IN VITRO DIFFERENTIATION OF STEM CELLS TOWARDS ISLET-LIKE CELLS NGO KAE SIANG (B.Sc., National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2011 i ii ACKNOWLEDGEMENTS First of all, I want to thank my supervisors and advisors Professor Lee Kok Onn, Dr Gan Shu Uin, Professor Roy Calne, Dr Susan Lim, Dr Kerrie Tang, Dr Fong Chui Yee, and Associate Professor Phan Toan Thang Prof Lee has taught me the scientific way of thinking I am grateful for the invaluable time he spent to read and edit this thesis I want to thank Dr Gan Shu Uin It has been an honor to be her student I appreciate all her support, guidance and encouragement, patience and understanding during moments of personal difficulties She has taught me, both consciously and unconsciously, how good experiments are done I thank her for delivering knowledge selflessly, and spending time to read and edit my thesis She has been more than a supervisor She’s been an encouraging mentor, warm colleague, and caring friend The enthusiasm Professor Roy Calne has for research has always inspired me to see science with interest and curiosity I appreciate his contributions of ideas and funding to make my graduate experience productive and stimulating I would like to thank Dr Susan Lim and Dr Kerrie Tang for supplying the adipose tissue derived stem cells for the experiments of this project Dr Fong has generously supplied the Wharton’s Jelly derived stem cells for all the experiments of this research project Prof Phan also generously supplied the cord lining stem cells for the experiments In addition, I want to thank Dr Ma FengJuan and Jayavani D/O Karuppasamy for isolating and culturing adipose tissue derived stem cells I would like to thank Jeyakumar Masilamani, who did the primary isolation and culture for cord-lining cells with Prof Phan I would also like to thank Arjunan Subramanian who cultured the Wharton’s Jelly iii derived stem cells I would also like to thank Division of Regenerative Medicine, Pfizer for help with analysis of Figure 4.13 I would like to thank my labmates in Phoenix group: Diane Tan Ai Lin, Ooi Shu Qin, Zhou Yue, Loke Wan Ting and Fu ZhenYing for their assistance, friendship and happy memories in the past few years Diane and Shu Qin have given me precious mental support during my hard times I am also very grateful to my friend, Teo Peck Lian, for her love, listening ears, support, care, and understanding Last but not least, I want to thank my family, for their love and encouragement, and supporting me unconditionally iv TABLE OF CONTENTS TITLE PAGE ………………………………………………… …………………… i ACKNOWLEDGEMENTS iii TABLE OF CONTENTS v SUMMARY .xi LIST OF FIGURES AND TABLES xiii LIST OF PRESENTATIONS .xv LIST OF ABBREVIATIONS xvi CHAPTER Introduction and Literature Review .1 1.1 Background 1.1.1 1.2 1.3 1.4 What is diabetes? .1 Currently available Diabetes Treatments .3 1.2.1 Whole organ transplantation 1.2.2 Human pancreatic islet transplantation Gene therapy 1.3.1 In vitro gene therapy 1.3.2 In vivo gene therapy Stem cell therapy 10 1.4.1 Pancreas development 11 1.4.2 Human embryonic stem cells 14 1.4.3 Induced pluripotent stem cells 16 1.4.4 Mesenchymal stem cells 18 v 1.4.4.1 Bone marrow derived mesenchymal stem cells 18 1.4.4.2 Pancreatic stem cells 19 1.4.4.3 Amnion-derived stem cells 21 1.4.4.4 Adipose tissue derived mesenchymal stem cells 22 1.4.4.5 Wharton’s jelly derived mesenchymal stem cells and cord-lining stem cells 24 1.5 Objectives of the present study 26 CHAPTER Materials and Methods 27 2.1 Materials 27 2.1.1 Primary cells and cell lines 27 2.1.1.1 Human embryonic kidney 293T cells 27 2.1.1.2 Cord lining stem cells 27 2.1.1.3 Wharton’s jelly derived mesenchymal stem cells……………… 27 2.1.1.4 Adipose tissue derived mesenchymal stem cells……………….28 2.2 2.1.2 RT-PCR primers 29 2.1.3 Antibodies and assay kits 30 Methods 31 2.2.1 In vitro differentiation towards insulin-producing cells 31 2.2.1.1 3-step differentiation protocols ………………………… 31 2.2.1.2 1-step 7-day differentiation protocols .………………….31 2.2.2 Reverse transcription-polymerase chain reaction (RT-PCR) 32 2.2.3 Agarose gel electrophoresis .33 vi 2.2.4 Flow Cytometry .33 2.2.5 Enzyme-linked immunosorbent assay (ELISA) 33 2.2.6 Cytospin 34 2.2.7 Immunocytochemistry .34 2.2.8 Dithizone Staining 34 2.2.9 Microscopy……………………………………………………… … 35 CHAPTER Characterization of Cord Lining Stem Cells (CLSCs), Wharton’s Jelly Derived Mesenchymal stem cells (WJSCs), and Adipose Tissue Derived Mesenchymal Stem Cells (ADSCs) .36 3.1 Introduction 36 3.2 Results 37 3.3 3.2.1 Morphology of CLSCs .37 3.2.2 Morphology of WJSCs 38 3.2.3 Morphology of ADSCs 39 3.2.4 Fluorescence-activated cell sorting (FACS) analysis for MSC markers 40 3.2.5 Sox17 expression of WJSCs 45 3.2.6 Nestin expression of CLSCs, WJSCs, and ADSCs 48 3.2.7 Nanog, Oct4, SOX2 expression of CLSCs, WJSCs, and ADSCs 50 Summary 53 vii CHAPTER In Vitro Differentiation of Stem Cells towards Islet-like Cells using the 3-step differentiation protocol 54 4.1 Introduction 54 4.2 Results 56 4.2.1 Differentiation of CLSCs into islet-like cells 56 4.2.1.1 3-step differentiation protocol………………………… ………56 4.2.1.2 Comparison of pancreatic lineage markers of CLSCS before and after differentiation 58 4.2.1.3 Comparison of pancreatic lineage markers of differentiated CLSCs derived from different donors 60 4.2.2 Differentiation of ADSCs into islet-like cells 62 4.2.2.1 3-step differentiation protocol……………………………………62 