FORMATION AND FUNCTION OF INVASIVE SYNCYTIOTROPHOBLAST FROM HUMAN EMBRYONIC STEM CELLS NANI DJUNAIDI NATIONAL UNIVERSITY OF SINGAPORE 2014 FORMATION AND FUNCTION OF INVASIVE SYNCYTIOTROPHOBLAST FROM HUMAN EMBRYONIC STEM CELLS NANI DJUNAIDI (B.Sc (Cum laude), ATMAJAYA CATHOLIC UNIVERSITY OF INDONESIA) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2014 ACKNOWLEDGEMENT My journey in writing this doctoral thesis has been one of the most challenging processes in my life and obviously it would not have finished without supports from many people that were sincerely put everything to help me in this “academic marathon”. Above all, I dedicate this works to The Holy Trinity, which have prepared me this very challenging yet beautiful journey in my life. I also would like to give my utmost gratitude to A*STAR, NUS, and GIS for giving me this precious opportunity being able to pursue and finish my Doctor of Philosophy in this amazing and scientific- conducive country, Singapore. Never ending appreciation and gratitude also given to my astounding and supportive supervisor, Dr. Paul Robson, who without his precious advises and continuous supports, I would not have confidence to pursue and reach this point. Because of his invaluable guidance, I would be able to develop into one independent researcher. Also, most thankful to my scientific advisor panel in GIS- NUS and my graduate advisory committee, Prof. Neil Clarke, Prof. Tara Huber, Dr. Liou Yih-Cherng, Dr. Lawrence Stanton, and Dr. Wa Xian, which their critical evaluation and supportive advices, had made me to appreciate the complex beauty of science. I would like to thank my lab-mates in Developmental Cellomics Laboratory, especially Dr. Wishva Herath for his valuable bioinformatics guidance and help, which one of my thesis foundation bricks based on his interesting work. Also my gratitude goes to Dr. V Sivakamasundari, who even I recently know, but has been very nice and helpful towards me in giving advices and initiating inspiring talks. I would to extend my gratitude to my PhD-mates in the group for their continuous advices and scientific talks, Dr. Erlyani Abdul Hamid and Mehran Rahmani. I would i also thank Sun LiLi and Jameelah Sheik Mohammed for their bountiful help in executing my project in this lab. For priceless supports and keeping me up when I had my (many) down in this PhD journey, Rani D/O Ettikan my “mother” in GIS, without your caring and supports, I am not sure if I had survive when the experiments and environment failing me. I also would like to thank Dr. Irene Aksoy for the priceless and useful advices when I have trouble in my works. For my cheerful and supportive peers, Chen JiaXuan, Sathiyanathan Padmapriya, Lavanya Adusumilli, Wong WanQing, and LinLin, which without your presence, this lab or level 8 would never be the same and thank you for all your kind ears and shoulders when I felt at lost. My works also would not be possible without the support from my more than best friend, Patrick Tahya, thank you for all your kind encouragement and companion during this long PhD. Outside of the lab, I also would like to dedicate this thesis to my spiritual community friends and my second family in Singapore, AmoreDio, thank you for keeping me on the ground and my faith strong in perseverance. Thank you for making my life in Singapore far more better with your presences. At last but not least, I fully dedicate this doctoral thesis to my beloved family, my dad, mom, and my brother. Never ending gratitude for always there for me, support me when I am down and feel like giving up everything. Thank you for your endless support and love towards your daughter. Hopefully, after this entire journey, I could make both of you proud at the end of the day. I love you. “I have fought the good fight, I have finished the race, I have kept the faith.” - 2 Timothy 4:7 - ii TABLE OF CONTENTS Acknowledgements i Table of Contents iii List of Tables vi List of Figures vii List of Abbreviations ix List of Publication xii Summary xiii Chapter 1: Introduction 1 1.1 The Human Placenta and Pregnancy Pathology in Early Implantation 1 1.2 Origin and Development of the Human Placenta 7 1.2.1 Hourglass Model of Evolution and Development 13 1.2.2 Unique Characteristics and Functions of the Human Placenta 18 1.2.3 Trophoblast-derived Cell Types in the Placenta 30 1.2.4 Human Embryonic Stem Cells as a Tool to Study the Trophoblast Lineage 31 1.3 Trophoblast Lineage Derived from Human Embryonic Stem Cells 34 1.3.1 Human Embryonic Stem Cells (hESC) 34 1.3.2 Human Embryonic Stem Cell-Derived Trophoblast 36 1.4 The Syncytiotrophoblast 38 1.4.1 Types of Human Syncytiotrophoblast 39 1.4.2 The Invasive Syncytiotrophoblast at Implantation 40 1.4.3 Suggested Molecular Pathways in Syncytiotrophoblast 41 1.4.4 Mouse Syncytiotrophoblast in Relation to Human 43 iii 1.5 Purposes and Hypothesis of Study 45 