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Development and optimalization of sectioning technique for the study of migration and differentiation potential of testicular stem cells in X. tropicalis tadpoles
Bláhová, Monika ; Krylov, Vladimír (advisor) ; Pšenička, Martin (referee)
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...
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The study of Xenopus tropicalis testis-derived stem cells
Nguyen, Thi Minh Xuan ; Krylov, Vladimír (advisor) ; Bartůněk, Petr (referee) ; Hovořáková, Mária (referee)
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...
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Development and optimization of sectioning technique for the study of migration and differentiation potential of testicular stem cells in X. tropicalis tadpoles
Bláhová, Monika ; Krylov, Vladimír (advisor) ; Soukup, Vladimír (referee)
The rapid development of regenerative medicine and the urgent need for cells which are able to modulate the immune system, or even differentiate into variable cell types, have led to the research of mesenchymal stem cells. The Sertoli cells, which are essential for the proper development of sperm in the testis, have a strikingly similar character. In the previous research, a cell culture expressing markers of mesenchymal stem cells and Sertoli cells from juvenile male testes of X. tropicalis was established. At the same time, the cells were modified by the introduction of gene for the red fluorescent protein (RFP) in their genome. The aim of this diploma thesis was to clarify their characteristics after microinjection to X. tropicalis tadpoles (allogeneic transplantation). For these experiments, it was necessary to develop a reliable technique for the preparation of sections, which won't be harmfull for the samples. Using the vibratome sectioning method along with immunohistochemical labeling, the cell culture has been found to contain precursors of Sertoli and peritubulare myoid cells.
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Induction of Xenopus tropicalis testicular stem cell differentiation in vitro.
Strnadová, Karolína ; Tlapáková, Tereza (advisor) ; Javorková, Eliška (referee)
Origin of mammalian somatic cells in the developing testes remains unclear. This origin could be explained by established cell culture derived from testes of Xenopus tropicalis juvenile male. The expression profile of the cell culture showed transcription of some pluripotency genes, somatic Sertoli and peritubular myoid cell markers and last but not least, the mesenchymal stem cell markers. Conversely, germ cell genes were downregulated. Immunocytochemical analysis revealed expression of Vimentin, Sox9 and α-smooth muscle actin, indicating that the testicular cell culture is a common mesenchymal progenitor of the Sertoli and peritubular myoid cells and that the cell culture did not arise from spermatogonial stem cells undergoing incomplete reprogramming in vitro. Testing of X. tropicalis cell culture during induction of differentiation in vitro revealed that these cells are probably multipotent with the ability to differentiate into adipocytes, chondroblasts and osteoblasts. The ability to derive multipotent stem cells from the juvenile testes opens new possibilities of using these cells for biotechnology and medicine. Keywords: Testicular somatic cells, Xenopus tropicalis, progenitor, mesenchymal stem cells, induction of differentiation, multipotency
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Genetic mapping in Xenopus
Seifertová, Eva
The diploid amphibian Xenopus tropicalis represents a significant model organism for studies of early development, genes function and evolution. Such techniques as gynogenesis, injection of morpholino antisense oligonucleotide into fertilized eggs or transgenesis were established. In the recent ten years, many efforts have been made to complete the sequence information. X. tropicalis genome has been sequenced but the completion of its assembly only on the basis of sequence data has been impossible. Therefore, our first work was focused on one of approaches for a genome completing- genetic mapping. First of all, the genetic map of Xenopus tropicalis was established pursuant linkage and physical positions of markers. Since the map contained gaps, we developed a new method for genetic mapping based on the next generation sequencing of laser microdissected arm. Using Illumina next generation sequencing of fifteen copies of a short arm of chromosome 7, we obtained new insights into its genome by localizing previously unmapped genes and scaffolds as well as recognizing mislocalized portions of the genome assembly. This was the first time laser microdissection and sequencing of specific chromosomal regions has been used for the purpose of genome mapping. These data were also used in the evolution study of...
