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The study of Xenopus tropicalis testis-derived stem cells Nguyen Thi Minh Xuan Abstract The substances secreted by Sertoli cells (SCs) are crucial to determine male sex characteristics in embryos and regulate spermatogenesis in adulthood. The failure in SC maturation can cause sterility in men. Before puberty, SCs keep the ability to proliferate and have been considered as immature cells. They differ remarkably from mature cells in connection with their morphology and biochemical activity and thus they probably play a part in maintaining spermatogonia stem cells in an undifferentiated stage. The transient presence of cytokeratin in immature SCs has been reported in many species, but not in Xenopus yet. We investigated which molecules are expressing only in immature Sertoli cells of X. tropicalis testes. The regulation of cytokeratin and β-catenin was revealed by fluorescent immunostaining. Cytokeratin and membrane β-catenin co- expressed in X.tropicalis juvenile testes and in cultured SC progenitors, called XtiSCs, but they were absent in adulthood. There was no signal of cytokeratin in migrating SCs (pre-SCs) located outside the seminiferous tubules. The suppression of cytokeratin along with the breakdown of β-catenin-based cell contacts have been observed in XtiSCs after the treatment with a small...

Thanks to their ability to differentiate into variable cell types and migrate to the site of an injury mesenchymal stem cells (MSC) are broadly used in regenerative medicine. Their relative easy availability together with the property to control the immune system determines them as a cure of autoimmune diseases or a recovery of wounded tissues. Similar features posses Sertoli cells which take place in the seminiferous tubule of testis. Cell culture of testicular stem cells from juvenile male testes of X. tropicalis (XtTSC) was established in supervisor's laboratory. This cell culture showing both MSC's and SeC's properties was transformed to carry red fluorescent protein RFP. The aim of this diploma thesis was to investigate an behavior of transformed XtTSC in living organism, therefore cells were transplanted into the X. tropicalis tadpoles in stage 41. Subsequently, their migration potential was explored. To study of XtTSC's differentiation potential it was necessary to introduce a reliable sectioning techniques for the subsequent immunohistochemical analysis. Based on our experiments, we found that the XtTSC's cell culture contains precursors of SeC and peri-tubular myoid cells, however in vivo these cells turned into the dedifferentiated MSC-like state allowing a strong migration through the...

Testicular stem cells (TSCs) are relatively accessible potential source of pluripotent cells, which are particularly important for their application in regenerative medicine. Xenopus tropicalis is a useful model organism to study the migration and differentiation potential of stem cells. This amphibian is characteristic by outer fecundation and embryonic development of a great amount of embryos after fertilization. Oocytes and embryos are large enough (about 1 mm) to be suitable for micromanipulation micromanipulations. Laboratory of Developmental Biology, Faculty of Science, Charles University in Prague succeeded in the establishment of a mixed cell culture of TSCs growing on feeder layer of pre- Sertoli cells. This culture was derived from the testes of juvenile Xenopus tropicalis male. In the study of their differentiation potential it was found, that leukemia inhibitory factor (LIF) is the decisive factor allowing rapid proliferation of stem cells and their forming into characteristic colonies. This protein is produced by both types of cells which are present in the culture. The mouse LIF has the same positive effect on the proliferative potential of stem cells, which points at the evolutionary conservation of metabolic pathways associated with the maintenance of the stemness. RT-PCR analysis...

Because of their possible therapeutic potential stem cells are one of the most promising fields of study in biology. The aim of this thesis was to study the migration potential of testicular stem cells of Xenopus tropicalis, whose culture was established from the testes of juvenile males in the laboratory of my supervisor. Due to external fertilization and embryonic development Xenopus represents an ideal model organism for transplantation and microinjection experiments in-vivo. Transplantation of vital marked (PKH26) testicular stem cells into blastula and peritoneum of tadpoles showed their wide migratory potential including intestine (entoderm), heart, pronephros, genital groove (mesoderm) and epidermis (ectoderm). Based on my experiments, I found that the ideal number of cells for transplantation ranges between 250-500 per tadpole. To further characterize the stem cells, I constructed a plasmid vector carrying a gene for a red fluorescent protein. This plasmid was then used for preparation of frogs with whole-body expression of Katushka RFP under control of CAG promoter. The next aim is to gain the RFP positive offspring by crossing the transgenic individuals with the wild type. Male offspring can be used for establishing culture of testicular stem cells stably expressing the reporter gene. In this way...