| dc.contributor.advisor | Nolte-Kaitschick, Jessica Dr. | |
| dc.contributor.author | Mellies, Nadine | |
| dc.date.accessioned | 2015-06-24T08:30:31Z | |
| dc.date.available | 2015-06-24T08:30:31Z | |
| dc.date.issued | 2015-06-24 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0022-6035-B | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-5158 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | From stem cells to male germ cells: Experimental approaches for the in vitro generation of mouse and human spermatogonial stem cells | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Hoyer-Fender, Sigrid Prof. Dr. | |
| dc.date.examination | 2015-05-29 | |
| dc.description.abstracteng | Currently different in vitro experimental approaches make use of the unique potential of pluripotent cells in order to produce functional sperm aiming at the establishment of new treatment opportunities for male infertility.
The establishment of proliferating human spermatogonial stem cell (SSC) lines and their in vitro differentiation into haploid male germ cells represents one of these approaches and was tried to succeed in the presented thesis. The isolation of human SSCs from testicular biopsies from infertile men was performed using the Magnetic Activated Cell Separation technique and succeeded several times in the enrichment of putative SSCs, characterized by their grape-like structures characteristically for SSCs and a positive SSC marker staining. Despite of numerous attempts and testing of different culture conditions, a stable and proliferating human SSC cell line could not be established. Because other studies also revealed great difficulties in the establishment of long term human SSC cultures, a further experimental approach plans to use human pluripotent stem cells for in vitro differentiation of human SSCs. In the presented thesis finally three human induced pluripotent stem cell (hiPSC) lines derived from infertile men revealing different spermatogenesis phenotypes could be established in culture, were characterized by the common tools to proof their pluripotency and are now suitable for the start of in vitro differentiation experiments. For the generation of these hiPSCs from moderate proliferating scrotal human fibroblasts a modified so called “Feeder Free” mRNA/miRNA reprogramming was successfully developed.
Various mouse studies proved the derivation of haploid male germ cells from pluripotent stem cells, but revealed especially improper genomic imprinting often yielding in unhealthy offspring. Here, the in vitro differentiation strategy established by Nayernia et al. (2006) should be improved by the overexpression of the premeiotic gene Stra8 (Stimulated by Retinoic Acid Gene 8). These experiments were planned to be carried out with the original cell line SSC 12 derived from Nayernia and colleagues (2006) as well as with a here newly generated embryonic stem cell (ESC) line derived from double transgenic Stra8 EGFP/Sycp3 DsRed mice. Finally, in vitro spermatogenesis experiments using SSC 12 cells with a two times increased stable Stra8 overexpression indicated an earlier entry into and progress through the meiotic process analyzed on the mRNA level, whereat no meiotic or postmeiotic structures could be detected on the protein level using immunostaining analyses and electron microscopy. Stra8 overexpressing cells revealed an overall increased methylation level of imprinting genes indicating that Stra8 indeed has an effect on epigenetic reprogramming. Paternally imprinted genes tend to an androgenic imprinting pattern, while the maternally imprinted genes failed to undergo the essential demethylation in differentiating Stra8 overexpressing cells.
A further part of the presented thesis focused on the development of a reproducible and robust co-culture system for the derivation of mouse SSCs. For this purpose transgenic mouse ESCs were co-cultured with human testicular feeder (HTF) cells, which were characterized to represent predominantly human peritubular cells by using markers for testicular somatic cells on mRNA and protein level. The co-culture-derived cells were called putative mouse SSCs because of their grape-like cell morphology, which is characteristic for SSCs according to the literature. These SSC-like cells could also be generated using mouse wild type ESCs and iPSCs suggesting a general suitability of the co-culture system for the derivation of putative SSCs. The as viable proved putative mouse SSCs expressed several in the literature accepted markers for SSCs and primordial germ cells (PGCs) on mRNA and protein level. The induction of differentiation of these SSC-like cells revealed an up-regulation of postmeiotic markers indicating the onset of the meiotic process detectable on the mRNA level. The analyzed imprinted genes revealed rather a somatic than androgenic methylation pattern. Transplantations of cells into germ cell depleted mice did not yield in a repopulation of the seminiferous tubules, but the detection of a reporter construct, contained in the transgenic ESCs used for co-culture experiments, indicated the presence of transplanted cells for at least 15 days within the testis. These data leads to the hypothesis that the co-culture of mouse ESCs with HTF cells yielded in a mixed cell population with a predominantly PGC character and presumably a small fraction of undifferentiated and differentiating spermatogonia. | de |
| dc.contributor.coReferee | Burfeind, Peter Prof. Dr. | |
| dc.subject.eng | in vitro spermatogenesis | de |
| dc.subject.eng | spermatogonial stem cells | de |
| dc.subject.eng | embryonic stem cells | de |
| dc.subject.eng | induced pluripotent stem cells | de |
| dc.subject.eng | co-culture | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0022-6035-B-0 | |
| dc.affiliation.institute | Biologische Fakultät für Biologie und Psychologie | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 828185433 | |