4.2.2.2 Comparison of pancreatic lineage markers of ADSCs before and after differentiation 64 4.2.2.3 Comparison of pancreatic lineage markers of differentiated ADSCs derived from different donors 66 4.2.3 Differentiation of WJSCs into islet-like cells 68 4.2.3.1 3-step differentiation protocol………………………………….68 4.2.3.2 Comparison of pancreatic lineage markers of WJSCs before and after differentiation 70 4.2.3.3 Comparison of pancreatic lineage markers of differentiated WJSCs derived from different donors……… 72 viii 4.2.3.4 Reproducibility of differentiation of the WJSCs from the same donors……… .74 4.2.4 Expression of PC1/3, PC2, glucagon and somatostatin of CLSCs, ADSCs, and WJSCs after differentiation .76 4.2.5 Expression of Glut2, PC1/3, PC2, glucagon and somatostatin at different stages along the 3-step differentiation pathway .78 4.2.6 Real-time PCR analysis of expression of glucagon and PC1/3 of differentiated WJSCs……………………….……………………………80 4.2.7 Dithizone staining of differentiated islet-like clusters .82 4.2.8 C-peptide release of differentiated CLSCs, WJSCs, and ADSCs……….84 4.2.9 GLP-1 expression of differentiated WJSCs .86 4.2.10 Glucagon immunostaining of uninduced and differentiated WJSCs .87 4.3 Summary 89 CHAPTER In Vitro Differentiation of Stem Cells Towards Islet-like Cells using 1step 7-day differentiation protocol 91 5.1 Introduction 91 5.2 Results 92 5.2.1 Islet-like cells differentiation of WJSCs, CLSCs, and ADSCs using 1-step 7-day differentiation protocol 92 5.2.2 PC1/3, PC2, glucagon, and somatostatin expression of uninduced and differentiated CLSCs, ADSCs, and WJSCs 94 5.3 Summary .96 ix CHAPTER Discussion and Conclusion 97 6.1 Characterization of WJSCs, CLSCs, and ADSCs 97 6.2 In vitro differentiation of CLSCs, ADSCs, and WJSCs towards islet-like cells using 3-step differentiation protocol 100 6.3 In vitro differentiation of CLSCs, ADSCs, and WJSCs towards islet-like cells using 1-step differentiation protocol 104 6.4 Conclusion 104 6.5 Future work 105 REFERENCES .106 x PC1/3 and thus likely to produce GLP-1 (Wilson, Kalamaras et al 2002) However, GLP-1 was not detected using ELISA after subjecting the CLSCs, WJSCs or ADSC to the stage protocol This might be due to the cells were not directed to intestinal fate along differentiation Besides, dipeptidyl peptidase (DPP-4) is an enzyme expressed on the surface of most cell types and plays a major role in GLP-1 degradation The ELISA kit used was to detect the presence of bioactive GLP-1 but not the degraded GLP-1 Presence of DPP-4 in the culture supernatant may lead to GLP-1 degradation resulting in the failure to detect intact GLP-1 In this project, C-peptide was assayed through ELISA to reflect the insulin secretion of differentiated islet-like clusters to into the media instead of measuring insulin available directly The possible insulin uptake from surrounding media that lead to detection of false-positive insulin secretion has been reported in studies of insulin producing cell differentiation from embryonic stem cells (Hansson, Tonning et al 2004; Paek, Morgan et al 2005) C-peptide is the by-product of proinsulin cleavage Proinsulin is cleaved into c-peptide and insulin during proinsulin processing by prohormone convertase Since C-peptide is co-secreted with insulin, concentration of C-peptide was measured to reflect the insulin secretion in the media Dithizone is a zinc-chelating agent that selectively stains for insulin producing cells The possible insulin uptake of cells from media may lead to false positive results In this study, C-peptide was only detected in supernatant of differentiated sample Although there was insulin expression at transcript level, C-peptide was rarely detected through ELISA This might be due to the low expression of insulin of the cells and the level was below the detection level of the kit This finding indicated that the 3- 103 step differentiation protocol will require significant modification in the case of CLSCs, WJSCs and ADSCs for them to produce insulin sufficient for treatment of diabetes 6.3 In vitro differentiation of CLSCs, ADSCs, and WJSCs towards islet-like cells using 1-step differentiation protocol The present study also subjected ADSCs, WJSCs and CLSCs to the 1-step differentiation protocol described by Chiou et al to determine if a shorter period of differentiation could be achieved However, the result was not satisfactory as there were only little clusters formed Both CLSCs and ADSCs could not tolerate this protocol and started to detach and die Furthermore, the pancreatic lineage profiles of these differentiated ADSCs and CLSCs were not comparable to the differentiated clusters underwent 3-stage differentiation protocol Upregulation of expression of glucagon, somatostatin was not detected in ADSCs and upregulation of expression of PC2, and somatostatin was not detected in CLSCs underwent 7-day differentiation protocol The results suggested that the 7-day differentiation protocol was not suitable and further optimization will be required 6.4 Conclusion In conclusion, this study has investigated the potential of WJSCs, CLSCs, and ADSCs to differentiate into islet-like clusters The 3-step differentiation protocol described by Sun et al was shown to upregulate the expression of glucagon in WJSCs in a consistent manner but not in CLSCs and ADSCs However, expression of insulin was not 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