Final scientific assignment international dental course program

IDENTIFICATION OF SURFACE MARKERS OF DENTAL EPITHELIAL-LIKE CELLS DERIVED FROM INDUCED PLURIPOTENT STEM CELLS FINAL SCIENTIFIC ASSIGNMENT INTERNATIONAL DENTAL COURSE PROGRAM PHAM DO THANH THANH DEPARTMENT OF BIOMATERIALS FACULTY OF DENTISTRY HIROSHIMA UNIVERSITY 2016 i APPROVAL SHEET We certify that we have approved the final scientific assignment: Prepared by : Pham Do Thanh Thanh Entitled : Identification of surface markers of dental epithelial-like cells derived from induced pluripotent stem cells …………………………………… Professor Koichi Kato Department of Biomaterials Institute of Biomedical & Health Sciences Hiroshima University i This final scientific assignment has been presented at Faculty of Dentistry, Hiroshima University through the teleconference system with the sister universities (University of Medicine and Pharmacy in Ho Chi Minh City, Vietnam and Airlangga University, Indonesia) on June 4, 2016 JUDGES: Prof Koichi Kato, PhD Prof Kotaro Tanimoto, DDS., PhD Assistant Prof Isao Hirata, PhD Assoc Prof Ngo Thi Quynh Lan, DDS., PhD Assoc Prof Nguyen Thi Hong, DDS., PhD ii TABLE OF CONTENTS APPROVAL SHEET i TABLES OF CONTENTS iii LIST OF FIGURES v LIST OF TABLE v ACKNOWLEDGEMENT vi ABBREVIATIONS x ABSTRACT xiii CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEW 2.1 Induced pluripotent stem cells (iPSCs) 2.2 Embryoid body (EB) 2.3 Dental epithelial cell 2.3.1 Characteristics 2.3.2 Function in tooth development 2.4 Tumor protein p63 2.4.1 Location and isoforms 2.4.2 Functions 2.5 Cytokeratin 14 2.6 CD49f 2.7 Epithelial cadherin iii CHAPTER 3: CONCEPTUAL MAPPING AND HYPOTHESIS 10 3.1 Conceptual mapping 10 3.2 Hypothesis 11 CHAPTER 4: RESEARCH METHODOLOGY 12 4.1 Research type 12 4.2 Experimental design 12 4.3 Materials and methods 12 4.3.1 Cell culture 12 4.3.1.1 Feeder layer 12 4.3.1.2 Maintenance of undifferentiated state of miPSCs 12 4.3.2 Formation of EBs 13 4.3.3 Dental epithelial differentiation of miPSCs 14 4.3.4 Immunofluorescence and surface labelling (two steps) 14 4.3.5 Flow cytometry 15 CHAPTER 5: RESULTS AND DISCUSSION 17 5.1 Derivatization of DE-like cells from miPSCs 17 5.2 Correlation between intracellular markers (p63, CK14) and surface antigens (CD49f, E-cadherin) studied by immunofluorescent staining 21 5.3 Correlation between p63 and surface antigens (CD49f, E-cadherin) studied by flow cytometry 24 CHAPTER 6: DISCUSSION 26 CHAPTER 7: CONCLUSION 30 REFERENCES 31 iv LIST OF FIGURES Figure 5.1.1 Page Maintenance of undifferentiated state of miPSCs on MMC-treated SNL feeder layer……………………………………………………………………… 18 5.1.2 EB formation………………………………………………………………… 19 5.1.3 Differentiation of DE-like cells……………………………………………… 20 5.2.1 Fluorescent micrographs of cells stained with antibodies against p63 and E-cadherin or CD49f………………………………………………… 5.2.2 Fluorescent micrographs of cells stained with antibodies against CK14 and E-cadherin or CD49f……………………………………………… 5.3 23 The results of flow cytometry for quantitatively analyzing correlation between p63 and surface antigens (CD49f, E-cadherin) …………………… 6.1 22 25 Scheme for the relationship between integrin α6β4 and laminin-5 and -10/11…………………………………………………………………………… 29 LIST OF TABLE Table Page Summary of double staining immunofluorescence findings………………… v 24 ACKNOWLEDGEMENT This final scientific assignment was carried out to fulfill the requirement for completion of the bachelor degree from Faculty of Odonto-stomatology, University of Medicine and Pharmacy at Ho Chi Minh city, Vietnam The contents of this final scientific assignment were obtained from research work at Department of Biomaterials, Faculty of Dentistry, Hiroshima University, Japan during participating in the International Dental Course