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Differential Modulation of BMP Signaling by Activin/Nodal and FGF

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Differential Modulation of BMP Signaling by Activin/Nodal and FGF Differential modulation of BMP Signaling by Activin/Nodal and FGF pathways in lineage specification of Human Embryonic Stem Cells Inaugural – Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy, Free University, Berlin by Smita Sudheer from India May, 2010 1st Reviewer: Prof. Dr. Hans Lehrach Max Planck Institute for Molecular Genetics 2nd Reviewer: Prof. Dr. Petra Knaus Free University Berlin Date of PhD Defense: 29-11-2010 Acknowledgement Acknowledgement I extend my appreciation to Prof. Hans Lehrach for admitting me as a PhD student in his department. I would like to thank Dr. James Adjaye for giving me an opportunity to venture into the immensely interesting field of human embryonic stem cell research and also for the helpful comments and corrections. I am grateful to Prof. Heiner Niemann for the fruitful collaborative project on Bovine preimplantation development. I thank Boris for motivating me during my initial days of PhD and whose work has been the basic motivation behind this research work. I am deeply thankful to Prof. Petra Knaus for being my second supervisor and for her critical comments and constructive discussions. I also thank her for conducting biannual symposiums for all her external PhD students, setting a platform for interactions between each other and also with her. Special thanks to Ingo and Karol for helping me in learing mouse preimplantation embryo handling. I thank Beatrix and Rudi for the electron microscopy experiments. I thank Aydah for helping me with Illumna microarray experiments and also being very understanding in terms of urgency. I also thank other animal facility staff as well as the library staff for their support. I extend my gratitude to Aditi, Lukas, Anja, Julia, Thomas, Masoud, Sahar, Marc, Wibke, Anne, Bodo, Sigmar, Pamela, Janett, Sebastian, Anna, Uli, Ingrid and Kathleen for the all the help they have extended, either concerning work or administrative formalities. Thanks to Birgit for taking care of the orders in such an efficient way that sometimes, during the time of urgency, she used to take extra efforts to make sure that the product reaches soon. I thank Frank and Sven for helping me solve computer problems. Both good and bad times were a part of my stay in this laboratory. I thank my colleagues, Justyna, Marc, Guifre and Thore for all their help, especially duing my initial days and all the times we spent together, sharing our PhD experiences and making the work environment very conducive. I would like to thank all my lab members, also including Mei-Chih, Raed, Katharina, Ying, Bjorn, Mathias and Alessandro for extending a helping hand whenever I needed and also for the enjoyable times. Not knowing german language was never a barrier for me in the lab, just because of all my considerate colleagues and I thank all of them for the same. Acknowledgement For a person to perform his best, his happiness in life matters, which is brought about by surroundings, the loved ones, family and friends. I express my deepest gratitude to my beloved parents, brothers and other family members, for their constant support, love and encouragement. I run short of words, in extending my immense gratitude to my life partner and best friend, Raghu Bhushan, whose great support, motivation, love and understanding were the driving forces for me ever since we met, without which accomplishing this goal would have been impossible for me. Also, Raghu has been immensely helpful to me in terms of subject-oriented discussions, critical analysis of the experiments and also in helping me in some experiments in the times of urgency. Special thanks to Antjew, Sandra and Mitko for making the surroundings such a nice place to live, with a beautiful garden as a bonus. I thank all my friends and well-wishers in Berlin and elsewhere for all the moral support and pleasant times together. Contents Table of Contents Abbreviations ....................................................................................................................................... I-III List of Figures ......................................................................................................................................IV-VI List of Tables .......................................................................................................................................... VII Abstract ................................................................................................................................................ VIII Abstract in German ................................................................................................................................ IX 1. Introduction .................................................................................................................................... 1-26 1.1: Human and mouse embryonic stem cells and associated signaling pathways…………………………..3 1.2: Blastocyst formation, development and gastrulation .................................................................. 3 1.2.1: Blastocyst formation ................................................................................................................. 3 1.2.2: Extraembryonic membranes ..................................................................................................... 5 1.2.3: Blastocyst development and Gastrulation ................................................................................ 7 1.2.4: Epithelial mesenchymal transition (EMT) ................................................................................. 8 1.3: Placenta ...................................................................................................................................... 10 1.3.1: Human and mouse (rodent) placenta ..................................................................................... 10 1.3.2: Human Placental development ............................................................................................... 11 1.4: Trophoblast formation and differentiation: ............................................................................... 13 1.4.1: In vitro differentiation of ESCs into trophoblast ..................................................................... 13 1.4.2: Trophoblast stem cells (Cytotrophoblasts) ............................................................................. 14 1.4.3: Syncytiotrophoblast ................................................................................................................ 15 1.4.4: Invasive extravillous cytotrophoblasts (EVTs) ......................................................................... 18 1.5: Signalling pathways: ................................................................................................................... 19 1.5.1: The TGF-β super-family: Activin, Nodal TGF-β and BMPs ....................................................... 19 1.5.1.1: BMP signalling pathway ....................................................................................................... 21 1.5.1.2: ACTIVIN/NODAL signaling pathway ...................................................................................... 22 1.5.2: FGF signaling pathway ............................................................................................................. 23 2. Methods: ...................................................................................................................................... 27-43 2.1: Cell culture and experimental set-up ......................................................................................... 27 2.1: In vitro culture of Human embryonic stem cells (hESCs) ....................................................... 27 2.1.1: Mouse Embryonic Fibroblasts Culture ................................................................................ 27 2.1.1.1: Isolation of Mouse Embryonic Fibroblasts (MEFs) ........................................................... 27 2.1.1.2: Cryopreservation (freezing) of MEFs ................................................................................ 28 Contents 2.1.1.3: Thawing and maintaining of MEFs ................................................................................... 28 2.1.1.4: Inactivation and plating of MEFs (Feeders preparation) .................................................. 29 2.1.2: Preparation of FGF2, matrigel and media ........................................................................... 29 2.1.2.1: Preparation of FGF2 (bFGF) stock solution ...................................................................... 29 2.1.2.2: Preparation of Matrigel stock solution and Matrigel-coated plates ................................ 29 2.1.2.3: Conditioned medium preparation .................................................................................... 30 2.1.2.4: Defined medium preparation ........................................................................................... 30 2.1.3: Handling and manipulation of hESCs .................................................................................. 30 2.1.3.1: Human embryonic stem cell lines .................................................................................... 30 2.1.3.2: Handling and manipulation of hESCs ............................................................................... 31 2.1.3.3: Thawing human ES cells ..................................................................................................
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