UNIVERSIDADE DE LISBOA FACULDADE DE CIÊNCIAS DEPARTAMENTO DE BIOLOGIA ANIMAL Testing a mesendoderm protocol in Smad5 and Smad1/5 depleted embryonic stem cells for primordial germ cell induction Mestrado em Biologia Evolutiva e do Desenvolvimento Sara Mariana Eugénio Ferraz Mendes Dissertação orientada por: Professora Dra. Susana Marina Chuva de Sousa Lopes e Professora Dra. Maria Gabriela Rodrigues 2015 Contents i. Acknowledgements ................................................................................................................................ I ii. Abbreviations .................................................................................................................................... II iii. Abstract ............................................................................................................................................ III iv. Resumo ............................................................................................................................................ IV I. Introduction .............................................................................................................................. 1 1. Embryonic Stem Cells in mouse ............................................................................................ 2 2. BMP signalling: BMP4 and Smads 1/5 .................................................................................. 3 3. Primordial germ cells (PGCS)................................................................................................. 4 4. Primordial Germ Cells from Embryonic Stem Cells ............................................................... 7 II. Project Objective ....................................................................................................................... 9 I. Material and Methods ............................................................................................................ 10 1. Cell Culture .......................................................................................................................... 10 1.1 Cell lines .............................................................................................................................. 10 1.1.1 Non-transgenic cell line E14 ............................................................................................ 10 1.1.2 Smad5-/- Blimp:RFP .......................................................................................................... 10 1.1.3 Smad5fl/fl Smad1fl/fl and Smad5-/- Smad1-/- ...................................................................... 10 1.2 Mouse Embryonic Stem Cell (mESC) line Derivation .......................................................... 10 1.2.1 Genotyping ...................................................................................................................... 12 1.2.2 Agarose gel Electrophoresis ............................................................................................ 13 1.3 Splitting ............................................................................................................................... 13 1.4 Cryopreservation ................................................................................................................. 13 1.5 Thawing ............................................................................................................................... 13 1.6 Differentiation Media experimental design ........................................................................ 14 2. Alkaline Phosphatase staining ............................................................................................ 15 3. Analysis of gene expression ................................................................................................ 16 3.1 RNA isolation and cDNA synthesis ...................................................................................... 16 3.2 Quantitative reverse-transcriptase PCR (QPCR) ................................................................. 16 4. Immunostaining .................................................................................................................. 17 5. Imaging ................................................................................................................................ 18 6. Statistical Analysis ............................................................................................................... 18 II. Results ..................................................................................................................................... 19 1. Effects of cell density during mesendoderm (ME) differentiation ..................................... 19 2. ME markers are upregulated in E14 after 6 days ME-differentiation................................. 20 3. PGC markers are upregulated in E14 after ME differentiation ........................................... 20 4. Derivation of Smad5 KO Blimp1:rfp mESCs lines ................................................................ 22 5. Derivation of Smad5 and Smad 1 KO mESCs lines .............................................................. 23 5.1 ME markers are upregulated in Smad1fl/fl Smad5fl/fl WT mESCs ......................................... 23 5.2 ME markers are upregulated in Smad1-/- Smad5-/- KO mESCs ............................................. 23 III. Discussion ................................................................................................................................ 27 1. ME-differentiation protocol vs Hayashi-protocol ............................................................... 27 2. Induction of PGCLCs using the ME-differentiation protocol ............................................... 27 3. Role of (BMP) Smad1/5 signalling during PGC development ............................................. 29 IV. Final Remarks .......................................................................................................................... 31 V. Supplemental Information ...................................................................................................... 32 III. References .............................................................................................................................. 35 i. Acknowledgements First, I would like to thank Prof. Gabriela Rodrigues for allowing the connection with Dr. Susana Chuva de Sousa Lopes, for being so patient and understanding throughout the development of my final master student phase. To Professor Christine Mummery and Dr. Susana Chuva de Sousa Lopes for giving me the opportunity to perform my master internship at the department of anatomy and embryology of the Leiden University Medical Center, I am grateful. I am also thankful for the Erasmus scholarship, which facilitated this experience. Also for the international office from my home college, that was always understanding and helpful with all the paperwork and along the whole process in general, I am grateful. Thank you to all the members of Susanas’ group, both for showing me what a real scientist is all about and for having taken the time to teach me and support me with laboratory practices. In particular I would like to recognise Ana Bernardo for the helpful discussions of project ideas. A very special thank you goes to Maria Fernandes, for teaching me, for the patience, for the moral support, for showing me nice places to go and eat in the Netherlands, for reviewing my thesis, for more things than I could not possibly fit here, and also for the best home-made Francesinha I ever tasted! Thank you to my friends of all times, that accompanied me through my little adventure in breezy Dutch lands, and diligently heard about my daily life stories, making me feel whole far from home. To the ones that helped boost my motivation and love for science, I thank you. To my family that supported me, not only financially, but also with piece of mind, remaining calm at all times and allowing me to understand things and carve my own path by myself. To all the friends and acquaintances I made along this journey with whom I shared a talk or even just a fleeting moment, I am grateful to you for the memories, and for, in a very particular way, opening up my mind to other points of view, to other cultures and to the realisation of an amazingly infinite set of possibilities awaiting to be taken. I ii. Abbreviations AP Alkaline phosphatase ASCs Adult stem cells bFGF Basic fibroblast growth factor BME 2-Mercaptoethanol BMP Bone morphogenetic protein BP Base pairs BSA Bovine serum albumin CHIR CHIR99021 E Embryonic day EGF Epidermal Growth Factor EpiLCs Epiblast-like cells EpiSC Epiblast stem cells ESCs Embryonic stem cells ExE Extra-embryonic ectoderm FACS Fluorescence activated cell sorting FCS Fetal calf serum hESCs Human embryonic stem cells ICM Inner cell mass IPSCs Induced pluripotent stem cells IVF In vitro fertilization LIF Leukaemia inhibitory factor ME Mesendoderm MEF Mouse embryonic fibroblast mESCs Mouse embryonic stem cells NEAA Non-essential amino acids PBS Phosphate-buffered saline PCR Polymerase chain reaction PD PD0325901 PFA Paraformaldehyde PGCLCs Primordial germ cell-like cells PGCs Primordial germ cells qPCR quantitative polymerase chain reaction RFP Red fluorescent protein SCF Stem Cell Factor T Brachyury-T TGF-β Transforming growth factor beta VE Visceral endoderm WT Wild-type II iii. Abstract Mouse; PGCs (Primordial Germ Cells); Mesendoderm; Smad1/5; CHIR99021 The developmental pathway of Primordial Germ