(m/h) PSC (murine/human) Pluripotent stem cells 2D / 3D Two / three dimensional
AA Amino acids
Actb Ac$n, beta
ANOVA Analysis of Variance
Ascl1 Achaete-scute family bHLH transcrip$on factor 1 Bmp Bone morphogene$c proteins
bp Base pair
BS3 (Bis(sulfosuccinimidyl)suberate c-Myc Myelocytomatosis oncogene CGE Caudal ganglionic eminence CMV Cytomegalovirus promotor
Cre Cre recombinase expression cassele
CRISPR/Cas Clustered regularly interspaced short palindromic repeats / CRISPR associated proteins Dkk1 Dickkopf WNT signaling pathway inhibitor 1
DMEM Dulbecco's Modified Eagle Medium
DMSO Dimethylsulfooxid
DSB Double strand break
EB Embryoid body
ECM Extracellular matrix
EDTA Ethylenediaminetetraace$c acid e.g. exempli gra$a (la$n), for example Emx1 Empty spiracles homeobox 1 EpiSC Epiblast stem cells
ESCs Embryonic stem cells
EtBr Ethidium bromide
FCS Fetal calf serum Fgf Fibroblast growth factor FH Forkhead DNA binding domain Foxg1 Forkhead box transcrip$on factor G1 GABA Gamma-Aminobutyric acid
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-Gapdh Glyceraldehyde-3-phosphate dehydrogenase GFP Green fluorescent protein
Gli3 GLI-Kruppel family member 3 GMEM Glasgow Minimum Essen$al Medium
gRNA Guide RNA
Gsx2 GS homeobox 2
HDR Homology directed repair
HEK293T Human embryonic kidney cells, line 293 that expresses a mutant version of the SV40 large T an$gen
HR Homologous recombina$on
i.e. Id est (la$n), that is
ICM Inner cell mass
Indels Inser$ons and dele$ons iPSC Induced pluripotent stem cells IRES Internal ribosome entry site Klf4 Kruppel-like factor 4
KO Knockout
LacZ Lactose operon Z expression cassele, gene product: β-galactosidase
LB Lysogeny broth medium
LC-MS Liquid chromatography combined with mass spectrometry LGE Lateral ganglionic eminence
Lif Leukemia inhibitory factor
Lin28 Lin-28 homolog
MECP2 Methyl-CpG binding protein 2 MGE Medial ganglionic eminence Nam Neural cell adhesion molecule 1
Nanog Nano homeobox
Nes Nes$n
NHEJ Non-homologous end joining
Nkx2.1 NK2 homeobox 1
Oct4 Octamer binding transcrip$on factor 4, alterna$ve name: POU5F1 (POU domain, class 5, transcrip$on factor 1)
PAM Protospacer adjacent mo$f Pax6 Paired box protein 6
PBS Phosphate-buffered saline
PBS-T Phosphate-buffered saline supplemented 0.05 % Tween-20 PVDF Polyvinylidenfluorid
qPCR Quan$ta$ve real $me polymerase chain reac$on RG Radial glial cells
RT PCR Reverse transcrip$on polymerase chain reac$on
SD Standard devia$on
SDS PAGE Polyacrylamide gel electrophoresis with sodium dodecyl sulfate as denaturant SFEB(q) Serum free and morphogen reduced embryoid body like culture (with quick
reaggrega$on)
SHH Sony Hedgehog
SMAD SMAD family member
Sox2 Sex determining region Y-box 2 SVZ Subventricular zone
TALEN Transcrip$on ac$vator-like effector nucleases Tgfß Transforming growth factor beta 1
TRIS Tris(hydroxymethyl)-aminomethan Tubb3 Tubulin, beta 3 class III
VGlut1 Vesicular glutamate transporter 1, alterna$ve name: Slc17a7: solute carrier family 17, member 7
Wnt Integra$on/Wingless family
WT Wildtype
ZFN Zink finger nucleases
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-8.4 List of tables
Table 1: Overview about the applied pluripotent stem cell lines in these experiments...27
Table 2: Mouse pluripotent stem cell culture media composi$on...28
Table 3: HEK293T cell culture media composi$on...29
Table 4: Sequences to guide Cas9 to Foxg1 coding exon...30
Table 5: Primer sequences for PCR analysis...35
Table 6: Media for neuronal differen$a$on culture...37
Table 7: List of applied an$bodies...44
Table 8: Overview of the marker genes analyzed...46
Table 9: Primer combina$ons applied in RT PCR...49
Table 10: Primer combina$ons applied in qPCR...50
