations of bone morphogenetic protein 4 (BMP4), retinoic acid (RA) and lithium chloride (LiCl) for 7 days, hESCs adopted cobble-stone epithelial phenotype (hESC-derived epithelial cells (ES-ECs)) and expressed cytokeratin 14. Compared with ALCs and oral epithelial cells (OE), ES-ECs expressed amelogenesis-associated genes similar to ALCs. ES-ECs were compared with human fetal skin epithelium, human fetal oral buccal mucosal epithelial cells and human ALCs for their expression pattern of cytokeratins as well. ALCs had relatively high expression levels of cytokeratin 76, which was also found to be upregulated in ES-ECs. Based on the present study, with the similarity of gene expression with ALCs, ES-ECs are a promising potential cell source for regeneration, which are not available in erupted human teeth for regeneration of enamel. International Journal of Oral Science (2013) 5, 1–6; doi:10.1038/ijos.2013.14; published online 29 March 2013 Keywords: ameloblast; cytokeratin; dental epithelial cells; human embryonic stem cells; odontogenesis INTRODUCTION tion and matrix mineralization is initiated by secretory ameloblasts. Regenerative medicine promises novel therapies for tissue regenera- Secreted matrix proteins self-assemble to form the unique structure of tion, including the potential to replace missing teeth with bioengi- mineralized enamel. neered tissues. Tooth formation is guided by reciprocal signaling Previous studies have shown that mouse embryonic stem cells and interactions between the cranial neural crest-derived mesenchymal c-kit-positive mouse bone marrow cells can be differentiated into cells (forming dentin and pulp) and ectoderm-derived dental epithe- ameloblast-like cells.4,11–13 These studies of stem cells from mouse lial cells (forming enamel). Classic tissue recombination experiments suggest that human embryonic stem cells (hESCs) could be a viable have been used to successfully regenerate mouse, rat and pig tooth cell source for regeneration of dental epithelial cells. organs,1–6 and demonstrate that both epithelial and mesenchymal hESCsarederivedfromtheinnercellmassofblastocyst-stagehuman components are required for tooth organ regeneration. Dental embryos.hESCscanself-renewandgiverisetoalltypesofcellinthe mesenchymal stem cells from either human exfoliated deciduous human body.14 When induced with bone morphogenetic protein 4 or adult dental pulp are available for dentin/pulp complex bioen- (BMP4) and a-retinoic acid (RA), hESCs differentiate toward cells with gineering,7–10 while a dental epithelial component does not exist in an epithelial morphology.15 Cytokeratin expression is a characteristic of the erupted human tooth for enamel tissue regeneration. Therefore, epithelial cells, and the cytokeratin expression pattern of hESC-derived identification of alternative cell sources for dental epithelial cell epithelial cells (ES-ECs) has not been characterized and compared with regeneration remains a challenge. the cytokeratin expression patterns of other epithelia. Mammalian dental epithelial cells are unique in that they are Cytokeratins (CKs) are the typical intermediate filament proteins of derived from oral ectoderm and are responsible for the formation of epithelia, which are known to be expressed in an epithelial type- and the highly mineralized enamel. The ectoderm-derived oral epithelium stage-specific manner. CKs are chemically very stable, long and un- located at sites where teeth will form, proliferates and differentiates to branched filaments of ,10 nm in diameter, which are important for form a dental epithelial band (also referred as dental placode), which is maintaining mechanical stability and integrity of epithelial cells and the first sign of tooth formation. In communication with adjacent tissues at both the single cell and epithelial sheet levels. The CK gene dental mesenchyme, dental epithelial cells progress through multiple family consists of 54 distinct functional genes in humans. CKs are stages of differentiation in bud and cap stages of tooth morphogenesis. heterodimers by pairing one type I keratin and one type II keratin In the subsequent bell stage, cytodifferentiation of dental epithelial (1 : 1) molecule.16 cells results in ameloblast-lineage cells (ALCs), including presecretory, In this study, we characterized the expression patterns of CKs and secretory and maturation ameloblasts. Enamel matrix protein secre- genes associated with early enamel organ epithelial differentiation in 1Department of Orofacial