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Differentiation of mouse germline stem cells into meiotic germ cells

In vorliegender Dissertation sind folgende Studien ausgeführt. Spermatogoniale Stammzellen (english: spermatogonial stem cells, SSCs) sind als in vitro Zelllinien etabliert und diese werden als Maus „Germline stem cells (GSCs)“ bezeichnet. Die Maus GSCs können von einem unipotenten Zustand in einen pluripotenten Zustand konvertiert werden, dieser Prozess wird als „Reprogrammierung“ bezeichnet. Die entstehenden pluripotenten Maus Embryonale Stammzelle-ähnlichen Zellen sind als Maus „Germline-derived pluripotent stem cells (gPSCs)“ bezeichnet. Die in vitro kultivierten Maus GSCs können zu meiotisch-ähnlichen Zellen differenziert werden, dieser Prozess wird als „in vitro Spermatocytogenese und Meiose“ bezeichnet.

Titel: Differentiation of mouse germline stem cells into meiotic germ cells
Verfasser: Kim, Ju Lee GND
Gutachter: Schöler, Hans Robert GND
Organisation: FB 13: Biologie
Dokumenttyp: Dissertation/Habilitation
Medientyp: Text
Erscheinungsdatum: 13.11.2013
Publikation in MIAMI: 13.11.2013
Datum der letzten Änderung: 27.07.2015
Schlagwörter: Spermatogoniale Stammzelle; Embryonale Stammzelle; Unipotenz; Pluripotenz; Reprogrammierung; Spermatocytogenese; Meiose
Fachgebiete: Biowissenschaften; Biologie
Sprache: Englisch
Format: PDF-Dokument
URN: urn:nbn:de:hbz:6-35319658590
Permalink: https://nbn-resolving.org/urn:nbn:de:hbz:6-35319658590
Onlinezugriff:
Inhalt:
1. Summary 1
2. Introduction 3
2.1 Germ cell development 3
2.1.1 Spermatogonial stem cells 4
2.1.2 Spermatogenesis 6
2.2 In vitro culture of spermatogonial stem cells 10
2.2.1 Role of growth factors in spermatogonial stem cell proliferation 11
2.2.2 Restoration of spermatogenesis by spermatogonial stem cell transplantation 13
2.3 Reprogramming of germline stem cells 14
2.4 Differentiation of germline stem cells 15
3. Aim 19
4. Material and methods 21
4.1 Animals 21
4.2 Generation and culture of different cells 21
4.2.1 Derivation of mouse germline stem cells 21
4.2.2 Derivation of mouse embryo fibroblasts 22
4.2.3 Culture of mouse embryo fibroblasts 22
4.2.4 Culture of mouse germline stem cells 23
4.2.5 Culture of mouse embryonic stem cells 23
4.2.6 Culture of mouse germline-derived pluripotent stem cells 24
4.2.7 Culture of OP9 cells 24
4.2.8 Culture of human embryonic stem cells 25
4.2.9 Culture of Sertoli cells 25

II
4.2.10 Differentiation of mouse germline-derived pluripotent stem cells into
endothelial cell-like cells 26
4.2.11 In vitro differentiation of germline stem cells into meiotic germ cells 26
4.3 Characterization of mouse germline-derived pluripotent stem cells 27
4.3.1 Alkaline phosphatase staining 27
4.3.2 In vitro differentiation of germline-derived pluripotent stem cells 28
4.3.3 Teratoma formation 28
4.3.4 Chimera formation 28
4.3.5 Analysis for potency and functionality of mouse germline stem cells 29
4.3.5.1 Testicular transplantation 29
4.4 Analyses 29
4.4.1 Messenger RNA expression 29
4.4.1.1 RNA isolation 29
4.4.1.2 Complementary DNA synthesis 30
4.4.1.3 RT-PCR analysis 30
4.4.1.4 Real-time RT-PCR analysis 32
4.4.1.5 Micro array analysis 34
4.4.1.5.1 Whole genome expression analysis 34
4.4.1.5.2 Microarray data processing 34
4.4.2 Protein expression 34
4.4.2.1 Flow cytometry 34
4.4.2.2 Immunocytochemistry 35
4.4.2.3 Immunocytochemistry of spermatocytes with synaptonemal complex protein 3
35
4.4.3 Transmission electron microscopy 36
4.4.4 Round spermatid injection 36
4.4.5 Derivation of embryonic stem cell lines 37

III
4.4.6 Karyotyping of round spermatid injection-derived embryonic stem cell lines ... 37
4.4.7 Genotyping of round spermatid injection-derived embryonic stem cell lines .... 38
4.4.8 DNA methylation analysis 38
5. Results 40
5.1.1 Derivation of germline stem cells form mouse testes 40
5.1.2 Derivation of germline stem cells from human testicular biopsies 42
5.2 Conversion of unipotent germline stem cells into pluripotent germline-derived
stem cells 48
5.2.1 Conversion of germline stem cells from autologous testicular biopsy into
germline-derived pluripotent stem cells 48
5.2.2 Characterization of germline stem cells from autologous testicular biopsy into
germline-derived pluripotent stem cells 49
5.2.3 Differentiation ability of germline-derived pluripotent stem cells 60
5.2.3.1 In vitro and in vivo differentiation ability analysis 60
5.2.3.2 Differentiation of germline-derived pluripotent stem cells into endothelial celllike cells 62
5.3 In vitro differentiation of germline stem cells 70
5.3.1 Differentiation of germline stem cells into meiotic cells 70
5.3.2 Characterization of meiotic cells 71
5.3.2.1 Flow cytometry analysis of in vitro-generated cells 71
5.3.2.2 Immunofluorescence staining 73
5.3.2.3 Transmission electron microscopy 75
5.3.2.4 Real-time RT-PCR analysis 81
5.3.2.5 Round spermatid injection, embryo transfer, and derivation of embryonic stem
cells 82
5.3.2.6 Flow cytometry analysis 85

IV
5.3.2.7 Karyotyping 86
5.3.2.8 Methylation analysis 86
5.3.2.9 Characterization of round spermatid injection-derived Acrosin embryonic stem
cells 87
5.4 Derivation of germline stem cells from OG2 x Acrosin EGFP mouse testes 88
6. Discussion 90
6.1 Characteristics of germline stem cells 90
6.1.1 Potency and functional analysis of germline stem cells 91
6.1.2 Self-reprogramming ability of germline stem cells into pluripotent cells 92
6.1.3 Differentiation ability of germline-derived pluripotent stem cells into
endothelial-like cells 92
6.2 In vitro spermatogenesis of germline stem cells 94
7. References 100
8. Abbreviations 111
9. List of figures 115
10. List of tables 118
11. Appendix 119
11.1 Companies 119