Table 11: Overview of CRISPR/Cas9 mediated knockout of Foxg1 in different mPSC lines...52
Table 12: In silico analysis and verifica$on of clonality of derived Foxg1 wildtype and knockout mPSCs...54
Table 13: Overview about tested FOXG1 an$bodies and op$miza$ons...69
Table 14: Overview of mPSC lines used for comparison of Foxg1 WT and KO amer in vitro differen$a$on....77
Table 15: Buffer prepara$ons for Western blot analysis...115
Table 16: Size of derived embryoid body like structures grouped for PSC deriva$on and Foxg1 status. ...116
Table 17: Neuronal gene expression values of Foxg1 wildtype mESCs and miPSCs ...118
Table 18: Neuronal gene expression values of Foxg1 knockout mESCs and miPSCs ...120
8.5 List of figures
Figure 1: Schema$c view of project context...3
Figure 2: Different possibili$es to generate pluripotent stem cells from mouse (Pauklin et al. 2011)...6
Figure 3: Different pluripotent states in spa$otemporal order in vivo and in vitro (Wu et al. 2015)...8
Figure 4: Genome edi$ng using induced double strand breaks (Komor, Badran et al. 2016)...10
Figure 5: Key pathways in the early telencephalon palerning (Hébert and Fishell 2008)...14
Figure 6: Default model of differen$a$on of PSCs to cor$cal progenitors (Van den Ameele et al. 2014)...15
Figure 7: Differences between 2D and 3D differen$a$on with regard to spa$al organiza$on (Van den Ameele et al. 2014)...17
Figure 8: Foxg1 gene structure...19
Figure 9: Expression palern of Foxg1 in the telencephalon (Danesin and Houart 2012)...20
Figure 10: Role of Foxg1 in the developing telencephalon (Yip et al. 2012)...20
Figure 11: Overview of the project workflow with main analyses....25
Figure 12: Guide RNA binding sites within Foxg1....30
Figure 13: Schema$c view of Foxg1 knockout workflow...32
Figure 14: Schema$c view of preliminary neuronal differen$a$on protocol. ...38
Figure 15: Schema$c view of final neuronal differen$a$on protocol....39
Figure 16: Verifica$on of introduced Foxg1 muta$ons by PCR...53
Figure 17: Embryoid body forma$on and growth over $me with preliminary differen$a$on protocol...55
Figure 18: Analysis of neuronal gene expression Venus Foxg1 wildtype and knockout miPSCs with reverse transcrip$on end-point PCR at different $me points of differen$a$on...56
Figure 19: Analysis of pan-neuronal gene expression amer differen$a$on of the Venus miPSCs using the preliminary protocol...57
Figure 20: Analysis of telencephalic gene expression amer differen$a$on of Venus miPSCs using the preliminary protocol...58
Figure 21: Comparison of area and diameter of GFP miPSC derived embryoid like structures differen$ated with preliminary and final protocol. ...59
Figure 22: Size of embryoid body like structures derived from three different Foxg1 knockout miPSCs compared to Foxg1 wildtype GFP miPSCs at day 25 of differen$a$on...60
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-Figure 23: Analysis of pan-neuronal gene expression amer differen$a$on of the Foxg1 wildtype and
knockout GFP miPSCs using the final protocol....61
Figure 24: Analysis of telencephalic gene expression amer differen$a$on of the Foxg1 wildtype and knockout GFP miPSCs using the final protocol....62
Figure 25: Size of embryoid body like structures derived from Foxg1::Venus reporter mESCs....63
Figure 26: Venus expression in Foxg1::Venus reporter mESC at days 1, 15, and 25 of differen$a$on...64
Figure 27: Quan$fica$on of Venus expression in Foxg1::Venus reporter mESC amer differen$a$on using flow cytometry...65
Figure 28: Western blot analysis of Venus expression amer differen$a$on of Foxg1::Venus mESCs....65
Figure 29: Analysis of pan-neuronal gene expression amer differen$a$on of the Foxg1::Venus mESCs...66
