Study of human nasal epithelial stem or progenitor cell growth and differentiation in an in vitro system

STUDY OF HUMAN NASAL EPITHELIAL STEM OR PROGENITOR CELL GROWTH AND DIFFERENTIATION IN AN in vitro SYSTEM LI YINGYING (Master of Medicine, Wuhan University, P.R. China) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF OTOLARYNGOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2014 i Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously Li Yingying Aug 2014 ii Acknowledgement First and foremost I want to thank my supervisor, Assoc. Prof. Wang De Yun. It has been an honor to be his PhD student. He has taught me not only knowledge, but also the philosophy of life. I still remember the scene that he asked me to spell the full name of PhD and showed me how to understand the meaning of PhD when we first met. During these four years, the joy and enthusiasm he has for his research was contagious and motivational for me. I am also thankful for the excellent example he has provided as a successful scientist and professor. The members of the ENT research lab have contributed immensely to my personal and professional time at NUS. This group has been a source of friendships as well as good advice and collaboration. I am especially grateful for my senior, Dr. Li Chun Wei. He helped me be familiar to many of research works with great patience and selflessly shared with me some tips he picked up during his experience. I would like to acknowledge the senior research fellow, Dr. Yu Feng Gang. I very much appreciated his enthusiasm, intensity, willingness to share his knowledge on stem cell culture and inspired me with amazing ideas. In addition, I would also thank other members in this lab: Ms. Liu Jing, Dr. Yan Yan and Dr. Louise Tan Soo Yee. Without their generous help and support, I could not fulfill my PhD work. I must specially thank Assoc. Prof. Thomas Loh, our head of department, who gives me a help and support in my study. I would also like to appreciate the help from all the doctors in our department, especially Assoc. Prof. Chao Siew Shuen, iii who gives us a great support with clinic samples supply. I need to thank the sharing of clinic ideas from Dr. Lim Chwee Ming and Dr. Ng Chew Lip. Throughout my PhD study, we have many collaboration works with exchangestudents from China and I have had the pleasure moment to work with them. The optimization of components and description of characteristics for hNESPCs was mainly finished by Dr. Zhao Xue Ning from Shandong University (Shandong, China). The differentiation study was also collaborated with her. For comparison study for hNESPCs from NP and healthy controls, Dr. Yu Xue Ming from Shandong University (Shandong, China) helped me to trace the proliferation of progenitor cell. In my later work of cilia impairment in hyperplasia epithelium in NP, Dr. Gao Tian from Haerbin medical University (Heilongjiang, China), Dr. Jin Peng and Dr. Duan Chen from Shandong University (Shandong, China) help me a lot in staining works. Other peoples, who are not our major collaborators, also have given me a great help in my work for these years. My lab neighbors, Ms. Wen Hong Mei, Dr. Huang Chiung Hui, Dr. Kuo I-Chun, and Dr. Seow See Voon from Department of Pediatrics always give me assistance and let me share the facilities with them. Ms. Li Chun Mei from Department of Anaesthesia always lent me a hand with my routine lab work. I should like to express my gratitude for your kindness iv I also thank National University of Singapore for giving me the chance to pursue PhD and offering me the scholarship. And last but not least, this dissertation is dedicated to my family. My parents are always standing with me and giving me endless love and encouragement. My husband is always showing his patience and thoughtfulness to my work, and teaching me how to simplify works by using software. Without their help and support, it’s hard for me to persist during tough times in the PhD. It’s my fortune to be your daughter/ wife. Yours, Li Yingying v Table of Contents Title i Declaration ii Acknowledgement . iii Publications and Presentations at Conferences . 231 Publications . 231 Presentations at Conferences 234 Table of Contents . vi Summary . xii List of Tables xv List of Figures xvi List of Abbreviations . xxi Chapter 1. Literature review . 1.1. Chronic rhinosinusitis with NP 1.1.1. Epidemiology 1.1.1. Histopathology 1.1.2. Pathogenesis 1.1.3. Treatment 16 1.2. Nasal epithelium 16 1.2.1. Structure and bio-physiology 16 1.2.2. Epithelial impairment in NP . 