Chapter 2: Material and methods 48 2.1 Human Embryonic Stem Cell Culture 49 2.2 Human Embryonic Stem Cell Differentiation towards Trophoblast Lineage 50 2.3 Signaling Molecules Screening from RNA Sequencing 51 2.4 Human Embryonic Stem Cells-derived Trophoblast Differentiation towards Invasive Syncytiotrophoblast 52 2.4.1 High Content Screening (HCS) 53 2.4.2 Fusion Rate Quantification using immunofluorescence Confocal Microscopy 57 2.4.3 Validation of Invasive Syncytiotrophoblast Markers using Immunofluorescence Microscopy 58 2.4.4 Validation of Invasive Syncytiotrophoblast Markers using Western Blot 60 2.4.5 Detection of Human Chorionic-Gonadotrophin (hCG) Production 61 2.5 Molecular Validation of Human Trophoblast Lineage 62 Chapter 3: Results 1: Signaling Molecules Selection from High Content Screening 65 3.1 High Content Screening Background 66 3.2 Cell Culture Optimization 71 3.3 Culture Growth Media Optimization 80 3.4 HCS Software and Measurement Parameters Optimization 83 3.5 HCS Results Analysis and Selection of Growth Factor(s)/ Cytokine(s) 91 Chapter 4: Results 2: Qualitative and Quantitative Validation of Syncytiotrophoblast 118 iv Chapter 5: Discussions 136 4.1 Signaling Molecules in Invasive Syncytiotrophoblast Formation 137 4.2 Molecular Pathways in Syncytiotrophoblast Invasiveness 148 Conclusions and Future Works 155 References 160 v LIST OF TABLES Table 2.1. Morphology features selected for measurement in HCS Cellomics® ArrayScan® VTI Reader. Table 2.2. List of antibodies used to validate human invasive syncytiotrophoblast markers. Table 2.3 Reverse transcriptions PCR master mix composition (a) and thermal cycler machine condition (b). Table 2.4 List of TaqMan Assay probes that were used in the RT-PCR. Table 3.1. Selection of various basal medias for signaling molecules trophoblast differentiation in HCS system. Table 3.2. Various morphology features detection and measurement based on channel selection in HCS system. Table 3.3. Short-listed potential signaling receptors and the respective ligands. vi LIST OF FIGURE Figure 1.1. The timeline of human embryonic development prior to implantation. Figure 1.2. The Barnes embryo from Carnegie Institute collection Figure 1.3. The comparison of four widely accepted embryonic conservation models. Figure 1.4. The timeline and morphology comparison between mouse (top) and human (bottom) embryo. Figure 1.5. The schematic comparison between mouse (top) and human (bottom) embryonic implantation process. Figure 1.6. in vitro human blastocyst implantation (left) and in vivo of macaque blastocyst implantation (right). Figure 1.7. A tissue section of a human blastocyst implanting into the maternal uterine wall. Figure 1.8. The Barnes embryo tissue section. Figure 2.1. The 96 wells culture plate arrangement of various signaling molecules treatment for HCS platform. Figure 2.2. The calculation formula to quantify the percentage of the fusion rate. Figure 3.1. The High Content Screening (HCS) experimental pipeline from addressing biological question to generate cellular knowledge. Figure 3.2. The experimental outline in hESC differentiation for further trophoblast sub-types derivation. Figure 3.3. The morphology of H1 pluripotent stem cells (a) and H1-differentiated trophoblast population (b). Figure 3.4. The morphology of H1-differentiated trophoblast by SB treatment for 4 days. Figure 3.5. The morphology comparison of H1-differentiated trophoblast using SB- mTeSR (a) and SB standard differentiation protocol (b). Figure 3.6. The comparison of H1-differentiated trophoblast cells upon sub-culture, ROCK inhibitor incubation prior to sub-culture (a) and without ROCK inhibitor incubation (b). Figure 3.7. The comparison of different seeding number of H1-differentiated trophoblast cells 24 hours upon sub-culture. Figure 3.8. The comparison of cells quantity between cultures that were grown 72 hours in serum-containing media and serum-free media. Figure 3.9. The systematic outline of the process of defining cellular processes in HCS system. Figure 3.10. The faults in cell identification due to incorrect parameter values input. Figure 3.11. The optimized summary of assay parameters value for proper both cell- nuclei identification and measurement. Figure 3.12. The schematic Bounding Box for cell area, length, and width measurement. vii Figure 3.13. The schematic for cell area and nuclei area measurements. Figure 3.14. The Isoform switch of FGFR2 in H1 differentiation towards trophoblast lineage. Figure 3.15. Summary of quantitative and qualitative results of HCS for each signaling molecule and combination on H1-differentiated trophoblast culture treatment (a) – (n). Figure 3.16. Representative quantitative measurement of morphology features from 24 hours (a) and 48 hours (b) signaling molecules treatment. Figure 4.1. The fusion rates measurement by confocal microscopy. Figure 4.2.