Program from 20122016 I would like to dedicate my acknowledgement of gratitude towards the following people for all their support and encouragement during my academic study and research assignment at Hiroshima University Firstly, I would like to express my deep gratitude to Professor Koichi Kato, Dean of Faculty of Dentistry, Hiroshima University; to Professor Takashi Takata, former Dean and Founder of International Dental Course program and to Professor Motoyuki Sugai, former Dean; for giving me a chance to study abroad in the dental field It is also with immense gratitude that I acknowledge Associate Professor Ngo Thi Quynh Lan, Dean of Faculty of Odonto-Stomatology, University of Medicine and Pharmacy at Ho Chi Minh City and Associate Professor Le Duc Lanh, former Dean, for the development of collaboration between the two universities to implement this program and thank you for selecting me as a candidate for this program and continuously supporting me vi I also want to extend my appreciation to Faculty of Dentistry, Hiroshima University and NGO Hiroshima for the scholarship they provided me monthly during four years Without this grant, it would have been difficult for me to live and study in Japan I owe my deepest gratitude to Professor Koichi Kato, Chair of Department of Biomaterials, for giving me the chance to join his department and experiments on the stem cell field He continuously supported me throughout my research His professional guidance, encouragement, recommendation and vision helped me a lot in designing and developing my research work He also spent a lot of time to read and provide helpful comments and correction for my research proposal, presentation and thesis writing even though he was very busy Without his precious patience and support, I could have not accomplished my research My sincere thank also goes for Assistant Professor Isao Hirata, who taught me the fundamental knowledge about research from the very first day that I entered the Biomaterials Laboratory He kindly provided me with a concrete overview of all the research themes in the department so that I could choose the one that I liked the most Besides that, he also kindly provided technical support and answered the questions from me Especially, I would like to express my warmest love and very great appreciation to my supervisor, Aimi Naim Abdullah, PhD She spent a lot of time to teach me patiently and dedicatedly and also give helpful comments for my proposal, presentation and thesis Additionally, she always tried to find the best solutions for all the difficulties I encountered during research Not only did she provide the insightful comments and evaluation for my research activity but also enthusiastically encourage, motivate and inspire me to my best vii With her help, I could deepen my knowledge to be able to go through and accomplish the research work I am very happy to be guided by a patient and caring supervisor like her Besides that, I would like to sincerely thank Professor Kotaro Tanimoto, DDS, PhD and Associate Professor Nguyen Thi Hong, DDS, PhD who stimulated me to gain insights into my research and widen my prospective through their insightful questions as a judge They also encouraged me a lot for my thesis defense I also want to thank the members in Biomaterials department, iPS cells research groups including Satoshi Miyauchi, MA and Assistant Professor Ryo Nishikiori, who kindly and patiently helped me with flow cytometry procedure and analysis and Azusa Onishi, who always helped me when I had any difficulties during research Additionally, they always encouraged and motivated me a lot during research I also owe a great debt of gratitude to department of International Collaboration Development for Dentistry: Professor