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. Koichi Kato, PhD 2. Kotaro Tanimoto, DDS. Isao Hirata, PhD 4.
Ngo Thi Quynh Lan, DDS. Nguyen Thi Hong, DDS., PhD ii TABLE OF CONTENTS APPROVAL SHEET. i TABLES OF CONTENTS. iii LIST OF FIGURES.
v LIST OF TABLE. xiii CHAPTER 1: INTRODUCTION. 1 CHAPTER 2: LITERATURE REVIEW. Induced pluripotent stem cells (iPSCs).
Dental epithelial cell. Function in tooth development. Location and isoforms. 9 iii CHAPTER 3: CONCEPTUAL MAPPING AND HYPOTHESIS.
11 CHAPTER 4: RESEARCH METHODOLOGY. Materials and methods. Maintenance of undifferentiated state of miPSCs. Formation of EBs.
Dental epithelial differentiation of miPSCs. Immunofluorescence and surface labelling (two steps). 15 CHAPTER 5: RESULTS AND DISCUSSION. Derivatization of DE-like cells from miPSCs.
Correlation between intracellular markers (p63, CK14) and surface antigens (CD49f, E-cadherin) studied by immunofluorescent staining. Correlation between p63 and surface antigens (CD49f, E-cadherin) studied by flow cytometry. 31 iv LIST OF FIGURES Figure Page 5.1 Maintenance of undifferentiated state of miPSCs on MMC-treated SNL feeder layer……………………………………………………………………… 18 5.3 Differentiation of DE-like cells……………………………………………… 20 5.1 Fluorescent micrographs of cells stained with antibodies against p63 and E-cadherin or CD49f………………………………………………….2 Fluorescent micrographs of cells stained with antibodies against CK14 and E-cadherin or CD49f……………………………………………….3 The results of flow cytometry for quantitatively analyzing correlation between p63 and surface antigens (CD49f, E-cadherin) …………………… 25 6.1 Scheme for the relationship between integrin α6β4 and laminin-5 and -10/11……………………………………………………………………………. 29 LIST OF TABLE Table Page 1 Summary of double staining immunofluorescence findings………………… 24 v 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 2012- 2016. 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 do 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 do 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 ABBREVIATIONS ∆N Lack of N-terminal transactivation domain AMBN Ameloblastin AMELX Amelogenin BMP-4 Bone morphogenetic protein 4 CD104 Integrin beta 4 CD29 Integrin beta 1 CD49f, ITGA6 Integrin alpha 6 CK5 Cytokeratin 5 CK14 Cytokeratin 14 CK19 Cytokeratin 19 c-Myc Avian myelocytomatosis virus oncogene cellular homolog DE Dental epithelial DE-miPSCs Dental epithelial like cells derived from mouse induced pluripotent stem cells Dnmt3a DNA methyltransferase 3A Dnmt3b DNA methyltransferase 3B Dnmt3l DNA methyltransferase 3L EB Embryoid body E-cadherin, CD324, CDH1 Epithelial-cadherin ENAM Enamelin FACS Fluorescence activated cell sorting x Gdf3 Growth differentiation factor 3 iPSCs Induced pluripotent stem cells IRES Internal ribosomal entry site Klf4 Kruppel-like factor 4 KRT14 Keratin 14 LIF Leukemia inhibitory factor MEM Minimum Essential Medium miPSCs Mouse induced pluripotent stem cells MMC Mitomycin-C Myb Myeloblastosis Nanog-GFP Nanog promoter-driven green fluorescent protein NT-4 Neurotrophin-4 Oct 3/4 Octamer-binding transcription factor ¾ p53 Tumor suppressor protein that in humans is encoded by the TP53 gene p63 Tumor protein p63 PITX2 Paired-like homeodomain transcription factor 2 RUNX2 Runt domain transcription factor 2 Shh Sonic hedgehog SNL 76/7 Clonally-derived from a STO cell line that expresses both G418 resistance and leukemic inhibitory factor (LIF) at an abundant level Sox2 Sex determining region Y-box 2 xi TA N-terminal transactivation domain TACs Transit amplifying cells Tcl1 T-cell leukemia/lymphoma protein 1 Utf1 Undifferentiated embryonic cell transcription factor 1 Wnt7b Wingless-type MMTV integration site family, member 7B Zic3 Zinc finger protein of the cerebellum xii ABSTRACT [Purpose] Induced pluripotent stem cells (iPSCs) have potential to differentiate into dental epithelial-like cells (DE-like cells) in the presence of neurotrophic factor as an inducer under defined culture condition. In order to improve the differentiation efficiency, a method to measure the efficiency should be established. The present study was carried out to investigate the correlation between intracellular markers (p63, CK14) and cell surface markers (CD49f, E- cadherin) for specific identification of DE-like cells derived from iPSCs. After 6 days, EBs were collected and cultured on gelatin-coated dishes or 2-well glass slides for another 9 days to induce differentiation.
The correlation between intracellular markers (p63, CK14) and cell surface markers (CD49f, E-cadherin) was analyzed by double staining immunofluorescence and flow cytometry. [Results] The results showed that there is no correlation between the expression of intracellular markers (p63, CK14) and that of cell surface markers (CD49f, E-cadherin) in the control sample. The NT-4 treated sample showed a weak correlation for p63/E-cadherin and CK14/CD49f and no correlation for CK14/E-cadherin. A higher correlation was observed between p63 and CD49f in NT-4 treated cells.
Flow cytometry results showed higher percentage of p63/CD49f compared to p63/E-cadherin in both NT-4 treated and untreated cells. [Discussion] This study revealed that CD49f served as a better surface marker for DE-like cells derived from iPSCs, being in accordance with the previous study. E-cadherin is classified as a general marker for all types of epithelium cells, thus it may not be a suitable candidate for the specific isolation of DE-like cells. This finding provides a new approach to identify and enhance the homogeneity of DE-like cells for further applications in regenerative dentistry.
xiii CHAPTER 1: INTRODUCTION Dealing with a tooth loss is one of the biggest challenges in the field of dentistry. Several therapeutic approaches such as full dentures, removable partial dentures, implant surgery and tooth transplantation can be offered to handle this issue. Nonetheless, these treatments come with a baggage of complications. It has been reported that denture therapies may cause traumatic ulcers, hyperplasia, and denture-induced stomatitis while the implant surgery may lead to peri- implantitis, neurosensory disturbance, maxillary sinus perforation, bone fracture as well as osseointegration failure.1-5 Likewise, tooth transplantation may also result in chronic root resorption.6 In order to develop optimal treatment for the tooth loss, tooth bioengineering may offer a promising alternative medical application.
Many researchers have used stem cells as a starting material for the tooth bioengineering based approach on the fundamental concept of mimicking the tooth developmental process in the embryonic stage. Induced pluripotent stem cells (iPSCs) served as a great candidate for tooth bioengineering as they may hinder the risk of immune rejection along with ethical and clinical controversies.