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Modeling Cortical Development and Microcephaly in Human Pluripotent Stem Cells Using Combinatorial Pathway Inhibition

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Modeling Cortical Development and Microcephaly in Human Pluripotent Stem Cells Using Combinatorial Pathway Inhibition Modeling cortical development and microcephaly in human pluripotent stem cells using combinatorial pathway inhibition Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Naresh Mutukula 2019 The research work for this dissertation was performed from July 2014 to May 2019 under the supervision of Dr. Yechiel Elkabetz at the Max Planck Institute for Molecular Genetics in Berlin, Germany. The dissertation was submitted in May 2019 to the Department of Biology, Chemistry and Pharmacy of the Freie Universität Berlin, Germany. 1st Reviewer: Dr. Yechiel Elkabetz Max Planck Institute for Molecular Genetics, Berlin. 2nd Reviewer: Prof. Dr. Sigmar Stricker Freie Universität Berlin Date of disputation: 18th Nov, 2019 Acknowledgements First and foremost, I would like to express my deep gratitude to my supervisor Dr. Yechiel Elkabetz for giving me the opportunity to work in his lab and introducing me to the very exciting world of pluripotent and neural stem cell biology research. I am very grateful to him for his excellent supervision, constant support and immense patience he has shown in both good and bad times during all these years of my PhD. I would like to thank him for all the teachings and discussions including non- academics during the last few years. Secondly, I would like to thank Prof. Dr. Sigmar Stricker for accepting to be the second reviewer of my PhD dissertation. I am very thankful to my good friend Rotem Volkman from Tel Aviv University, who was there for me during the initial phase of my PhD. I always admired his views and suggestions. I would like to thank Dr. Yakey Yaffe for teaching pluripotent and neural stem cell culture techniques and sharing his everlasting infectious enthusiasm towards science. It was a pleasure working with both of them and will always remember the great times we have shared. Toda raba!! I would like to thank Daniel Rosebrock for all the help with the computational analysis for the manuscript and this dissertation. I would also like to thank Amèlia Aragonés Hernández for sharing her views and thoughtful discussions. I would like to thank my wife and colleague Sneha Arora for helping me with the generation of microcephaly mutant cell lines. A huge hug and special thanks for putting up with me all these years of my PhD. Dhanyavaad!! I would like to thank FACS, RNA-Sequencing and Imaging facility of MPIMG, Berlin and Tel Aviv University for all the help and support. I would also like to thank all the previous members of Elkabtez’s group for all the support whenever needed and for being such great people. 3 Finally, I would like to thank my family — my father – Ananda Rao, my mother – Swaroopa Rani and my brothers – Mahesh Kumar and Suresh Kumar for their unconditional love and support during the ups and downs of my life. This wouldn’t have been possible without your constant support. Words fall short to express my love and gratitude to you. 4 Table of Contents Acknowledgements ........................................................................................................................ 3 Table of contents ............................................................................................................................ 5 Abstract (English) ........................................................................................................................... 8 Abstract (Deutsch)........................................................................................................................ 10 1. Introduction ................................................................................................................................ 13 1.1 Early mammalian embryonic development .............................................................. 13 1.2 Neurulation ......................................................................................................................... 13 1.3 In vivo neural induction and patterning ...................................................................... 15 1.4 Role of morphogens in neural induction and patterning ...................................... 18 1.4.1 Transforming Growth Factor (TGF)-β Superfamily ............................................ 18 1.4.1.1 Bone morphogenetic protein (BMP) Signaling ................................................. 19 1.4.1.2 Activin/Nodal TGF- β Signaling ............................................................................ 20 1.4.2 Fibroblast growth factor (FGF) Signaling .............................................................. 20 1.4.3 WNT Signaling .............................................................................................................. 21 1.4.4 Retinoic acid (RA) Signaling ..................................................................................... 21 1.4.5 Sonic Hedge Hog (SHH) Signaling ......................................................................... 21 1.5 Pluripotent stem cells (PSCs) – a powerful platform to study embryogenesis ...................................................................................................................................................... 22 1.6 Neural stem cells (NSCs) .............................................................................................. 23 1.7 NSCs in cerebral cortex development ...................................................................... 24 1.8 Recapitulating in vivo neural induction and patterning in vitro using PSCs .. 27 1.9 Notch signaling and rosette formation - a tool for measuring efficiency of cortical specification ............................................................................................................... 31 1.10 Modeling cortical neuro developmental disorders .............................................. 32 2. Materials and methods .......................................................................................................... 35 2.1 Cell lines ............................................................................................................................. 35 2.2 Reagents ............................................................................................................................ 35 2.3 Molecular biology application kits................................................................................ 37 2.4 Reagents preparation .................................................................................................... 37 2.5 Generation and culture of human embryonic stem cells (hESCs) .................... 39 2.6 Generation and culture of induced PSCs (iPSCs) ................................................. 40 5 2.7 Neural induction and rosette formation of PSCs using the small EBs (sEBs) protocol ....................................................................................................................................... 41 2.8 Derivation of cerebral organoids from human pluripotent stem cells ............... 44 2.9 RNA isolation ..................................................................................................................... 48 2.10 Preparation and Sequencing of RNA Sequencing (RNA-Seq) Libraries ...... 49 2.11 Description of processed RNA-Seq datasets of human brain transcriptomes ...................................................................................................................................................... 49 2.12 RNA-Seq data processing and normalization for single and pooled organoids ................................................................................................................................... 50 2.13 Combined RNA-Seq data analysis for organoid and human brain datasets ...................................................................................................................................................... 50 2.14 Correspondence analysis ............................................................................................ 51 2.15 Differential Expression and Gene Set Enrichment analysis of organoid data sets .............................................................................................................................................. 51 2.16 Single cell RNA-Seq (scRNA-Seq) procedures.................................................... 52 2.16.1 Organoid dissociation ............................................................................................... 52 2.16.2 Single cell library preparation ................................................................................. 52 2.16.3 Single cell RNA-Seq data processing .................................................................. 52 2.16.4 Single cell gene mapping and UMI counting ..................................................... 53 2.16.5 Single cell dataset alignments, tSNE analysis and cell-subpopulation identification .............................................................................................................................. 53 2.17 Immunostaining and confocal imaging .................................................................... 54 2.18 Quantitative PCR (qPCR) analysis .......................................................................... 56 2.19 Measurements and statistical analysis ................................................................... 57 3. Results ........................................................................................................................................
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