IMMUNOLOGICAL CHARACTERIZATION OF HUMAN UMBILICAL CORD LINING DERIVED CELLS AND THEIR THERAPEUTIC APPLICATION IN A DIABETIC MOUSE MODEL ZHOU YUE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010   i   ii ACKNOWLEDGMENTS The work of this thesis has been done in several laboratories, with the support of many people. I feel lucky to have known and worked with these people and I would like to express my gratitude here. First of all, I owe my deepest gratitude to my supervisors and advisors who have constantly guided, supported and encouraged me: Professor K O Lee, Doctor Paul A. MacAry, Assoc Professor Phan Toan Thang, Doctor Gan Shu Uin and Professor Roy Calne. Prof. Lee has taught me the scientific way of thinking and more importantly, has always inspired me to see science with interest and curiosity, which was my major motivation through these years of research. I am also grateful for the invaluable time he has spent to read and edit this thesis. Dr. MacAry, though not officially my supervisor, has led the immunological studies, which contributed to a large part of this thesis. Prof. Phan generously supplied the cord lining cells for all the experiments of this research project and guided me when I started with animal experiments. Dr. Gan performed all the lentiviral transduction for the cells used in this project and also read and edited this thesis. She has been more than a supervisor but also a caring friend. Prof. Calne has provided invaluable advice on my project every time he visited Singapore. I am also indebted to all the fellow students and colleagues that I have worked with on this thesis project. It is impossible to name them all here. But I feel especially grateful to Lin Gen who worked with me on the immunological experiments and also spent his time going through the first few drafts of this thesis. I benefited a lot from working with Phua Meow Ling, Cindy on the diabetic mouse model and from discussion with Dr. Laura Rivino. I would also like to thank other people from immunology program, including Lim Yan Ting, Fatimah Bte Mustafa, Too Chien Tei and Desmond Chan. Co  iii workers from Prof. Phan’s lab, especially Audery, Khoo Ying Ting and Dr. Anandaroope Mukhopadhyay, taught me the basic molecular biology techniques, and Jeyakumar Masilamani did the primary isolation and culture for cord lining cells together with Prof. Phan. Co-workers from Dr. Gan Shu Uin’s lab, including Diane, Tan Ai Lin, Ngo Kae Siang, Ooi Shu Qin and Fu Zhen Ying, gave me precious mental support during my difficult times and shared with me some of the happiest moments in these past years. Ngo Kae Siang and Fu Zhen Ying also helped me with the animal experiments. In addition, I have received technical assistance from people from adjacent laboratories: Han Hwan Chour helped me troubleshoot the immunohistochemistry experiments. Pradeep Paul Panengad did cryosection for my cord lining cell graft. It was great pleasure to have spent my research years with these people because they were not only supportive at work but also pleasant to spend time with. Finally, I want to thank my mother who has brought me to this world and shaped me as what I am. It was her who planted in my mind the idea of being a scientist and led me all the way to fulfill this goal up to this day.   iv TABLE OF CONTENTS TITLE PAGE …………………………………………………………………….…… .i ACKNOWLEDGMENTS………………………………………………………….… .iii TABLE OF CONTENTS………………………………………………………….…….v SUMMARY…………………………………………………………………………….xiii LIST OF FIGURES AND TABLES………………………….……………………… xv LIST OF PUBLICATION AND PRESENTATIONS…………………………… xix LIST OF ABBREVIATIONS…………………………………………………………xxi CHAPTER 1.1 1.2 Introduction………………………………………………………1 Background………………………………………………………….