42 1.2.3. Update in research . 49 vi Chapter 2. Objectives of this study 59 2.1. Research questions . 59 2.2. Objectives 62 2.3. Significance 62 Chapter 3. Material and Methods . 64 3.1. Study subjects 64 3.2. Immunohistochemical staining (IHC) 65 3.2.1. Staining procedures for paraffin embedded tissues 66 3.2.2. Evaluation of IHC staining patterns 66 3.3. Cell preparation and fixed for Immunofluorescence . 68 3.3.1. Progenitor cells . 68 3.3.2. Differentiated cells in ALI culture 68 3.3.3. Cytospin 68 3.4. Immunofluorescence (IF) . 68 3.5. Scanning electron microscopy . 71 3.6. RNA extraction 71 3.6.1. Extract from solid tissues 71 3.6.2. Extract from cells 72 3.7. Microarray analysis 72 3.8. Real-Time quantitative reverse transcription PCR (RT- qPCR) 73 3.9. Tissue IF staining and mRNA evaluation in tissue section . 75 3.9.1. Evaluation of staining patterns of p63 and Ki67 in nasal tissues …………………………………………………………………75 vii 3.9.2. Measurement of cilia length, cilia area and ciliated cell percentage …………………………………………………………………75 3.9.3. Total fluorescence intensity (TFI) evaluation of ciliogenesis associated genes 76 3.9.4. mRNA evaluation . 76 3.10. Human nasal epithelial stem or progenitor cells (hNESPCs) culture and evaluation . 77 3.10.1. Medium 77 3.10.2. Cell preparation 77 3.10.3. Cell passage . 79 3.10.4. Doubling time 80 3.10.5. Colony forming efficiency . 80 3.10.6. Cell proliferation assay 81 3.10.7. Senescence-associated β -galactosidase staining . 82 3.10.8. Staining evaluation . 82 3.10.9. mRNA evaluation 83 3.11. Air-liquid interface (ALI) culture and evaluation of differentiated cells …………………………………………………………………… .83 3.11.1. ALI culture . 83 3.11.2. Transepithelial electrical resistance (TEER) measurement 84 3.11.3. Ciliary beat frequency (CBF) . 85 3.11.4. Staining evaluation of transwell . 86 3.11.5. mRNA evaluation 87 viii 3.12. Statistic analysis . 87 3.12.1. Comparing the proliferation rate between hNESPCs from healthy controls and nasal polyps . 88 3.12.2. Comparing the differentiation change between hNESPCs from healthy controls and nasal polyps 88 3.12.3. Comparing the impairment cilia in nasal polyps to healthy controls in vivo 89 Chapter 4. Establishment of an in vitro culture system of human nasal epithelial stem or progenitor cells (hNESPCs) . 91 4.1. Results 91 4.1.1. Stem cell marker screening in nasal epithelium . 91 4.1.2. Formulation of serum-free media . 93 4.1.3. hNESPCs express stem cell markers 94 4.1.4. Proliferation and division of hNESPCs 96 4.1.5. Capacity of differentiation of hNESPCs . 98 4.2. Discussion 103 4.3. Summary 105 Chapter 5. Pathology changes of human nasal epithelial stem or progenitor cells (hNESPCs) from in vitro and air-liquid interface (ALI) culture system in nasal polyp (NP)…………………………………………………………………………… 107 5.1. Pathology change of hNESPCs in vitro culture system in NP . 107 5.1.1. Results . 107 5.1.2. Discussion . 119 ix 5.2. Pathology change of hNESPCs from NP biopsies in ALI culture system 123 5.2.1. Results . 123 5.2.2. Discussion . 153 5.3. Summary 159 Chapter 6. Impairment of ciliary architecture and ciliogenesis in hyperplasic nasal epithelium from NP biopsies . 160 6.1. Results 160 6.1.1. Patients’ histopathological characteristics 160 6.1.2. Cilia morphology and function . 163 6.1.3. Evaluation of cilia related marker . 168 6.1.4. Cilia morphology and ciliogenesis associated markers in the adjacent inferior turbinate from NP patients . 175 6.1.5. Correlation between cilia length and cilia related markers . 176 6.1.6. Sub-group analysis 182 6.1.7. 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Appendices Publications and Presentations at Conferences Publications  Yunfeng Ma, Hai-Dan Chen, Yubin Wang, Qilong Wang, Yingying Li, Yinglan Zhao, Xiao-lian Zhang. Interleukin 24 as a novel potential cytokine immunotherapy for the treatment of Mycobacterium tuberculosis infection. Microbes and infection. 2011 July; 13(2011): 1099-1110. (Impaction factor: 2.82)  C. W. Li, K. K. Zhang, T. Y. Li, Z. B. Lin, Y. Y. Li, M. A. Curotto de Lafaille, L. Shi, D. Y. Wang. Expression profiles of regulatory and helper T-cell-associated genes in nasal polyposis. Allergy. 2012 Feb; 67: 732–7400. (Impaction factor: 5.98)  Fenggang Yu, Xuening Zhao, Chunwei Li, BM, Yingying Li, BM, Yan Yan, Li Shi, Bruce R. Gordon, MD; De-Yun Wang. Airway stem cells: review of potential impact on understanding of upper airway diseases. Laryngoscope. 