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|a urn:nbn:de:hbz:6-33309473736
|2 urn
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|a eng
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|a 570 Biowissenschaften; Biologie
|2 23
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|a Sugawa, Fumihiro
|0 http://d-nb.info/gnd/1052028659
|4 aut
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|a Universitäts- und Landesbibliothek Münster
|0 http://d-nb.info/gnd/5091030-9
|4 own
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|a Generation and molecular analysis of in vitro germ cells from mouse and human pluripotent stem cells
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|a [Electronic ed.]
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|c 2014
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|b Universitäts- und Landesbibliothek Münster
|c 2014-06-10
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|a Acknowledgement ......................................................................................................... i -- Table of figures and tables ......................................................................................... ii -- Abbreviations .............................................................................................................. v -- Zusammenfassung .................................................................................................... vii -- Abstract ....................................................................................................................... ix -- 1. Introduction ............................................................................................................ 1 -- 1.1. Germ cells ......................................................................................................... 1 -- 1.1.1. Primordial gem cell specification .......................................................... 2 -- 1.1.2. Germ cell migration ............................................................................... 4 -- 1.1.3. Epigenetic reprogramming ..................................................................... 5 -- 1.1.4. Sex differentiation and meiosis .............................................................. 7 -- 1.1.5. Folliculogenesis and oocyte maturation ................................................. 8 -- 1.2. Germ cell differentiation from mouse and human embryonic stem cells ......... 9 -- 1.2.1. In vitro differentiation of mouse germ cells ........................................ 10 -- 1.2.1.1. In vitro differentiation of post-migratory PGCs ...................... 10 -- 1.2.1.2. Differentiation by co-culture with animal-origin somatic cells -- ......................................................................................................... 12 -- 1.2.1.3. In vitro differentiation of pre-migratory PGCs ........................ 14 -- 1.2.2. In vitro differentiation of human germ cells ........................................ 15 -- 1.2.2.1. The generation of post-migratory PGC-like cells .................... 16 -- 1.2.2.2. The generation of putative gametes ......................................... 17 -- 1.2.2.3. Differentiation by co-culture with animal-origin somatic cells -- ......................................................................................................... 18 -- 2. Materials and methods ........................................................................................ 22 -- 2.1. Materials ......................................................................................................... 22 -- 2.1.1. Reagents ............................................................................................... 22 -- 2.1.2. Equipment ............................................................................................ 24 -- 2.2. Methods .......................................................................................................... 24 -- 2.2.1. Animals ................................................................................................ 24 -- 2.2.2. Cell culture medium ............................................................................. 25 -- 2.2.3. Cell culture ........................................................................................... 28 -- 2.2.3.1. Derivation of mouse embryo fibroblasts (MEF) ...................... 28 -- 2.2.3.2. Mouse Embryonic Fibroblast (MEF) culture .......................... 28 -- 2.2.3.3. Preparation of MEF-conditioned medium (CM) ..................... 28 -- 2.2.3.4. Preparation of coated-plates ..................................................... 29 -- 2.2.3.5. Mouse ESC culture .................................................................. 29 -- 2.2.3.6. Mouse PGC differentiation ...................................................... 30 -- 2.2.3.7. Human ESC and iPSC culture ................................................. 30 -- 2.2.3.8. Human PGC precursor and PGC differentiation ..................... 31 -- 2.2.4. Amplification of STELLA promoter sequence ................................... 31 -- 2.2.5. Ligation of the STELLA promoter sequence into pEGFP-1 vector ... 32 -- 2.2.6. Transformation of competent bacterial cells ........................................ 32 -- 2.2.7. Plasmid DNA preparation ................................................................... 