…………….1 1.1.1 Prevalence of diabetes mellitus……………………………………………1 1.1.2 Pancreas and islet transplantation……………………………………… 1.1.3 Cell therapy……………………………………………………………… 1.1.4 Gene therapy………………………………………………………………5 1.1.5 Immunosuppression……………………………………………………….5 Present study………………………………………………………………………6 CHAPTER Literature Review……………………………………………… 2.1 Embryonic stem cells…………………………………… ……………………….9 2.2 Induced pluripotent stem cells………………………………………………… .13 2.3 Bone marrow derived mesenchymal stem cells………………………………….14 2.3.1   MHC expression…………………………………………………………16 v 2.4 2.3.2 T lymphocytes……………………………………………………………16 2.3.3 Dendritic cells………………………………………………………… 18 2.3.4 B lymphocytes and natural killer cells………………………………….19 Fetal/neonatal stem cells…………………………………………………………20 2.4.1 Background………………………………………………………………20 2.4.1.1 Immune tolerance in pregnancy………………………………….21 2.4.1.2 HLA-G in immune tolerance…………………………………….23 2.4.1.2.1 HLA-G receptors……………………………………………… .23 2.4.1.2.2 HLA-G functions……………………………………………… .24 2.4.1.2.3 In vivo evidence of HLA-G function…………………………….27 2.4.1.3 HLA-E……………………………………………………………28 2.4.1.4 Pregnancy related tissues in cell therapy……………………… .28 2.4.2 Amniotic fluid stem cells……………………………………………… .29 2.4.3 Placental stem cells………………………………………………………34 2.4.3.1 Amniotic epithelial cells…………………………………………35 2.4.3.2 Amniotic mesenchymal cells…………………………………….37 2.4.4 Umbilical cord blood stem cells…………………………………………39 2.4.5 Wharton’s jelly stromal cells…………………………………………….40 2.4.6 Umbilical cord perivascular cells……………………………………… .44 2.4.7 Umbilical cord lining cells……………………………………………….45 CHAPTER 3.1 Materials…………………………………………………………………………49 3.1.1   Materials and Methods…………………………………………49 Primary cells and cell lines………………………………………………49 vi 3.2 3.1.1.1 Cord lining epithelial and mesenchymal cells………………… .49 3.1.1.2 Choriocarcinoma cell lines………………………………………52 3.1.1.3 Primary keratinocytes……………………………………………52 3.1.1.4 Conditioned culture supernatants……………………………… .52 3.1.2 PCR primers…………………………………………………………… .53 3.1.3 Gene transfer constructs………………………………………………….55 3.1.3.1 Gene expression………………………………………………….55 3.1.3.2 Gene silencing……………………………………………………55 3.1.5 Animals………………………………………………………………… 56 3.1.6 Antibodies and kits………………………………………………………57 Methods………………………………………………………………………… 58 3.2.1 Colony formation and rhodamine staining………………………………58 3.2.2 Molecular biology techniques……………………………………………58 3.2.2.1 Protein extraction and quantification…………………………….58 3.2.2.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE)………………………………………………………………………………… 59 3.2.2.3 Western blot…………………………………………………… .60 3.2.2.4 Reverse transcription-polymerase chain reaction (RT-PCR)……60 3.2.2.5 Agarose gel electrophoresis…………………………………… .61 3.2.3 Flow cytometry………………………………………………………… 62 3.2.4 Isolation of the immune cell populations……………………………… .63 3.2.4.1 Density gradient centrifugation………………………………… 63 3.2.4.2 Magnetic-activated cell sorting (MACS)……………………… .64 3.2.5   Stimulated T cell proliferation………………………………………… .64 vii 3.2.5.1 Mitogen stimulation assay…………………………………… .64 3.2.5.2 Mixed leukocyte reaction (MLR)…………………………….64 3.2.5.2.1 MLR in 96-well plate setting…………………………………….65 3.2.5.2.2 MLR in 24-well plate setting…………………………………….65 3.2.5.2.3 CLEC titration experiments…………………………………… .66 3.2.6 [3H] thymidine incorporation assay…………………………………… .66 3.2.7 CFSE cell proliferation assay…………………………………………….66 3.2.8 Multiplex cytokine assay……………………………………………… .68 3.2.9 Stable transduction using lentivial constructs……………………………69 3.2.10 In vivo studies……………………………………………………………69 3.2.10.1 Xenotransplantation for survival study………………………… 69 3.2.10.2 CLEC-based ex vivo gene therapy………………………………70 3.2.10.3 Anesthesia, euthanasia and necropsy…………………………….71 3.2.11 In vitro differentiation toward insulin-producing cells………………… 71 3.2.12 Immunohistochemistry………………………………………………… 72 3.2.12.1 Paraffin sections………………………………… .