Sciences, University of California, San Francisco, USA and 2Department of Biology, Humboldt State University, Arcata, USA Correspondence: Dr Y Zhang, Department of Orofacial Sciences, University of California, San Francisco PO Box 0422, 521 Parnassus Avenue, Room S704, San Francisco CA 94143-0422, USA E-mail: [email protected] Received 2 December 2011; accepted 14 January 2013 Characterization of hESC-derived epithelial cells LW Zheng et al 2 ES-ECs, as compared to human fetal skin epithelium (SE), fetal oral Table 1 Sequences of primers used to amplify genes associated to buccal mucosal epithelial cells (OEs) and ALCs. These results will odontogenesis direct our further studies to explore the potential for using ES-ECs Genes Sequences of primers Size/bp to regenerate ALCs. Amg Forward GGCTGCACCACCAAATCATCC 383 Reverse CCCGCTTGGTCTTGTCTGTCG Fgf8 Forward GTTGCACTTGCTGGTCCTCT 220 MATERIALS AND METHODS Reverse GAGTTGGTAGGTCCGGATGA Maintenance of hESCs Msx1 Forward TCCTCAAGCTGCCAGAAGAT 342 The hESC line (WA09) was purchased from the WiCell Research Reverse TCTCCAGCTCTGCCTCTTGT Institute (Madison, WS, USA). The cells were maintained on a layer Shh Forward CCAATTACAACCCCGACATC 339 of mitosis inactivated mouse embryonic fibroblasts (MEFs) in uncon- Reverse CCGAGTTCTCTGCTTTCACC ditioned medium: Dulbecco’s modified Eagle’s medium (DMEM)/ Msx2 Forward ACACAAGACCAATCGGAAGC 222 Reverse GCAGCCATTTTCAGCTTTTC Ham’s F-12 medium containing 20% Knockout serum replacement Pitx2 Forward ACTTTACCAGCCAGCAGCTC 369 (Life Technologies, Carlsbad, CA, USA), non-essential amino acids Reverse GTGGGGAAAACATGCTCTGT 21 21 (Millipore, Billerica, MA, USA), 1 mmol?L L-glutamine, 0.1 mmol?L GAPDH Forward ACCACAGTCCATGCCATCAC 452 21 beta-mercaptoethanol and 4 ng?mL basic fibroblast growth factor, Reverse TCCACCACCCTGTTGCTGTA 50 mg?mL21 penicillin and streptomycin (Sigma-Aldrich, St Louis, MO, USA).17 fold change in mRNA expression levels between the two conditions, Epithelial induction of hESCs assuming a doubling of the amplified product with each PCR cycle. As described by Metallo and co-workers,15 BMP4 and a-RA were used to induce differentiation of hESCs to an epithelial lineage. In brief, hESCs were grown for 2 days on Matrigel (BD Biosciences, Franklin Characterization of amelogenesis-associated genes in differentiated Lakes, NJ, USA) coated culture-ware in MEF-conditioned medium. cells The MEF-conditioned medium was then supplemented with varying Conventional PCR was used to analyze the gene expression of Fgf8,a concentrations of BMP4 (Life Technologies, Carlsbad, CA, USA) and signaling molecule upregulated in placode stage of dental epithelial RA (Sigma-Aldrich, St Louis, MO, USA). In parallel studies, lithium cells; Msx1, Msx2, Shh and Pitx2 have been identified to be important chloride (LiCl), a Wnt signaling activator,18 was added to the induc- factors in early stages of amelogenesis. In addition, gene expression of tion medium. amelogenin, the characteristic enamel matrix protein marker for hESCs were induced for 7 days using the following conditions: secretory ameloblasts was determined. The gene of glyceraldehyde- Group A, 25 ng?mL21 BMP411 mmol?L21 RA; Group B, 25 ng?mL21 3-phosphate dehydrogenase (GAPDH) was used as an endogenous BMP411 mmol?L21 RA1100 mmol?L21 LiCl; Group C, 25 ng?mL21 control. A cDNA library of human fetal tooth organs was used as a BMP411 mmol?L21 RA11mmol?L21 LiCl; Group D, 12.5 ng?mL21 positive control for amelogenin amplification. PCR was performed at 6 6 6 6 BMP411 mmol?L21 RA; Group E, 12.5 ng?mL21 BMP411 mmol?L21 95 C for 5 min, 34 cycles of 95 C for 30 s, 57 C for 30 s and 72 C for 6 RA1100 mmol?L21 LiCl; Group F, 12.5 ng?mL21 BMP411 mmol?L21 1 min, followed by 72 C for 5 min. Primer sequences for target genes RA11 mmol?L21 LiCl. Upon induction, cell morphology was recorded are shown in Table 1. After amplification, equal volumes of PCR daily using a phase contrast microscope. After 7 days’ induction, cells products were separated by electrophoresis on agarose gels. were collected and total RNA was extracted using RNeasy Mini kit (Qiagen, Dusseldorf, Germany) according to manufacturer’s instruc- Characterization of CKs in differentiated cells tion. SuperScript III First-Strand Synthesis System (Life Technologies, All fetal tissues were collected from 16- to 20-week-old human fetal 19 Carlsbad, CA, USA) was used to synthesize cDNA. In brief, 2 mgtotal cadavers under guidelines approved by Committee on Human 21 RNA,
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