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Evaluation of Small Molecules for Neuroectoderm Differentiation & Patterning Using Factorial Experimental Design

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Evaluation of Small Molecules for Neuroectoderm Differentiation & Patterning Using Factorial Experimental Design Evaluation of Small Molecules for Neuroectoderm differentiation & patterning using Factorial Experimental Design Master Thesis in Applied Physics For the degree of Master of Science in Biotechnology DIMITRIOS VOULGARIS Department of Physics, Division of Biological Physics CHALMERS UNIVERSITY OF TECHNOLOGY Göteborg, Sweden 2016 Master thesis in Applied Physics Evaluation of Small Molecules for Neuroectoderm differentiation and patterning using Factorial Experimental Design Dimitrios Voulgaris Department of Physics Division of Biological Physics CHALMERS UNIVERSITY OF TECHNOLOGY Göteborg, Sweden 2016 Evaluation of Small Molecules for Neuroectoderm differentiation and patterning using Factorial Experimental Design DIMITRIOS VOULGARIS © DIMITRIOS VOULGARIS, 2016 Supervisor: Anders Lundin, Industrial PhD candidate, Astra Zeneca and Karolinska Institutet Examiner: Julie Gold, Associate Professor, Division of Biological Physics, Department of Physics, Chalmers University of Technology Master thesis for the degree of M.Sc. in Biotechnology Division of Biological Physics Department of Physics Chalmers University of Technology SE-142 96 Göteborg Sweden Telephone +46 (0)31-722 1000 Cover: hiPSCs differentiated for 4 days on LN-521 in neural induction N2B27 medium stained with DAPI (blue) and the intermediate filament Nestin (green). Printed by Chalmers Reproservice Göteborg, Sweden 2016 Evaluation of Small Molecules for Neuroectoderm differentiation and patterning using Factorial Experimental Design DIMITRIOS VOULGARIS Department of Physics Chalmers University of Technology Evaluation of Small Molecules for Neuroectoderm differentiation and patterning using Factorial Experimental Design DIMITRIOS VOULGARIS Department of Physics Chalmers University of Technology ABSTRACT Screening for therapeutic compounds and treatments for diseases of the Brain does not only encompass the successful generation of iPS-derived homogenous neural stem cell populations but also the capacity of the differentiation protocol to derive on-demand region-specific cells. Νoggin, a human recombinant protein, has been extensively used in neural induction protocols but its high production costs and batch-to-batch variation have switched the focus to utilizing small molecules that can substitute noggin. Resultantly, the aim of this study was to optimize neuroepithelial stem cell generation in a cost-efficient fashion as well as to evaluate the impact that patterning factors (i.e. small molecules or proteins that enhance the emergence of type-specific neuronal populations) have on the regionality of the neural stem cell population. Findings in this study suggest that DMH1 is indeed a small molecule that can replace noggin in neural induction protocols as previously documented in literature; DMHI appears also to have a ventralizing effect on the generated neural population. Keywords: induced pluripotent stem cells, neuroectoderm differentiation, small molecules, factorial experimental design. Acknowledgements Firstly, I would like to express my gratitude to my thesis advisor, PhD candidate, Anders Lundin (Karolinska Institutet and AstraZeneca), for his continuous guidance, motivation and excellent support throughout this thesis, his inspirational tutoring was pivotal to the successful completion of this thesis. I would also like to thank my academic thesis advisor Associate Professor, Julie Gold (Department of Physics, Chalmers University of Technology), for her guidance in the writing of this thesis as well as her comments which always posed a source for improvement of my thought process and academic writing. I am deeply grateful to Cecilia Boreström and Anna Jonebring for all the scientific discussions throughout my thesis. Moreover I would like to express my sincere appreciation to Louise Stjernborg, Anette Kry-Persson and Anna Svensson for their advice and support in the lab. I would like to thank Josefina Kristensson, Märta Jansson, Louise Delsing and Cecilia Graneli for the wonderful company in the office during my stay at AstraZeneca and Ryan Hicks and the rest of the iPSC team for welcoming me into the team. Last but not least I would like to thank my family for believing in me and for all the support they have provided me throughout the years, the greatest gift I have ever and will ever receive. I dedicate this thesis to my father. Abbreviations hiPSCs: Human Induced pluripotent stem cells hESCs: Human embryoid stem cells TGFβ: Transforming growth factor beta BMPs: Bone morphogenetic proteins SMAD: Portmanteau of the protein SMA (Caenorhabditis Elegans) and the Drosophilia protein Mothers Against Decapentaplegic (MAD) R-SMAD: Receptor-regulated SMAD co-SMAD: Common-mediator SMAD ICC: Immunocytochemistry PCR: Polymerase Chain Reaction EB: Embryoid Body ECM: Extracellular Matrix ICM: Inner Cell Mass GW: Gestational week PNS: Peripheral Nervous System SHH: Sonic Hedgehog MHP: Median Hinge Point NES cells: Neuroepithelial stem cells Wnt: Wingless-related integration site NI: Neural Induction SMs: Smalls Molecules FED: Factorial Experimental Design ALK: activin receptor-like kinase Fz: Frizzled Table of Contents Chapter 1: Purpose and Significance of the Study ......................................................... 1 1.1 Introduction ......................................................................................................... 1 1.2 Aim ....................................................................................................................... 2 1.3 Limitations............................................................................................................ 2 Chapter 2: Background & Literature Overview .............................................................. 3 2.1 Embryogenesis ..................................................................................................... 3 2.2 Induced Pluripotent Stem Cells ........................................................................... 6 2.3 Brain development .............................................................................................. 7 2.3.1 Ectodermal fate ................................................................................................ 7 2.3.2 Primary & Secondary neurulation .................................................................... 8 2.3.3 Dorsal-ventral polarization ............................................................................... 9 2.4 Neuroectoderm Induction ................................................................................. 13 2.5 TGFβ signaling pathway ..................................................................................... 14 2.6 Wnt signaling pathway ...................................................................................... 16 2.7 Small Molecules ................................................................................................. 17 Chapter 3: Materials & Methods .................................................................................. 19 3.1 Cell culture ......................................................................................................... 21 3.1.1 Neuroectoderm Induction protocol ............................................................... 21 3.2 Immunocytochemistry ....................................................................................... 23 3.2.1 Data credibility and ICC controls .................................................................... 23 3.2.2 ICC in this study .............................................................................................. 23 3.3 Polymerase Chain Reaction ............................................................................... 25 3.3.1 Real-time quantitative PCR ............................................................................ 25 3.3.2 Sample Preparation ........................................................................................ 25 3.4 Factorial Experimental Design ........................................................................... 27 Chapter 4: Results......................................................................................................... 29 4.1 Evaluation of the standard noggin-containing protocol .................................... 29 4.1.1 Immunocytochemistry and qPCR ................................................................... 30 4.2 Factorial Experimental Design: Evaluation of alternative BMP-4 inhibitors. .... 35 4.3 Evaluation of optimized protocol and comparison to the standard protocol ... 44 Chapter 5: Discussion ................................................................................................... 50 5.1 Evaluation of the standard protocol .................................................................. 50 5.2 Evaluation of the optimized protocol ................................................................ 51 5.3 Factorial Experimental Design ........................................................................... 52 Chapter 6: Conclusion & Future Work ......................................................................... 53 References……………………………… ………………………………………………………………………………….54 Appendix 1 - Materials ....................................................................................................... 59 Appendix 2 - Reagents ....................................................................................................... 59 Appendix 3 - Seeding densities on day 4 for the standard protocol ................................. 60 Chapter 1: Purpose and
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