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Engineered in Vitro Models of Post-Implantation Human Development to Elucidate Mechanisms of Self-Organized Fate Specification During Embryogenesis

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Engineered in Vitro Models of Post-Implantation Human Development to Elucidate Mechanisms of Self-Organized Fate Specification During Embryogenesis Engineered In vitro models of post-implantation human development to elucidate mechanisms of self-organized fate specification during embryogenesis by Mukul Tewary A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto © Copyright by Mukul Tewary 2018 Engineered In vitro models of post-implantation human development to elucidate mechanisms of self-organized fate specification during embryogenesis Mukul Tewary Doctor of Philosophy Institute of Biomaterials and Biomedical Engineering University of Toronto 2018 Abstract During embryogenesis, cells in different positions of the embryo acquire different fates in a seemingly autonomous process called ‘fate-patterning’. Fundamental studies have identified important signaling molecules (morphogens) that play crucial roles in coordinating developmental fate-patterning, examples include members of the transforming growth factor beta family – like bone morphogenetic proteins (BMPs), and Nodals. However, mechanistic understanding of how these morphogens coordinate fate-patterning remains unclear. Here we aim to apply bioengineering strategies to develop an in vitro model of developmental fate-patterning and employ it to interrogate the underlying mechanisms that govern this critical process. We first developed a robust, high-throughput platform to enable geometric-confinement of adherent cell types and employed it to screen various BMP4 supplemented defined media to identify conditions that coaxed geometrically-confined human pluripotent stem cell (hPSC) colonies to undergo peri-gastrulation-associated fate-patterning. This screen resulted in identification of defined conditions that spatially segregated compartments in the differentiating hPSC colonies expressing fate markers of trophoblast-like, primitive-streak-like, endoderm-like, mesoderm-like, and ectoderm-like tissues. Using a combination of experimental and ii computational-modelling approaches, we identified a stepwise mechanism of reaction-diffusion and positional-information underlying the observed peri-gastrulation-like fate-patterning. Here, a BMP4-Noggin reaction-diffusion network self-organized BMP signaling gradient, and this gradient patterned peri-gastrulation-associated fates in a manner consistent with positional- information. Furthermore, we found that Nodal signaling was necessary to induce the expression of the primitive-streak compartment – the precursor of gastrulation-derived fates. Interestingly, we also observed that Nodal signaling dissected gastrulation-associated and neurulation-associated gene expression profiles in differentiating hPSC lines. Specifically, in differentiating hPSCs, upregulation of Nodal signaling was observed in cells that upregulated a gene profile associated with gastrulation whereas absence of Nodal signaling correlated with upregulation of a neurulation-associated gene profile. We hypothesized that treatment of geometrically-confined hPSC colonies with BMP4 in the absence of Nodal signaling would induce fate patterning associated with neurulation. We observed experimental results consistent with this hypothesis and identified a conserved underlying mechanism of a stepwise model of reaction-diffusion and positional-information underlying the pre-neurulation-associated patterning as well. Taken together this work provides deep insight into how morphogens regulate early developmental stages of human embryogenesis – which have been previously inaccessible for experimentation. iii Acknowledgments Over the course of this PhD project, I have been incredibly fortunate to be mentored by several amazing scientists who have shaped my progress both in this project, and as an early career scientist; and to whom I am deeply indebted. First and foremost, I would like to thank my supervisor, Dr. Peter Zandstra for not only providing me with the opportunity to be trained in one of the best stem cell bioengineering labs in the world, but also for the remarkable level of support and patience that he has displayed both for me, and for this project. Peter, thank you very much for being a shining example and role model of a great scientist and a great leader; and thank you for continuing to challenge and inspire me to be better in many different aspects of my life. I will forever cherish and look back fondly at my time spent in the Zandstra lab. I would also like to thank my committee members – Drs. Janet Rossant, Penney Gilbert, and Aaron Wheeler. Thank you for investing a large amount of your time and effort to provide me with the support and constructive scientific critique I needed, and for remaining invested in my development and progress. Your contributions have been of immense value to how this project has evolved. I would like to convey my deep gratitude to all my remarkably talented lab-mates – Dr. Celine Bauwens, Dr. Charles Yoon, Curtis Woodford, Dr. Elia Piccinini, Dr. Emanuel Nazareth, Jennifer Ma, Joel Ostblom, Dr. Laura Prochazka, Dr. Nafees Rahman, Dr. Nika Shakiba, Nimalan Thavandiran, visiting trainees – Dominika Dziedzicka, Dr. Hirokazu Akiyama, and other colleagues from various other labs whom I have been lucky enough to be around over the course of my graduate training. Being in the company of a group of such high caliber has always motivated me to strive to be a better scientist. In addition, as I have progressed through this project, I have made what I hope are lifelong friends. Finally, I would like to thank my family – who have encouraged and supported me in ways that are far too numerous to list. My mother, Kumudini; my aunt, Anita; my sister, Priyanka – thank you all so very much. iv Table of Contents Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Abbreviations .................................................................................................................. xvii Chapter 1 Introduction .....................................................................................................................1 Introduction .................................................................................................................................1 1.1 Why study fate patterning during embryogenesis? ..............................................................1 1.1.1 Self-organization in embryogenesis .........................................................................1 1.1.2 A fundamental question ...........................................................................................1 1.1.3 Applications in regenerative medicine.....................................................................2 1.2 Signaling pathways in development ....................................................................................2 1.2.1 TGF-beta pathway ...................................................................................................3 1.2.1.1 BMPs and Nodals ......................................................................................3 1.2.1.2 Signaling via SMAD family ......................................................................4 1.2.1.3 Extracellular inhibitors of BMP and Nodal signaling ...............................5 1.2.2 Wnt pathway ............................................................................................................7 1.2.3 FGF pathway ............................................................................................................8 1.3 Brief introduction of early mammalian development ..........................................................9 1.3.1 Pre-implantation development in mice and humans ................................................9 1.3.2 Post-implantation embryonic development ...........................................................11 1.3.2.1 Gastrulation .............................................................................................14 1.3.2.2 Fate patterning in ectoderm / getting set for Neurulation ........................15 v 1.3.2.3 Markers of gastrulation and pre-neurulation ...........................................17 1.4 Mechanisms of Developmental fate patterning .................................................................18 1.4.1 Reaction-Diffusion .................................................................................................18 1.4.2 Positional-Information ...........................................................................................20 1.5 Bioengineering technologies to control cellular environments .........................................22 1.6 In vitro models of early mammalian development ............................................................26 1.6.1 Mouse .....................................................................................................................27 1.6.2 Human ....................................................................................................................29 1.7 Thesis motivation, hypothesis, and approach
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