2012 July; 122:1463–1469. (Impaction factor: 2.27) 231  Xuening Zhao, Fenggang Yu, Chunwei Li, Yingying Li, Siew-Shuen Chao, Woei-Shyang Loh, Xinliang Pan, Li Shi, De-Yun Wang. The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cells in vitro. American Journal of Rhinology & Allergy. 2012 Sep; 26:345350. (Impaction factor: 2.06)  Yu FG, Sim AC, Li CW, Li YY, Zhao XN, Wang DY, Loh KS. Identification of a subpopulation of nasopharyngeal carcinoma cells with cancer stem-like cell properties by high aldehyde dehydrogenase activity. Laryngoscope. 2013 Aug; 123 (8):1903-1911. (Impaction factor: 2.27)  Yu FG, Li YY, Morshead CM. Induced pluripotent stem cells for the treatment of stroke: the potential and the pitfalls. Current stem cell research & therapy. 2013 Sep; 8(5):407-414. (Impaction factor: 2.96)  Xue Min Yu, Chun Wei Li, Ying Ying Li, Jing Liu, Zhi Bin Lin, Tian Ying Li, Li Zhao, Xin Liang Pan, Li Shi, De Yun Wang. Down-regulation of EMP1 is associated with epithelial hyperplasia and metaplasia in nasal polyps. Histopathology. 2013 Nov; 63(5):686-95. (Impaction factor: 3.51) 232  Peng Jin, Chun Wei Li, Zhi bin Lin, Tian Ying Li, Ying Ying Li, Li Shi, De Yun Wang. Down-regulation of EMP1 is associated with epithelial metaplasia in nasal inverted papilloma. Asian Rhinology Journal. 2013 Dec; 1:21-5.  Fenggang Yu, Wen-son Hsieh, Fredrik Petersson, Henry Yang, Yingying Li, Chunwei Li, Soon Wah Low, Jing Liu, Yan Yan, De –Yun Wang, Kwok Seng Loh. Malignant cells derived from 3T3 fibroblast feeder layer in cell culture for nasopharyngeal carcinoma. Experimental Cell Research. 2014 Mar; 10:322(Harvey; and Crystal.):193-201. (Impaction factor: 3.60)  Xue Min Yu, Chun Wei Li, Siew Shuen Chao, Ying Ying Li, Yan Yan, Xue Ning Zhao, Feng Gang Yu, Jing Liu, Liang Shen, Xin Liang Pan, Li Shi, De Yun Wang. Reduced growth and proliferation dynamics of nasal epithelial stem/progenitor cells in nasal polyps in vitro. Scientific Reports. 2014 Apr; 4:4619. (Impaction factor: 2.92)  Ying Ying Li, Chun Wei Li, Siew Shuen Chao, Fenggang Yu, Xue Min Yu, Jing Liu, Yan Yan, Liang Shen, William M. Gordon, Li Shi, De Yun Wang. Impairment of ciliary architecture and ciliogenesis in hyperplasic nasal 233 epithelium from nasal polyps. Journal of Allergy Clinical Immunology. 2014 Sep; 10.1016. (Impaction factor: 11.25) Presentations at Conferences  Yingying Li, Xuening Zhao, Fenggang Yu, Chunwei Li, Siew-Shuen Chao, Woei-Shyang Loh, Li Shi, Xinliang Pan, De-Yun Wang. A culture system for culturing human nasal epithelial stem cells. 2nd YLL-SOM Annual Graduate Scientific Congress Programme. Singapore, 15 Feb 2012. Poster No. 51.  Yingying Li, Siew Shuen Chao, Jing Liu, Chunwei Li, Xuening Zhao, Xuemin Yu, Fenggang Yu, Yan Yan, Li shi, De-Yun Wang. A culture model for in vivo human nasal epithelial self-renewal and differentiation. 15th Asian Reserch Symposium in Rhinology. Singapore, 24-26 May 2012.  Yingying Li, Jing Liu, Chunwei Li, Xueming Yu, Fenggang Yu, De-Yun Wang. Differentiation pattern of human nasal epithelia progenitor cell in ALI culture system. European Academy of Allergology and Clinical Immunology. Geneva, Switzerland, 16-20 June 2012. Poster No. 1273.  Yingying Li, Jing Liu, Chunwei Li, Xuening Zhao, Xuemin Yu, Fenggang Yu, Yan Yan, Siew-Shuen Chao, Li Shi, De-yun Wang. A culture model for in vivo human nasal epithelial self-renewal and differentiation. 3rd YLL-SOM Annual Graduate Scientific Congress Programme. Singapore, 30 Jan 2013. Poster No. 83. 234  Yingying Li, Jing Liu, Chunwei Li, Xuemin Yu, Fenggang Yu, Li Shi, DeYun Wang. Dynamic patterns of human nasal epithelia stem/progenitor cell differentiation in ALI culture system. 20th International Federation of Oto-Rhino-Laryngological Societies world Congress. Seoul, South Korea, 1-5 June 2013. Poster No. FP62-08.  Ying Ying Li, Chun Wei Li, Siew Shuen Chao, Jing Liu, Xuemin Yu, Zhao Li, Shi Li, De Yun Wang. Myrtol enhances ciliation and mucin secretion in human nasal epithelial cells in vitro. 20th International Federation of Oto-Rhino-Laryngological Societies world Congress. Seoul, South Korea, 1-5 June 2013. Poster No.EP176. 235 [...]