Makiko Fujii, Associate Professor Hiroko Oka, Assistant Professor Nguyen Thi Phuong Thao and Associate Professor Maretaningtias Dwi Ariani They kindly provided a lot of useful information for my academic and daily life in Japan They always try their best to understand, help the international dental students and make the International Dental Course program better day by day I wish to offer thanks to my lovely classmates and students of all generation of International Dental Course and Short Stay programs who shared great memories with me during four years For the lab mates in department of Biomaterials: Ms Prak Malina, Ms Chihiro Matsuda, Mr Eiji Imado and Mr Hiroki Yoshii, I am really thankful for all the discussion and the fun we have had I am so happy to be surrounded by supportive and kind friends like them viii They made me have a lot of fun during research activity I will never forget the good time we shared together Last but not least, I want to thank my family, my friends for their spiritual supports during my research and thesis writing My sincere thanks go to my beloved father and mother, who gave birth to me and unconditionally love me, continuously encourage and support me throughout my life regardless of the geographical distance I might have not included all the supporting people who have helped me in various aspects I sincerely apologized to whom whose name was not cited in the above lines Hiroshima, July 2016 Pham Do Thanh Thanh ix surface marker for dental epithelial stem cells, which give rise to dental epithelial cells, derived from cervical loops.64 The aforementioned findings prompted me to investigate the correlations between intracellular markers of DE cells (p63, CK14) and two surface antigens (CD49f, Ecadherin) by double staining immunofluorescence and flow cytometry for miPSC-derived DElike cells Based on the results, the control cells exhibited no correlation between p63 and CD49f or E-cadherin On the contrary, the NT-4 treated cells demonstrated a weak correlation between p63 and E-cadherin, while a strong correlation was seen between p63 and CD49f For the correlation between CK14 and E-cadherin, both control and NT-4 treated samples exhibited no correlation Although the expression of CK14 can be detected in both samples, there is a possibility that cells in both samples are different in nature One of the possibilities is that the control cells can be considered as transit amplifying cells (TACs) derived from asymmetric division of pluripotent cells On the other hand, the NT-4 treated cells can be regarded as DE-like cells but not TACs, because NT-4 is known to bind to p75 neurotrophin receptor and induce the apoptosis in TACs.73,74 The finding for CK14 and E-cadherin expression in control samples can be supported by the previous study reporting that strong expression was observed for CK14, but not Ecadherin in the TACs.75,76 In the case of correlation between CK14 and CD49f, the control cells, which had similar characteristics to TACs, showed no correlation, while the NT-4 treated cells, which could be considered as DE-like cells, showed weak correlation Although there is no previously reported data available regarding the expression of CD49f in TACs, several studies have demonstrated that in TACs there was no expression of integrin beta 1, a subunit that forms a heterodimer with CD49f.77 This also suggests the possibility of negative expression of CD49f in TACs, in accordance with the finding obtained in this study 27 Flow cytometry results showed that p63 had a better correlation with CD49f than Ecadherin for both control and NT-4-treated samples, being in accordance with the immunofluorescence results Nonetheless, the control cells demonstrated a slightly higher percentage of