Neural crest development in a human embryonic stem cell-based model system Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Sabine Münst, geb. Schenk aus Siegburg Bonn 2012 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter: Prof. Dr. Oliver Brüstle 2. Gutachter: Prof. Dr. Michael Hoch Tag der Promotion: 24.08.2012 Erscheinungsjahr: 2012 “the only interesting thing about vertebrates is the neural crest“ P. Thorogood, 1989 Table of content Table of content Abbreviations................................................................................................................................. III Summary............................................................................................................................................ 1 Zusammenfassung.......................................................................................................................... 2 1 Introduction............................................................................................................................... 4 1.1 Pluripotent stem cells........................................................................................................................4 1.1.1 Human embryonic stem cells............................................................................................................... 5 1.1.2 Induced pluripotent stem cells ........................................................................................................... 6 1.2 The neural crest...................................................................................................................................8 1.2.1 Neurulation and neural crest specification ................................................................................... 9 1.2.2 Fate of cranial neural crest cells.......................................................................................................11 1.2.3 Fate of trunk neural crest cells..........................................................................................................15 1.2.4 Environmental cues for in vitro neural crest cell differentiation.......................................17 1.2.5 Differentiation of neural rosettes and neural crest cells from pluripotent stem cells .....................................................................................................................................................19 1.2.6 Therapeutic potential of pluripotent stem cell‐derived neural crest cells ....................20 1.3 Aim of the project............................................................................................................................. 21 2 Materials and Methods.........................................................................................................22 2.1 Materials ............................................................................................................................................. 22 2.1.1 Cell lines......................................................................................................................................................26 2.1.2 Cell culture media...................................................................................................................................26 2.1.3 Coating materials ....................................................................................................................................28 2.1.4 Buffers, solutions and gels for immunological detection methods ...................................28 2.2 Methods............................................................................................................................................... 30 2.2.1 Cell culture and cell separation.........................................................................................................30 2.2.2 Electrophysiological recordings.......................................................................................................34 2.2.3 Time‐lapse imaging................................................................................................................................35 2.2.4 Immunocytochemical analysis..........................................................................................................35 2.2.5 Histological stains...................................................................................................................................37 2.2.6 Molecular analysis..................................................................................................................................37 2.2.7 Immunoblot analysis.............................................................................................................................42 I Table of content 3 Results........................................................................................................................................45 3.1 Formation of neural tube­like structures in plated embryoid bodies........................... 45 3.2 Human embryonic stem cell­derived neural tube­like structures display a dorsal­ ventral patterning............................................................................................................................ 46 3.3 Neural crest cells accumulate in distinct aggregates in the periphery of plated embryoid bodies............................................................................................................................... 47 3.4 Crescent­shaped aggregates form in the periphery of isolated neural tube­like structures............................................................................................................................................ 50 3.5 Switching Cadherin expression patterns in human embryonic stem cell­derived neural crest cells mimic in vivo Cadherin distribution ....................................................... 51 3.6 Spontaneous differentiation of peripheral neurons in the periphery of plated embryoid bodies............................................................................................................................... 53 3.7 Isolation of neural crest cells by manual picking.................................................................. 53 3.8 Microarray analysis shows substantial gene expression differences between neural crest cells and a human embryonic stem cell­derived neural stem cell population .......................................................................................................................................... 58 3.9 Isolated neural crest cells can readily be differentiated into different types of peripheral neurons ......................................................................................................................... 59 3.10 Isolated neural crest cells give rise to putative Schwann cells...................................... 62 3.11 Isolated neural crest cells give rise to mesenchymal derivatives ............................... 64 4 Discussion.................................................................................................................................67 4.1 Neural crest development can be recapitulated in vitro .................................................... 67 4.2 The human embryonic stem cell­based model as source for multipotent neural crest cells ....................................................................................................................................................... 72 4.3 Outlook ................................................................................................................................................ 82 5 References ................................................................................................................................84 6 Acknowledgement .................................................................................................................98 7 Appendix ...................................................................................................................................99 8 Declaration............................................................................................................................ 100 9 Curriculum Vitae ................................................................................................................. 101 II Abbreviations Abbreviations AA ascorbic acid APS ammonium persulfate BDNF brain-derived neurotrophic factor BMP bone morphogenetic protein BRN3A brain-specific homeobox/POU domain protein 3A cDNA complementary deoxyribonucleic acid CNS central nervous system DAPI 4′,6-Diamidino-2-phenylindol dcAMP dibutyl cyclic adenosine monophosphate EMT epithelial-to-mesenchymal transition EBs embryoid bodies EGF epidermal growth factor ES embryonic stem FACS fluorescence activated cell sorting FCS fetal calf serum FGF fibroblast growth factor Fn fibronectin GDNF glial-derived-neutrophic factor GFAP glial fibrillary acidic protein HNK-1 human natural killer-1 HRP horseradish peroxidase hES human embryonic stem ICM inner cell mass iPS induced pluripotent stem L liter Ln laminin lt-hESNSCs long term proliferated human embryonic stem cell-derived neural stem cells MEF murine embryonic fibroblasts mL milliliter NC neural crest p75NTR low affinity nerve growth factor receptor (p75NTR)