Figure 30: Analysis of telencephalic gene expression amer differen$a$on of the Foxg1::Venus mESCs....67
Figure 31: Western blot analysis using the Foxg1 C-Terminal An$body (ab18259, Lot GR237352-1)...70
Figure 32: Western blot analysis using the Foxg1 C-Terminal An$body (ab18259, Lot GR199183-1)...71
Figure 33: Western blot analysis using the Foxg1 N-Terminal An$body (Abcam ab86292, Lot GR90269-2) 73... Figure 34: Western blot analysis using the Foxg1 Central An$body (SAB1307246, Lot SA100608W)...74
Figure 35: Western blot analysis using the Foxg1 Central monoclonal an$body (MABD79, Lot: 2744121)....75
Figure 36: PCR analysis to validate the Foxg1 muta$ons and presence of transgenes in the mPSCs. ...78
Figure 37: Growth kine$cs of Foxg1 wildtype and knockout mPSCs. ...79
Figure 38: Morphology and size of derived embryoid body like structures. ...80
Figure 39: Size of derived embryoid body like structures grouped. ...80
Figure 40: Analysis of pan-neuronal gene expression in Foxg1 wildtype mPSCs over $me...82
Figure 41: Analysis of telencephalic gene expression in Foxg1 wildtype mPSCs over $me....83
Figure 42: Analysis of pan-neuronal gene expression in Foxg1 knockout mPSCs...84
Figure 43: Analysis of telencephalic gene expression in Foxg1 knockout mPSCs...85
Figure 44: Growth kine$cs in different Foxg1 wildtype and knockout mPSCs - single lines...116
Figure 45: Diameter of derived embryoid body like structures - single lines....117
Figure 46: Area of derived embryoid body like structures - single lines....117
Figure 47: Analysis of neuronal gene expression in Foxg1 wildtype mPSCs over $me - single lines...119
Figure 48: Analysis of neuronal gene expression in Foxg1 wildtype mPSCs over $me of neuronal differen$a$on - single lines...121
8.7 List of publica$ons
Oral and poster presenta$ons at interna$onal conferences
Feugang JM, Mall EM, Ryan PL 2013: Dynamic of relaxin and its receptor RXFP1 and RXFP2 proteins in boar spermatozoa. Society for the Study of Reproduc$on's 46th Annual Mee$ng, July 2013
Petersen B, Frenzel A, Lucas-Hahn A, Hassel P, Ziegler M, Hadeler KG,Mall EM, Nowak-Imialek M, Ol M, Niemann H: 2015. Efficient produc$on of GGTA1-/- /Fah+/- knockout pigs by CRISPR/
Cas9 and soma$c cell nuclear transfer. IPITA, IXA, CTS Joint Congress, 15.-19.11.2015 in Melbourne, Australia. Xenotransplanta$on 22, Suppl. S1:S39.
Mall EM, Burchardt B, Herrmann D, Talluri TR, Petkov S, Nowak-Imialek M, Niemann H: 2015.
Preven$ng brain contribu$on of primate-to-pig chimeras: Knock- out of Foxg1 and evalua$on of its func$onality with in vitro differen$a$on. 3rd Interna$onal Annual Conference of the German Stem Cell Network (GSCN), 09.- 11.09.2015, Frankfurt/Main, Abstract Book, p. 175.
Mall EM, Burchardt B, Herrmann D, Niemann H: 2016. Analysis of the Foxg1 knockout phenotype in murine iPSC using SFEBq differen$a$on. 2016 CRTD/ISSCR Interna$onal Symposium, 1.- 3.02.2016, Dresden, Abstract Book, p. 61
Mall EM, Burchardt B, Herrmann D, Niemann H: 2016. Analysis of the Foxg1 knockout phenotype in murine iPSC using neuronal differen$a$on. 4th Interna$onal Annual Conference of the German Stem Cell Network (GSCN), 12.- 14.09.2016, Hannover, Abstract Book, p. 228.
Mall EM, Herrmann D, Niemann H: 2017. Neuronal differen$a$on of mouse pluripotent stem cells in vitro does recapitulate important aspects of the Foxg1 knockout phenotype. 9th Interna$onal Mee$ng of the Stem Cell Network NRW, 16. - 17. 05. 2017, Münster
Scien$fic publica$ons
Handreck A,Mall EM, Elger D, Gey L, and Gernert M: 2015. Different Prepara$ons, Doses, and Treatment Regimens of Cyclosporine A Cause Adverse Effects but No Robust Changes in Seizure Thresholds in Rats. Epilepsy Research. doi:10.1016/j.eplepsyres.2015.02.006.