... tissue retain their abnormal behavior Therefore, this thesis focuses on the establishment of a human nasal epithelial stem or progenitor cells (hNESPCs) model in vitro, an investigation of biophysiology and pathophysiology of cell growth and differentiation of hNESPCs isolated from xii healthy nasal mucosa and NP biopsies, and the implication of this cell model in the study of the pathogenesis and molecular... this cell model could simulate the in vivo pathophysiological changes of NP in vitro In conclusion, we established an in vitro model for validating and comparing hNESPCs morphology and functional activity in the cells from NP and healthy controls, and demonstrated that the intrinsic factors related to cell growth and differentiation may explain the mechanism underlying the histopathological patterns of. .. regulation of Treg and Th cells may contribute to the development of NP As the results of microorganism stimulation and inflammatory cells disorder, epithelial damage and remodeling occurred in NPs 3 Epithelial repair and damage As the results of genetic predisposition and inflammatory cells disorder, the capacity of epithelial repair is decreased, leading to the imbalance of epithelial homeostasis in NPs... inflammation and damage the epithelium in NPs As followed, inflammatory cells involve into the damaged site and play as the major roles in the development of NPs 2) Inflammatory cells and cytokines NP could be separated into two sub-types based on the pattern of inflammatory cell infiltration in NP tissues: (i) increase eosinophil levels and Th2 cytokine skewing; (ii) less eosinophilic than first type... nasal epithelial stem or progenitor cells inner dynein arms immunofluorescence intraflagellar transport also Tg737, intraflagellar transport 88 intraflagellar transport protein A intraflagellar transport protein B immunohistochemical staining interleukins interleukins 13 interleukin 6 xxii IL-9 IT KRT14 KRT5 LEKT1 LTC4S Math1 MCPH MFI MMP MTOC MUC1 MUC16 MUC4 MUC5AC MUC5B NF-κB NOS2A NP ODAs Odf2 Ofd1... Identifying the causal genes and gene variants in NP is important in order to improve prevention, diagnosis, and treatment of NP 2 Inflammatory mechanisms Inflammatory cells disorder is one of the most important mechanisms in NP pathogenesis Based on the genetic defection, the atopic immunity response to microorganisms triggers the epithelial remodeling in NPs In the following parts, both the inflammatory... mechanisms leading to aberrant epithelial remodeling and impairment of mucociliary apparatuses such as cilia architecture and ciliogenesis in hyperplastic nasal epithelium from NP biopsies Initially, we tested the cultural components and conditions for hNESPCs growth and differentiation in vitro Primary cells from biopsies survived and more than 90% of cells proved to have properties of epithelial stem. .. of inflammatory cells (e.g., eosinophils and neutrophils), basal- or goblet cells hyperplasia, squamous metaplasia epithelial remodeling, and stromal edema Epithelium abnormalities in NP involved with stem cells and their progenitors might result from intrinsic (including epigenetic) alterations in their transcriptional and regulatory programs, which in turn affect proliferative and differentiation. .. growth factor forkhead box protein A2 also HFH4,Forkhead box protein J1 forkhead box P3 glyceraldehyde-3-phosphate dehydrogenase GATA binding protein 3 goblet cell hyperplasia goblet cell metaplasia glucocorticosteroids granulocyte-macrophage colony-stimulating factor guanosine-5'-triphosphate hematoxylin and eosin heparin-binding EGF-like growth factor hairy and enhancer of split-1 major histocompatibility... epithelial stem or progenitor cells in a chemical-defined medium Meanwhile, these cells successfully differentiated into functional cells (e.g., ciliated and goblet cells) in air-liquid interface (ALI) culture These results indicated that we had successfully isolated and expanded pure hNESPCs in vitro Secondly, we investigated whether the growth and differentiation properties of the nasal epithelial cells from . i STUDY OF HUMAN NASAL EPITHELIAL STEM OR PROGENITOR CELL GROWTH AND DIFFERENTIATION IN AN in vitro SYSTEM LI YINGYING (Master of Medicine, Wuhan University, P.R. China) A. establishment of a human nasal epithelial stem or progenitor cells (hNESPCs) model in vitro, an investigation of biophysiology and pathophysiology of cell growth and differentiation of hNESPCs. changes of NP in vitro. In conclusion, we established an in vitro model for validating and comparing hNESPCs morphology and functional activity in the cells from NP and healthy controls, and