double-positive cells (p63/CD49f and p63/E-cadherin) than NT-4-treated cells Namely, there is discrepancy between data from flow cytometry and immunofluorescence One of the reasons for this discrepancy may have implications in the fact that these two methods focused on different cell populations: In flow cytometry, total cells consisting of cells in EBs and those migrated from EBs were all used for the analysis Because most of the DE-like cells are likely present in migrated populations that seem to be a quite minor fraction in total cells Therefore, it is rather reasonable that the percentages of p63-positive DE-like cells determined by flow cytometry are quite small at the level comparable to the detection limit In addition, it may be probable that the localization of p63 has also influence on the flow cytometry data Because p63 transcription factor is synthesized in a cytosol and translocated into a nucleus, fluorescent labeling must be done intracellularly together with labeling of antigens present on a cell membrane (CD49f or E-cadherin) It is still challenging to detect both intracellular and surface proteins in flow cytometry with double staining technique, because of difficulties in preserving the integrity of surface antigens during membrane permeabilization using Triton-X and in detecting fluorescence emitted from the intracellular space These potential difficulties may cause the underestimation of intracellular and surface proteins Although the observed percentages were relatively low, the data obtained from this study clearly showed that there is a stronger correlation between p63 and CD49f compared to p63 and E-cadherin in the NT-4 treated cells This correlation may be due to the reason that p63 regulates the expression of cell adhesion molecules such as CD49f integrin in epithelial cells.78 Even so, 28 the mechanism is still unclear and thus further investigation is needed to understand the direct implications of p63 in CD49f expression With all the aforementioned evidences, p63 expression has strong correlation with CD49f expression in NT-4 treated cells, and CD49f may serve as a better surface marker than Ecadherin for DE-miPSCs E-cadherin is classified as a general marker for all types of epithelial cells, and thus may not be a suitable candidate as a specific marker of DE-like cells On the other hand, the presence of CD49f on DE-like cells led us to consider the important function of this protein in these cells CD49f forms integrin α6β4, a receptor for laminin and 10/11, and activates PI3K/AKI and downstream molecules Rac1/cdc42 and RhoA GTPases for the polarity, spreading and filopodia formation of DE cells.79-86 Laminin and 10/11 are abundantly found in a basement membrane and needed for growth and polarization of DE cells as well as development and shape of tooth bud.65 During tooth development, the dental epithelium and mesenchyme coordinately and reciprocally interact across the basement membrane Figure 6.1 Scheme for the relationship between integrin α6β4 and laminin-5 and -10/11 29 CHAPTER 7: CONCLUSION The present study demonstrates that p63 and CD49f in NT-4 treated cells exhibit higher correlation than any other combinations of intracellular markers and surface antigens (p63/Ecadherin, CK14/CD49f, or CK14/E-cadherin) Accordingly, it may be concluded that CD49f serves as a better surface marker for DE-miPSCs than E-cadherin In the light of these findings, it is worth testing CD49f as a surface marker to purify and enrich DE-like cells derived from iPSCs by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS) for further exploring their potentials in tooth regeneration 30 REFERENCES Budtz-Jørgensen E Oral mucosal lesions associated with the wearing of removable dentures J Oral Pathol 