…………… 72 3.2.12.2 Cytospin………………………………………………………….72 3.2.13 Microscopy………………………………………………………………73 3.2.14 Statistical analysis……………………………………………………….73 CHAPTER In Vitro Studies of Cord Lining Cells………………………….75 4.1 Introduction………………………………………………………………………75 4.2 Results………………………………………………………………………… 76 4.2.1   Morphology and growth properties of cord lining cells……………… 76 viii 4.3 4.2.2 HLA-G and HLA-E protein expression in CLECs………………………78 4.2.3 HLA-G splicing variants expressed in CLECs………………………… 80 4.2.4 HLA-G and HLA-E protein expression in CLMCs…………………… .82 4.2.5 Epithelial and MHC class I markers in CLECs………………………….84 Summary……………………………………………………………………… 86 CHAPTER In Vitro Analysis of Cord Lining Cell Immunogenicity……87 5.1 Introduction…………………………………………………………………… 87 5.2 Results……………………………………………………………………………88 5.2.1 Mitogen stimulated T cell proliferation………………………………….88 5.2.2 Mixed leukocyte reactions (MLR)……………………………………….90 5.2.2.1 Conditioned medium…………………………………………… 90 5.2.2.2 CD4+ versus CD8+ T cells……………………………………….92 5.2.2.3 Co-culture……………………………………………………… 94 5.2.3 T cell apoptosis…………………………………………………………102 5.2.4 CD4+CD25highFoxp3+ regulatory T cells……………………………….104 5.2.5 Other regulatory T cells…………………………… ………………….106 5.2.6 Cytokine profile of MLR……………………………………………….110 5.2.7 HLA-G functional studies…………………………………………… .116 5.2.7.1 shRNA inhibition……………………………………………….116 5.2.7.2 Blocking antibody………………………………………………118 5.2.8 5.3   LPS induced dendritic cell maturation………………………………….120 Summary……………………………………………………………………… 122 ix CHAPTER Xenotransplantation of Cord Lining Cells………………… 125 6.1 Introduction…………………………………………………………………… 125 6.2 Results………………………………………………………………………… 126 6.3 6.2.1 Composition of the cellular graft……………………………………….126 6.2.2 Detection of CLECs after xenotransplantation…………………………128 6.2.3 Neovascularization of CLEC graft…………………………………… .130 6.2.4 Control primary keratinocytes………………………………………….132 6.2.5 Prolonged survival of CLECs in contrast to keratinocytes…………… 134 6.2.6 Re-culturing of CLECs from graft…………………………………… .136 6.2.7 CLEC and keratinocyte co-transplantation…………………………… 138 6.2.8 Detection of CLMCs after xenotransplantation……………………… .140 Summary……………………………………………………………………… 144 CHAPTER Ex Vivo Insulin Gene Therapy using CLECs……………… 145 7.1 Introduction…………………………………………………………………… 145 7.2 Results………………………………………………………………………… 146   7.2.1 In vitro differentiation toward insulin-producing 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Mol Biol Cell 13(12): 4279-95. 202 [...]... 2 Immunological studies of human umbilical cord derived stem cells Zhou Y, Phan T T, Gan S U, Calne R, Lee K O and MacAry P A (2006) Abstract presented at National Healthcare Group (NHG) Annual Scientific Congress 2006 3 Analyzing the immunogenicity of human umbilical cord lining derived stem cells Zhou Y, Phan T T, Gan S U, Calne R, Lee K O and MacAry P A (2006) Abstract presented at World Transplant... sequences for HLA-G gene silencing…………………………… 56   xvii   xviii LIST OF PUBLICATION AND PRESENTATIONS 1 Characterization of human umbilical cord lining derived epithelial cells and transplantation potential Zhou Y, Gan S U, Lin G, Lim Y T, Masilamani J, Mustafa F, Phua M L, Rivino L, Phan T T, Lee K O, Calne R, MacAry P A Accepted 12/30/2010 for publication in “Cell Transplantation”, epub ahead of print 03/24/2011... SUMMARY This study describes the immunological characterization of a novel primary epithelial cell-type from the human umbilical cord Two