University of Helsinki Faculty of Medicine GENERATION OF GONADOTROPIN-RELEASING HORMONE NEURONS FROM HUMAN PLURIPOTENT STEM CELLS Carina Lund The Doctoral School in Health Sciences, Doctoral Programme Brain and Mind Stem cells and Metabolism Research Program, Research Programs Unit University of Helsinki Finland Academic dissertation To be presented for public discussion with the permission of the Faculty of Medicine of the University of Helsinki, in Aurora, Room 230, Siltavuorenpenger 10, on the 29th of May, 2020 at 12 o’clock. Helsinki 2020 University of Helsinki Faculty of Medicine Doctoral Programme Brain and Mind Department of Physiology Biomedicum Helsinki Supervised by Professor Taneli Raivio Stem Cells and Metabolism Research Program, Research programs unit Faculty of Medicine, University of Helsinki, Finland Pediatric Research Center, New Children’s Hospital Helsinki University Hospital, Finland Docent Timo Tuuri Department of Obstetrics and Gynecology Helsinki University Hospital, Finland Reviewed by Professor Johanna Ivaska Department of Biochemistry, University of Turku, Finland Professor Seppo Vainio, Department of Biochemistry and Molecular Medicine, University of Oulu, Finland Opponent Professor Marcelo N. Rivolta Centre for Stem Cell Biology, Department of Biomedical Science, University of Sheffield, United Kingdom ISBN 978-951-51-6111-6 (paperback) ISBN 978-951-51-6112-3 (PDF) Unigrafia Oy Helsinki 2020 https://ethesis.helsinki.fi/ The Faculty of Medicine uses the Urkund system (plagiarism recognition) to examine all doctoral dissertations. 2 ABSTRACT.............................................................................................................................................5 LIST OF ORIGINAL PUBLICATIONS....................................................................................................6 ABBREVIATIONS ..................................................................................................................................7 INTRODUCTION.....................................................................................................................................9 REVIEW OF THE LITERATURE............................................................................................................9 1. HUMAN PLURIPOTENT STEM CELLS AS A RESEARCH TOOL ................................................................9 Blastocyst stage embryos ...................................................................................................9 Embryonic stem cells in vitro...............................................................................................9 Human embryonic stem cells and induced pluripotent stem cells ....................................10 Development of culture conditions and differentiation techniques....................................11 Directing neuronal differentiation ......................................................................................12 2. THE HUMAN REPRODUCTIVE AXIS..................................................................................................15 Hormonal regulation of puberty and fertility ......................................................................15 Regulation of GnRH secretion in the hypothalamus .........................................................16 3. PUBERTY ....................................................................................................................................17 Congenital Hypogonadotropic Hypogonadism and Kallmann Syndrome.........................17 Foetal development of the HPG axis ................................................................................17 Minipuberty........................................................................................................................18 Delayed puberty ................................................................................................................19 Associated phenotypes and genetics of CHH...................................................................19 4. DEVELOPMENT AND ORIGIN OF GNRH NEURONS ...........................................................................20 Craniofacial development..................................................................................................21 Neural crest .......................................................................................................................21 Neural crest induction at the neural plate border ..............................................................22 4.3.1 BMP signalling pathway.............................................................................................................. 23 4.3.2 Wnt signalling pathway ............................................................................................................... 23 Neural crest cell multipotency ...........................................................................................23 Notch signalling and neural crest development ................................................................24 4.5.1 Notch signalling pathway ............................................................................................................ 25 Specification of preplacodal ectoderm ..............................................................................26 Development of the olfactory placodes.............................................................................27 Neurogenesis in the olfactory placode..............................................................................30 Origin of GnRH neurons?..................................................................................................31 Emergence and migratory path of GnRH neurons during embryogenesis .......................34 How is GnRH neuron migration regulated? ......................................................................36 AIMS .....................................................................................................................................................38 MATERIALS AND METHODS .............................................................................................................39 Ethical consideration .........................................................................................................39 5. ARTICLE I....................................................................................................................................39 Culture and maintenance of human pluripotent stem cells and DNA transfections..........39 shRNA-mediated knockdown of JAG1 and overexpression of human NICD1 .................39 Neural crest, neural lineage and mesenchymal derivative differentiation.........................39 Immunocytochemistry .......................................................................................................40 Flow cytometry ..................................................................................................................40 Migration assay .................................................................................................................40 Microarray transcription profiles ........................................................................................40 Reverse transcription and real-time PCR .........................................................................41 Chromatin immunoprecipitation ........................................................................................41 6. ARTICLE II...................................................................................................................................41 Cell lines............................................................................................................................41 Differentiation procedure...................................................................................................42 Analysis of GnRH Expression and Secretion....................................................................42 Human samples immunohistochemistry ...........................................................................42 3 7. ARTICLE III..................................................................................................................................43 Generation of GNRH1-TdTomato reporter cell line ..........................................................43 Electroporations and expansion of the reporter clones ....................................................43 Screening for reporter integration .....................................................................................43 Differentiation ....................................................................................................................44 Fluorescence-activated cell sorting...................................................................................44 Quantitative polymerase chain reaction............................................................................44 Immunocytochemistry .......................................................................................................44 Human tissue collection and immunohistrochemistry .......................................................45 RNA sequencing ...............................................................................................................45 7.10 RNA sequencing data analyses............................................................................................45 RESULTS..............................................................................................................................................46