32 -- 2.2.8. Generation of STELLA-GFP human ESCs and iPSC ......................... 32 -- 2.2.9. Lentivirus Production .......................................................................... 33 -- 2.2.10. Knock-down of BLIMP1 and PRDM14 .............................................. 33 -- 2.2.11. Fluorescence-Activated Cell Sorting Analysis ................................... 34 -- 2.2.12. Immunocytochemistry ........................................................................ 34 -- 2.2.13. Real-Time PCR (qPCR) ....................................................................... 34 -- 2.2.14. Microarray analysis ............................................................................. 36 -- 2.2.15. Microarray data processing .................................................................. 36 -- 2.2.16. Bisulfite Sequencing ........................................................................... 37 -- 2.2.17. Transplantation of reconstituted ovaries under the ovarian bursa ...... 38 -- 2.2.18. Electron microscopy ........................................................................... 39 -- 3. Results ................................................................................................................... 40 -- 3.1. PGC differentiation from human ESCs and iPSCs ........................................ 40 -- 3.1.1. Examination of in-house protocol and published protocol for PGC -- differentiation from human ESCs ........................................................... 40 -- 3.1.2. Search for a candidate gene for PGC identification ............................ 43 -- 3.1.3. Establishment of GFP reporter human pluripotent stem cells ............ 46 -- 3.1.4. Differentiation of STELLA-GFP human pluripotent stem cells ......... 46 -- 3.1.5. Differentiation toward mesoderm-committed germ cell precursors ... 49 -- 3.1.6. Differentiation of PGC precursors towards PGC-like cells ................ 54 -- 3.1.7. Characterization of PCG-like cells ..................................................... 56 -- 3.1.8. The molecular mechanism of PGC induction in vitro ........................ 59 -- 3.1.9. Global gene expression analysis of PGC precursors and PGC-like cells -- ................................................................................................................. 63 -- 3.2. PGC differentiation from mouse EpiSCs and ΔPE-Oct4-GFP+ EpiSCs ..... 80 -- 3.2.1. PGC differentiation from mouse EpiSCs ............................................ 80 -- 3.2.2. PGC differentiation from mouse EpiSCs and ΔPE-Oct4-G ............... 82 -- 3.3. Ultrastructural characterization of mouse ESC-derived oocytes and granulosa -- cells ................................................................................................................. 84 -- 3.3.1. Granulosa cells and the GC–oocyte interface of ESC-derived follicles -- ................................................................................................................. 84 -- 3.3.2. ESC-derived oocytes ........................................................................... 90 -- 4. Discussion ............................................................................................................. 94 -- 4.1. PGC differentiation from human ESCs and iPSCs ........................................ 94 -- 4.2. PGC differentiation from mouse EpiSCs and ΔPE-GFP-Oct4+ EpiSCs .... 101 -- 4.3. Ultrastructural characterization of mouse ESC-derived oocytes and granulosa -- cell ................................................................................................................. 102 -- 5. References ........................................................................................................... 106.
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|a free access
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|a Zusammengefasst zeigen unsere Daten, dass ESC-abgeleitete humane PGCs und in vitro generierte Oozyten der Maus ihren natürlichen Gegenstücken stark ähneln. Zusätzlich weisen unsere Studien auf bisher unbekannte Mechanismen während der humanen Keimzellspezifikation hin. Weitere Untersuchungen werden zeigen wie vorteilhaft solche in vitro Differenzierungsmodelle als alternative Ansätze für Untersuchungen in der reproduktiven Entwicklung tatsächlich sind.<br><br>
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|a specialized
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|a InC 1.0
|u https://rightsstatements.org/vocab/InC/1.0/
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|a Keimzellen
|a Stammzellen
|a Oozyten
|a Granulosazellen
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|2 DRIVER Types
|a Dissertation/Habilitation
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|2 DCMI Types
|a Text
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|a Schöler, Hans Robert
|u FB 13: Biologie
|0 http://d-nb.info/gnd/112747973
|4 ths
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|3 Zum Volltext
|q text/html
|u https://nbn-resolving.de/urn:nbn:de:hbz:6-33309473736
|u urn:nbn:de:hbz:6-33309473736
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|q application/pdf
|u https://repositorium.uni-muenster.de/document/miami/f4f9b7db-d12f-4695-a9e1-a479724a0f48/diss_sugawa.pdf
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