1981; 10:65-80 Albrektsson T, Isidor F Concensus report of session IV In: Lang NP, Karring T, eds Proceedings of the 1st European Workshop on Periodontology London, UK: Quintessence Publishing; 1994, p 365-369 Ziccardi VB, Assael LA Mechanism of trigeminal nerve injuries Atlas Oral Maxillofac Surg Clin North Am 2001; 9:1-11 Guler N, Delilbasi C Ectopic dental impants in the maxillary sinus Quintessence Int 2007; 38:e238-239 Esposito M, Hirsch JM, Lekholm U, Thomsen P Biological factors contributing to failures of osseointegrated oral implant (I) Success criteria and epidemiology Eur J Oral Sci 1998; 106:527-551 Robinson PJ, Grossman LI Tooth Transplantation In: Robinson PJ, Guernsey LJ, eds Clinical transplantation in dental specialties St Louis: C.V Mosby Co.; 1980, p 77-88 Abdullah AN Differentiation of induced pluripotent stem cells into dental epitheliallike cells [dissertation thesis], Hiroshima University; 2015 p 43 Liu P, Zhang Y, Chen S, Cai J, Pei D Application of iPS cells in dental bioengineering and beyond Stem Cell Rev Rep 2014; 10:663-670 Takahashi K, Yamanaka S Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 2006; 126:663-676 10 Ferreira LMR, Mostajo-Radji MA How induced pluripotent stem cells are redefining 31 personalized medicine Gene 2013; 520:1-6 11 Ebert AD, Liang P, Wu JC Induced pluripotent stem cells as a disease modeling and drug screening platform J Cardiovasc Pharmacol 2012; 60:408-416 12 Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner F Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease Proc Natl Acad Sci USA 2008; 105:58565861 13 Hanna J, Wernig M, Markoulaki S, Sun C W, Meissner A, Cassady J P Treatment of sicle cell anemia mouse model with iPS cells generated from autologous skin Science 2007; 318:1920-1923 14 Wada N, Wang B, Lin NH, Laslett AL, Gronthos S, Bartold PM Induced pluripotent stem cell lines derived from human gingival and periodontal ligament fibroblast J Periodont Res 2011; 46:438-447 15 Egusa H, Okita K, Kayashima H, Yu G, Fukuyasu S, Saeki M Gingival fibroblasts as a promising source of induced pluripotent stem cells PLoS ONE 2010; 5:e12743 16 Miyoshi K, Tsuji D, Kudoh K, Satomura K, Muto T, Itoh K Generation of human induced pluripotent stem cells from oral mucosa J Biosci Bioeng 2010; 110:345-350 17 Tamaoki N, Takahashi K, Tanaka T, Ichikasa T, Aoki H, Takeda-Kawaguchi T Dental pulp cells for induced pluripotent stem cell banking J Dent Res 2010; 89:773778 18 Arakaki M, Ishikawa M, Nakamura T, Iwamoto T, Yamada A, Fukumoto E, Saito M, Otsu K, Harada H, Yamada Y, Fukumoto S Role of epithelial-stem cell interactions during dental cell differentiation J Biol Chem 2012; 287:10590-10601 32 19 Otsu K, Kishigami R, Oikawa-Sasaki A, Fukumoto S, Yamada A, Fujikawa N, Ishizeki K, Harada H Differentiation of induced pluripotent stem cells into dental mesenchymal cells Stem Cells Dev 2012; 21:1156-1164 20 Ozeki N, Mogi M, Kawai R, Yamaguchi H, Hiyama T, Nakata K, Nakamura H Mouse-induced pluripotent stem cells differentiate into odontoblast-like cells with induction of altered adhesive and migratory phenotype of integrin PLoS ONE 2013; 8:e80026 21 Liu L, Liu YF, Zhang J, Duan YZ, Jin Y Ameloblasts serum-free conditioned medium: bone morphogenetic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells J Tissue Eng Regen Med 2016; 10:466-474 22 Itskovitz-Eldorv J, Schuldiner M, Krsenti D Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers Mol Med 2000; 6:88-95 23 Bratt-Leal AM, Carpenedo RL, McDevitt TC Engineering the embryoid body microenvironment to direct embryonic stem cell differentiation Biotechnol Prog 2009; 25:43-51 24 Kuroshawa H Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells J Biosci Bioeng 2007; 103:389-398 25 Peerani R, Rao