UCSF UC San Francisco Electronic Theses and Dissertations Title Human Trophoblast Differentiation and Invasion During Normal Pregnancy and Preeclampsia Permalink https://escholarship.org/uc/item/9vf1b2w9 Author Hunkapiller, Nathan Michael Publication Date 2011 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California ii Acknowledgments The success of my graduate work at UCSF is the result of an amazing network of people who have supported me throughout my time here. First, I would like to acknowledge all the members of Fisher lab who have been teachers, collaborators, and friends. Given the large size of our lab, the majority of my scientific training has come from our most experienced members; I recognize that I would never have come half as far as I have in this lab without learning techniques from and sharing ideas with Yan Zhou, Aka Prakobphol, Olga Genbacev, Kristy Red-Horse, and Miko Kapidzic. Because the leaps and bounds of scientific research tend to be few and far between, I am grateful to have worked with such personalities that lifted my spirits no matter the day. Kit Nazor, Matt Donne, and Gabe Goldfien were always in the mood to either talk science or just have a good time, whichever the situation required. My thesis committee members, Zena Werb and Rong Wang, were very generous to offer me their time and energy. I am thankful for their insightful project guidance and willingness to speak with me whenever I needed help. I doubt I would be a scientist if it were not for my family. There are many Hunkapillers that are thriving in science and, fortunately, I have always had them to look up to and emulate as models of how successful I wanted to be. I hope my Dad, Mike, knows how proud I am of his accomplishments and also that his career in science has had everything to do with mine. My big sister, Kathryn, was also clearly someone I wanted to take after, given that I followed her through every step of my education until now (elementary school, middle school, high school, college, and even working at Applied Biosystems after graduating college). As the only non-scientist in my immediate family, my mom, Beth, is responsible, among many other great things, for nurturing my iii social development in addition to my scientific development. I am grateful for such an amazing and generous family, unwavering in their support of me. I can’t say where I would be in life without my wife, Julie. No other person is more responsible for my happiness and my desire to be successful. As she is also a graduate student as UCSF, we have bolstered each other through the daily ups and downs of research. I am thankful that I can share every aspect of my life, including work, with her. Finally, I am thrilled to have chosen Susan Fisher as my graduate advisor (and that she let me join her lab!). Her warmth and passion for science struck me the first time I met her and has always been uplifting. As a former graduate student in the lab once said, ―Susan may not have much time to meet with you, but when you need her help, everything she tells you is scientific gold.‖ Nothing could have been more true. If I ever felt lost in my work, I need only go talk with Susan and soon I was back on track. I could not imagine a more brilliant, kind, generous, and encouraging science and life coach, and Susan knows how to have a good time, too! I am sure that we will maintain a great relationship throughout my career. iv Human Trophoblast Differentiation and Invasion During Normal Pregnancy and Preeclampsia By Nathan Hunkapiller Graduate Advisor: Susan J. Fisher, Ph.D. This work stems from our group’s interest in identifying the mechanisms that govern placental function in normal human pregnancy and in pregnancy complications. Cytotrophoblasts (CTBs) carry out many of the placenta’s most important functions. CTBs in contact with the uterus differentiate and subsequently invade the uterine stroma and blood vessels, a process that anchors the placenta to the uterus and redirects maternal blood toward the embryo/fetus. During differentiation/invasion, CTBs undergo phenotypic changes that result in vascular mimicry and display a marked arterial tropism. However, in preeclampsia (PE) CTB differentiation goes awry, and CTB invasion, particularly the arterial component, is shallow. First, we theorized that molecules involved in arterial differentiation/function might play a role during vascular invasion. We found that expression of Notch family members, which play important roles during arterial differentiation, was extensively modulated during human CTB invasion. Inhibition reduced CTB invasion and expression of an arterial marker. Using mouse models, we confirmed that Notch activity was highest in artery-associated