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-8.8 Author's statement of contribu$on
I, Eva Mall, planed and conducted all here presented experiments, performed the data analysis, and wrote the manuscript.
Significant contribu$ons to this thesis come from Doris Hermann and Prof. Dr. Heiner Niemann.
Doris Hermann performed the quan$ta$ve real-$me PCR and aided with the gene expression analysis.
Prof. Dr. Heiner Niemann supervised the development of the work, and aided with experimental design, data interpreta$on and manuscript evalua$on.
8.9 Declara$on
Herewith, I confirm that I have wrilen the present PhD thesis myself and independently, and that I have not submiled it or parts thereof at any other university worldwide.
Herewith, I agree that MHH can check my thesis by plagiarism detec$on somware as well as randomly check the primary data. I am aware that in case of suspicion, ombudsman proceedings according to § 9 of MHH ’Guidelines of Hannover Medical School to guarantee good scien$fic prac$ce and dealing with scien$fic fraud’ will be ini$ated. During such proceedings, the PhD process is paused.
Hannover, April 2017
_____________________________________
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-8.10 Acknowledgements
First and foremost I want to thank my supervisor Prof. Dr. Heiner Niemann for giving me the opportunity to work on this very interes$ng project. Thank you very much for the excellent supervision, fast correc$ons at every $me, and the constant mo$va$on over the last years.
Also, I would like to thank my co-supervisors, Prof. Dr. Michael Ol and Prof. Dr. Achim Gossler, for their construc$ve sugges$ons and helpful advice on the project design and occurring difficul$es.
I would like to express my gra$tude to Prof. Dr. Rüther and Prof. Dr. Thomas Moritz, who accepted to be my external and internal expert, respec$vely. Thank you for your precious $me.
I am also grateful for the financial support by the DFG through the Cluster of Excellence REBIRTH. Many thanks go to the organiza$on team of the PhD program "Regenera$ve Science", especially to Dr. Daniela Pelz, and Steffi Gomm for the great support.
I would like to thank the MS Core Facility Proteomics at the Hannover Medical School for performing the mass spectrometric analysis.
Many thanks to Prof. Dr. Ulrich Mar$n for the collabora$on and the opportunity to do the internship in his laboratory. And I want to thank Dr. Stephanie Wunderlich for supervision and for sharing her cells and her great exper$se. I did learn a lot and I am very thankful to you and all members of the LEBAO for making it an enjoyable $me.
At this point, I would like to thank all my colleagues at the ins$tute in Mariensee. Many thanks for the great support in the last years: if it was by prac$cal help with performing experiments / finding materials / preparing stock solu$ons / and so on, or by providing nice coffee breaks with various discussions (on- and off-topic). And of course for the constant coffee and cake supply, that omen rescued me over long days. Thanks for making the last years as enjoyable as they were.
My special thanks goes to Doris Hermann for performing the various qPCRs and sharing her exper$se on molecular biology. Thank you for every ques$on answered, every protocol shared, and every brilliant idea that helped to solve smaller and bigger problems I had. Without your constant support and the helpful discussions, I may have given up over some of the less pleasant experiments.
Many thanks also to Birgit Burchardt for the collabora$on with the mouse chimera experiments and the great $mes and talks we shared inside and outside the lab.
Dr. Monika Nowak-Imialek, thank you very much for the great support, for answering my countless ques$ons, and for proofreading the manuscript. Thank you both, Monika and Irene, for every most welcome distrac$on and all the fun we had. It was great to have you as roommates.
Many thanks to all my fellow doctoral students, especially Birgit, Irene, Hendrik, and Ronja for the shared problems and fun in the last years. Thanks you Hendrik for the very pely proofreading of this manuscript.
I want to appreciate the support of Antje Frenzel in performing the flow cytometry analysis, and of Patrick Aldag in the cell culture lab. And many thanks to Dr. Ulrich Baulain for sharing his exper$se on sta$s$cal analysis. Stoyan Petkov, I appreciated your help with the plasmid prepara$ons and the many helpful discussions. Your great sense of humor enlightened the one or the other long Saturday.
I dearly thank my family, especially my parents. Your love and support made me the person I am and helped me through this process. I am happy to know that I can always count on you.
Last but not least, I want to thank Helge. I deeply appreciate all the things you do for me, your constant support, that you always have my back, and never complain, even if I spend nearly every free minute with my horses. I love you with all my heart!
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