BM, Bauwens C, Yin T, Wood GA, Nagy A, Kumacheva E, Zandstra PW Niche-mediated control of human embryonic stem cell self-renewal and differentiation EMBO J 2007; 26:4744-4755 26 Park J, Cho CH, Parashurama N, Li Y, Berthiaume F, Toner M, Tilles AW, Yarmush ML Microfabrication-based modulation of embryonic stem cell differentiation Lab 33 Chip 2007; 7:1018-1028 27 Mohr JC, Zhang J, Azarin SM, Soerens AG, de Pablo JJ, Thomson JA, Lyons GE, Palecek SP, Kamp TJ The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells Biomaterials 2010; 31:1885-1893 28 Sarkar L, Cobourne M, Naylor S, Smalley M, Dale T, Sharpe PT Wnt/Shh interactions regulate ectodermal boundary formation during mammalian tooth development Proc Natl Acad Sci USA 2000; 97:4520-4524 29 Medawar A, Virolle T, Rostagno P, de la Forest-Divonne S, Gambaro K, Rouleau M, Aberdam D ∆Np63 is essential for epidermal commitment of embryonic stem cells PLoS ONE 2008; 3:e3441 30 Metllo CM, Ji L, de Pablo JJ, Palecek SP Directed differentiation of human embryonic stem cells to epidermal progenitors Methods Mol Biol 2010; 585:83-92 31 Wang B, Li L, Du S, Liu C, Lin, X., Chen, Y., Zhang, Y Induction of human keratinocytes into enamel-secreting ameloblasts Dev Biol 2010; 344:795-799 32 Tabata, MJ, Matsumura T, Liu JG, Wakisaka S, Kurisu K Expression of cytokeratin 14 in ameloblast-lineage cells of the developing tooth of rat, both in vivo and in vitro Arch Oral Biol 1996; 41:1019-1027 33 Domingues MG, Jaeger MM, Araújo VC, Araújo NS Expression of cytokeratins in human enamel organ Eur J Oral Sci 2000; 108:43-47 34 Dassule HR, Lewis P, Bei M, Maas R, McMahon AP Sonic hedgehog regulates 34 growth and morphogenesis of the tooth Development 2000; 127:4775-4785 35 Jernvall J, Thesleff I Reiterative signaling and patterning during mammalian tooth morphogenesis Mech Dev 2000; 92:19-29 36 Mina M, Kollar EJ The induction of odontogenesis in non dental mesenchyme combined with early murine mandibular arch epithelium Arch Oral Biol 1987; 32:123-127 37 Lumsden AG Spatial organization of the epithelium and the role of neural crest cells in the initiation of the mammalian tooth germ Development 1988; 103 Suppl:155169 38 Kollar EJ, Baird GB The influence of the dental papilla on the development of tooth shape in embryonic mouse tooth germs J Embryol Exp Morphol 1969; 21:131-148 39 Kollar EJ, Baird GR Tissue interactions in embryonic mouse tooth germ II The inductive role of the dental papilla J Embryol Exp Morphol 1970; 24:173-186 40 Vanio S, Karavanova I, Jowett A, Thesleff I Identification of BMP-4 as a signal mediating secondary induction between epithelial and mesenchymal tissue during early tooth development Cell 1993; 75:45-58 41 Yang A, KAghad M, Wang Y, Gillett E, Fleming MD, Dötsch V, Andrews NC, Caput D, McKeon F p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing, and dominant-negative activities Mol Cell 1998; 2:305-316 42 Mangiulli M, Valletti A, Caratozzolo MF, Tullo A, Sbisa E, Pesole G, D'Erchia AM Identification and functional characterization of two new transcriptional variants of 35 the human p63 gene Nucleic Acids Res 2009; 37:6092-6104 43 Serber Z, Lai HC, Yang A, Ou HD, Sigal MS, Kelly AE, Darimont BD, Duijf PH, Van Bokhoven H, McKeon F, Dötsch V A C-terminal inhibitory domain controls the activity of p63 by an intramolecular mechanism Mol Cell Biol 2002; 22:8601-8611 44 Donehower, LA, Harvey M, Slagle BL, McArthur MJ, Montgometry CA Jr, Butel JS, Bradley A Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours Nature 1992 356:215-221 45 Alea AM, Zheng B, Wang XJ, Vogel H, Roop DR, Bradley A p63 is a p53 homologue required for limb and epidermal morphogenesis Nature 1999; 398:708713 46 Annie Y, Schweiter R, Sun D, Kaghad M, Walker N, Bronson RT, Tabin