types of cells can be derived from the umbilical cord lining membrane, termed cord lining epithelial cells (CLECs) and cord lining mesenchymal cells (CLMCs) Preliminary immunophenotyping showed that CLECs but not CLMCs express significant levels of HLA-G isoforms and. .. major cause of premature illness and death in most countries, mainly through the increased risk of cardiovascular disease Other complications such as chronic nephropathy, retinal damage and neuropathy, lead to conditions as severe as kidney failure, blindness and limb amputation The frequency of diabetes is rising dramatically all over the world and has been recognized as a global epidemic In 2000, according... molecular marker expressions including the immunosuppressive molecules HLA-G and HLA-E 6 2 To study the immunological properties of cord lining cells using in vitro assays of T lymphocyte activation and dendritic cell maturation 3 To investigate the mechanisms of cord lining cell and T lymphocyte interactions 4 To study the immunogenicity of cord lining cells in vivo using an immunocompetent mouse model. .. organs/tissues and extreme shortage in their supply Additionally, cell transplantation has the versatility with ease of ex vivo culture and manipulation, potentially incorporating gene therapy into the transplantation approach Hence, novel strategies of using cell therapy and/ or gene therapy for the treatment of diabetes are being studied intensively For the treatment of type 1 diabetes, the areas of cell therapy... S U, Calne R, Lee K O and MacAry P A (2007) Oral presentation at 13th congress of the European Society for Organ Transplantation (ESOT)   xix   xx LIST of ABBREVIATIONS ABSL animal biosafety level AEC amniotic epithelial cell AF amniotic fluid AFSC amniotic fluid stem cell AHU animal holding unit AMC amniotic mesenchymal cell APC allophycocyanin balb bagg albino BLAST basic local alignment search tool... therapies Transplantation of isolated islets has also been used as an alternative to whole pancreas transplantation, which involves much less invasive surgical procedures However, both pancreatic and islet transplantations are limited by the lack of suitable donor organs/tissues The relatively new discipline of cell transplantation has the potential to fill the gap between increasing need of donor organs/tissues... success has been reported mainly with multipotent mesenchymal stem cells (MSCs) MSCs derived from bone marrow (Gabr et al 2008), Wharton’s jelly (Chao et al 2008) and adipose tissues (Chandra et al 2009) have been differentiated into insulin producing clusters Successful applications of those MSC -derived insulinproducing cells in treatment of diabetic animals have also been reported Problems that remain... hyperglycemia and is sometimes required in the treatment of type 2 diabetes as well However, patterns of physiologic insulin release are very hard to mimic with injections of exogenous insulin Even with the use of insulin premixes and analogs and close glucose monitoring, the risks of postprandial 1 hyperglycemia and hypoglycemia are present and long-term complications may still develop 1.1.2 Pancreas and .  i IMMUNOLOGICAL CHARACTERIZATION OF HUMAN UMBILICAL CORD LINING DERIVED CELLS AND THEIR THERAPEUTIC APPLICATION IN A DIABETIC MOUSE MODEL ZHOU YUE A THESIS SUBMITTED.  xviii  xix LIST OF PUBLICATION AND PRESENTATIONS 1. Characterization of human umbilical cord lining derived epithelial cells and transplantation potential. Zhou Y, Gan S U, Lin G, Lim Y T, Masilamani. CLECs has been tested in the treatment of a type 1 diabetes mouse model.  xv LIST OF FIGURES AND TABLES Figure 3.1 Schematic of human umbilical cord and the isolation of cord lining cells