trophoblasts. Analysis of mouse trophoblast stem cell differentiation in vitro suggested that Notch2 played an important role in remodeling the uterine arterioles in this species. Conditional deletion of Notch2 in invasive trophoblast lineages highlighted its central importance in coordinating increases in utero-placental blood flow; trophoblast invasion of maternal arterioles failed, the blood canals that supply the placenta were smaller, and placental perfusion was decreased. Lastly, we asked whether defects in CTB expression of Notch family members were evident in PE. An v absence of endovascular TB expression of the Notch ligand, JAG1, was frequently observed, suggesting that failures in Notch signaling are an important part of the pathogenesis of this condition. Second, we theorized that dysregulated CTB differentiation underlies preeclampsia. To test this hypothesis, we compared gene expression in differentiating CTBs derived from normal and PE placentas. Molecules known to be dysregulated in PE as well as novel candidate regulators were differentially expressed. Of these targets, we performed an expanded functional analysis on SEMA3B. The results helped to explain alterations in CTB and endothelial cell (EC) phenotypes that are commonly observed in PE. Last, we designed and tested high throughput methods to profile miRNA expression. These were applied in studies we initiated to probe the molecular mechanisms involved trophoblast differentiation and to identify novel serum biomarkers associated with PE. First, we performed experiments to optimize our experimental workflow, in which we measured the quantitative sensitivity of the Taqman miRNA expression assays in a variety of experimental conditions. Next, we identified miRNAs expressed by differentiating CTBs from normal and preeclamptic pregnancies. Lastly, we found many miRNAs that were differentially expressed in serum samples from women who experienced normal pregnancies or those complicated by PE. We believe that the results of the aforementioned experiments will enable many new research directions in the study of the maternal-fetal vascular interactions that occur during normal placentation and in pathological pregnancies. vi Table of Contents Page Chapter 1: Introduction………….…………………………………………………………….1 Early Embryonic Development and Uterine Attachment………………….………...1 Implantation and Placental Morphogenesis………………………………….………3 Interstitial and Endovascular Invasion at the Maternal-Fetal Interface……….…...5 Cellular and Molecular Aspects of CTB Differentiation and Invasion……….….....7 Preeclampsia, a Placental Pathology Associated with Faulty CTB Differentiation/Invasion…………………………………………………………………9 Notch Signaling, Vascular Development, and Disease-Associated Defects…...11 Mouse Placentation……………………………….…………………………………..15 Chapter 2: Review of Methods Useful in the Study of Cytotrophoblast Endovascular Invasion………………………………………………………………………24 Introduction…………………………………………………………………….……….24 Isolation and Culture of Human Cytotrophoblasts…………………………….…..24 Cytotrophoblast Isolation Procedure………………………………………………...25 Enzymatic Digestions, Time Considerations………………………………….…….29 Isolation and Culture of First Trimester Human Placental Villous Explants.…….30 Identification of Cytotrophoblast-Modified Blood Vessels in Tissue Sections…..31 Cytotrophoblast Migration and Induction of Endothelial Cell Apoptosis During Co-culture………………………………………………………………………34 In Vitro Models of CTB Endovascular Invasion Using Explanted Spiral Arterioles………………………………………………………………………………..36 In Vivo Models of Human Cytotrophoblast Vascular Remodeling………….……40 vii Chapter 3: A Role for Notch Signaling in Trophoblast Endovascular Invasion and in the Pathogenesis of Preeclampsia……………………………………………………..49 Abstract………………………………………………………………………………...49 Introduction….…………………………………………………………………………49 Results….………………………………………………………………………………53 CTBs Modulated Expression of Notch Molecules as they Differentiated/Invaded In Vivo and In Vitro..……………………………….53 Notch Signaling Regulated CTB Invasion and Expression of EFRNB2, an Arterial Marker………………………………….………………………….54 Placental Notch Activity is Largely Confined to the Mouse Ectoplacental Cone and Endovascular Trophoblasts..…………………..………………..56 Notch2 Deletion in Invasive Trophoblast Lineages Led to Litter-wide Lethality in Proportion to the Number of Mutant Offspring...……………..57 In the Absence of Notch2, TGCs and GlyTCs Failed to Invade Maternal Spiral Arterioles, which was Associated with Reduced Canal Size and Placental Perfusion……………………….…………………………………..60 Spiral Artery-Associated CTBs Failed to Express JAG1 in PE……….....62 Discussion…………….………………………………………………………………..63 Methods……………….………………………………………………………………..86 Chapter 4: CTB Gene Expression Profiling