C, Sharpe A, Caput D, Crum C, McKeon F p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development Nature 1999; 398:714-718 47 Senoo M, Pinto F,.Crum CP, McKeon F P63 is essential for the proliferative potential of stem cells in stratified epithelia Cell 2007; 129:523-536 48. Rufini A, Barlattani A, Docimo R, Velletri T, Niklison-Chirou M V, Agostini M, Melino G p63 in tooth development Biochem Pharmacol 2011; 82:1256-1261 49. Laurikkala J, Mikkola ML, James M, Tummers M, Mills AA, Thesleff I p63 regulates multiple signaling pathways required for ectodermal organogenesis and differentiation Development 2006; 133:1553-1563 50 Medawar A, Virolle T, Rostagno P, de la Forest-Divonne S, Gambaro K, Rouleau M, Aberdam D ∆Np63 is essential for epidermal commitment of embryonic stem cells 36 PLoS ONE 2008; 3:e3441 51 Metllo CM, Ji L, de Pablo JJ, Palecek SP Directed differentiation of human embryonic stem cells to epidermal progenitors Methods Mol Biol 2010; 585:83-92 52 Moll R, Franke WW, Schiller DL, Geiger B, Krepler R The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured ceils Cell 1982; 2:11-24 53 Nakai M, Tatemoto Y, Mori H, Mori M Distribution profiles of keratin proteins during rat amelogenesis Histochem 1986; 85:89-94 54 Nagle RB, Bocker W, Davis JR, Heid HW, Kaufmann M, Lucas DO, Jarasch ED Characterization of breast carcinomas by two monoclonal antibodies distinguishing myoepithelial from luminal epithelial cells J Histochem Cytochem 1986; 34:869-881 55 Sun P, Yuan Y, Li A, Li B, Dai X Cytokeratin expression during mouse embryonic and early postnatal mammary gland development Histochem Cell Biol 2010; 133:213-221 56 Kawano S, Saito M, Handa K, Morotomi T, Toyono T, Seta Y, Nakamura N, Uchida T, Toyoshima K, Ohishi M, Harada H Characterization of dental epithelial progenitor cells derived from cervical-loop epithelium in a rat lower incisor J Dent Res 2004; 83:129-133 57 Crivelini MM, de Araújo VC, de Sousa SO, de Araújo NS Cytokeratins in epithelia of odontogenic neoplasms Oral Dis 2003; 9:1-6 58 Sonnenberg A, Calafat J, Janssen H, Daams H, van der Raaij-Helmer LM, Falcioni R, Kennel SJ, Aplin JD, Baker J, Loizidou M, Garrod D Integrin alpha/beta complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement 37 membrane adhesion J Cell Biol 1991; 113:907-917 59 Mercurio A Receptors for the laminins: Achieving specificity through cooperation Trends Cell Biol 1995; 5:419-423 60 Hemler ME, Crouse C, Sonnenberg A Association of the VLA alpha subunit with a novel protein A possible alternative to the common VLA beta subunit on certain cell lines J Biol Chem 1989; 264:6529-6535 61 Dowling J, Yu QC, Fuchs E Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival J Cell Biol 1996; 134:559-572 62 Nievers MG, Schaapveld RQ, Sonnenberg A Biology and function of hemidesmosomes Matrix Biol 1999; 18:5-17 63 Sterk LM, Geuijen CA, Oomen LC, Calafat J, Janssen H, Sonnenberg A The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes J Cell Biol 2000; 149:969-982 64 Chang JY, Wang C, Jin C, Yang C, Huang Y, Liu J, McKeehan WL, D`Souza RN, Wang F Self-renewal and multilineage differentiation of mouse dental epithelial stem cells Stem Cell Res 2013; 11:990-1002 65 Fukumoto S, Miner JH, Ida H, Fukumoto E, Yuasa K, Miyazaki H, Hoffman MP, Yamada Y Laminin alpha is required for dental epithelium growth and polarity and the development of tooth bud and shape J Biol Chem 2006; 281:5008-5016 66 Hulpiau P, van Roy F Molecular evolution of the cadherin superfamily Int J Biochem Cell Biol 2009; 41:349-369 38 67 Chen YT, Stewart DB, Nelson WI Coupling assembly of the E-cadherin/β-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells J Cell Biol 1999; 144:687-699 68 Heymann R, About I, Lendahl U, Franquin JC, Obrink