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Genetic mapping in Xenopus
Seifertová, Eva ; Krylov, Vladimír (advisor) ; Ráb, Petr (referee) ; Marec, František (referee)
The diploid amphibian Xenopus tropicalis represents a significant model organism for studies of early development, genes function and evolution. Such techniques as gynogenesis, injection of morpholino antisense oligonucleotide into fertilized eggs or transgenesis were established. In the recent ten years, many efforts have been made to complete the sequence information. X. tropicalis genome has been sequenced but the completion of its assembly only on the basis of sequence data has been impossible. Therefore, our first work was focused on one of approaches for a genome completing- genetic mapping. First of all, the genetic map of Xenopus tropicalis was established pursuant linkage and physical positions of markers. Since the map contained gaps, we developed a new method for genetic mapping based on the next generation sequencing of laser microdissected arm. Using Illumina next generation sequencing of fifteen copies of a short arm of chromosome 7, we obtained new insights into its genome by localizing previously unmapped genes and scaffolds as well as recognizing mislocalized portions of the genome assembly. This was the first time laser microdissection and sequencing of specific chromosomal regions has been used for the purpose of genome mapping. These data were also used in the evolution study of...
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Preparation of Xenopus tropicalis transgenic testicular stem cell culture.
Vegrichtová, Markéta ; Tlapáková, Tereza (advisor) ; Drobná Krejčí, Eliška (referee)
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...
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Study of migration potential of testicular stem cells in Xenopus tropicalis
Fuxová, Helena ; Krylov, Vladimír (advisor) ; Černý, Robert (referee)
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...
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Preparation of X. tropicalis recombinant growth factors and their characterization in testicular tissue culture.
Borecká, Marianna ; Krylov, Vladimír (advisor) ; Drobná Krejčí, Eliška (referee)
In our Laboratory of Developmental Biology there was established a long term culture derived from Xenopus tropicalis testes. It contains pre-Sertoli cells mostly. They compose a feeder layer allowing cultivation of stem cells, revealing the morphology of spermatogonial stem cells. This diploma thesis was focused on a preparation of two growth factors, FGF2 (fibroblast growth factor 2) and GDNF (glial cell line-derived neurotrophic factor), with the subsequent characterization of their influence at cell culture mentioned above. Factors were selected on the basis of the microenvironmental niche theory, according which FGF2 and GDNF are the most important factors for spermatogonial stem cells proliferation and self-renewal. FGF2 recombinant factor was gained using the expression plasmid pET-15b. Its characterization in the testicular culture brought surprising result. Even a low concentration of FGF2 factor (2.5ng/ml) caused cell detaching and dying. Similar result was previously shown in differentiating osteoblast culture only. More experiments need to be done to prove if apoptose take place and why do testicular cells act this way. Key words: Xenopus tropicalis, FGF2, GDNF, SSC, pre-Seroli cells
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Physical mapping of genome regions without linkage map using BAC clones in Xenopus tropicalis
Špirhanzlová, Petra ; Krylov, Vladimír (advisor) ; Marec, František (referee)
Xenopus leavis was a favorite model organism during the 20th. century, but nowadays it has been replaced by diploid Xenopus tropicalis, which has not only shorter generation time, but also smaller genom. One of the disadvantages of Xenopus tropicalis is the lack of full physical and linkage map. According to JGI genome database (assembly 4.1) there are unmapped regions on short arm of the chromosome 2 and 7 . Several BAC clones ( with a single or dual-end sequence) has been found to be located within this region, according to a recent assembly 7.1. However , it isn't clear whether 100bp length of BAC ends is enough to place entire BAC clone into the genom of Xenopus tropicalis. In order to prove correct inclusion of these BAC clones into JGI database, several BAC clones, which are supposed to be located on short arm of chromosome 2, were picked. Using fluorescence in situ hybridisation, the signal of these BAC clones was localised on the short arm of chromosome 1 instead of chromosome 2 and in most cases they had opposite orientation. It means that the 100bp lenght of BAC ends propably isn't sufficient to place entire BAC clone on chromosome. New working protocol of BAC DNA isolation and labeling was established.
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