B, Mitsiadis TA E- and Ncadherin distribution in developing and functional human teeth under normal and pathological conditions Am J Pathol 2002; 160:2123-2133 69 Guan X, Bidlack FB, Stokes N, Bartlett JD E-cadherin can replace N-cadherin during secretory-stage enamel development PLoS ONE 2014; 9:e102153 70 Fukumoto S, Kiba T, Hall B, Lehara N, Nakamura T, Longenecker G, Krebsbach PH, Nanci A, Kulkarni AB, Yamada Y Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts J Cell Biol 2004; 167:973-983 71 Fibcham AG, Simmer JP Amelogenin proteins of developing dental enamel Ciba Found Symp 1997; 205:118-134 72 Martinez EF, da Silva LA, Furuse C, de Araujo NS, de Araujo VC Dentin matrix protein (DMP1) expression in developing human teeth Braz Dent J 2009; 20:365369 73 Schwab M Encyclopedia of cancer 3rd ed New York: Springer; 2012, p.3766 74 Truzzi F, Marconi A, Atzei P, Panza MC, Lotti R, Dallaqlio K, Tiberion R, Palazzo E, Vaschieri C, Pincelli C p75 neurotrophin receptor mediates apoptosis in transitamplifying cells and its overexpression restores cell death in psoriatic keratinocytes Cell Death Differ 2011; 18:948-958 75 Li CY, Cha W, Luder HU, Charles RP, McMahon M, Mitsiadis TA, Klein OD Ecadherin regulates the behavior and fate of epithelial stem cells and their progeny in 39 the mouse incisor Dev Biol 2012; 366:357-366 76 Sun XY, Liu HL, Fu XB Dedifferentiation of epidermal cells into transit amplifying cells induced by bFGF South Med J 2010; 30:2041-2046 77 Philip HJ, Fiona MW Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression Cell 1993; 73:713-724 78 Carroll DK, Carroll JS, Leong CO, Cheng F, Brown M, Mills AA, Brugge JS, Ellisen LW P63 regulates an adhesion programme and cell survival in epithelial cells Nat Cell Biol 2006; 8:551-561 79 Kikkawa Y, Sanzen N, Fujikawa H, Sonnenberg A, Sekiguchi K Integrin binding specificity of laminin-10/11: Laminin-10/11 are recognized by alpha beta 1, alpha beta and alpha beta integrins J Cell Sci 2000; 113:869-876 80 Yu H, Talts JF Beta1 integrin and alpha-dystroglycan binding sites are localized to different laminin-G-domain-like (LG) modules within the laminin alpha5 chain G domain Biochem J 2003; 371:289-299 81 Mercurio AM, Rabinovitz I, Shaw LM The alpha beta integrin and epithelial cell migration Curr Opin Cell Biol 2001; 12:541-545 82 Shaw LM, Rabinovitz I, Wang HH, Toker A, Mercurio Am Activation of phosphoinositide 3-OH kinase by the alpha6beta4 integrin promotes carcinoma invasion Cell 1997; 91:949-960 83 O’Connor KL, Shaw LM, Mercurio AM Release of cAMP gating by the alpha6beta4 integrin stimulates lamellae formation and the chemotacticmigration of invasive carcinoma cells J Cell Biol 1998; 143:1749-1760 40 84 Clark EA, King WG, Brugge JS, Symons M, Hynes RO Integrin-medicated signals regulated by members of the rho family of GTPases J Cell Biol 1998; 142:573-586 85 Price LS, Leng J, Schwartz MA, Bokoch GM Activation of Rac and Cdc42 by integrins mediates cell spreading Mol Biol Cell 1998; 9:1863-1871 86 Van Leeuwen FN, van Delft S, Kain HE, van der Kammen RA, Collard JG Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading Nat Cell Biol 1999; 1:242-248 41 ... help the international dental students and make the International Dental Course program better day by day I wish to offer thanks to my lovely classmates and students of all generation of International. .. this final scientific assignment were obtained from research work at Department of Biomaterials, Faculty of Dentistry, Hiroshima University, Japan during participating in the International Dental. .. Takata, former Dean and Founder of International Dental Course program and to Professor Motoyuki Sugai, former Dean; for giving me a chance to study abroad in the dental field It is also with immense