Cardiac failure is one of the leading cause of deaths worldwide. Potential therapeutic approach, which overcome invasive organ transplantation and delivery of immunosuppressive drugs, is lacking nowadays. However, research of mesenchymal stem cells (MSCs) therapy displays immunomodulation potential, which can further promote variety of organ regeneration without need of drug treatment. Xenopus tropicalis immature Sertoli cells (XtiSCs) culture was established in our laboratory from juvenile Xenopus tropicalis male. XtiSCs possess immunomodulatory capacity and differentiation to cardiomyocytes after the treatment with the inhibitor of glycogen synthase kinase-3 (GSK-3) CHIR99021. To test the survival rate of transplanted XtiSCs we firstly microinjected treated cells directly inside tadpole's heart. XtiSCs proliferated there for the whole tested time period (30 days). However, after direct heart XtiSCs injection and subsequent cardiac injury in adult frog, no cells were localized in wound area. Thus, we focused on remote control of cardiac regeneration using XtiSCs without CHIR99021 treatment. We injected cells inside skeletal muscle bed and confirmed their survival and proliferation. Moreover, if cells were transplanted 3 days before heart injury, it resulted in significant reduction of fibronectin...

Sertoli cells represent the only somatic cell type within the seminiferous tubules with direct contact to germ cells. Sertoli cells significantly contribute to the development of the testicular niche in a male embryo. Their role during postnatal life is in the regulation and nutrition of germ cells and the formation of the blood-testis barrier to protect these cells. In our laboratory, we have been successful in establishing a cell line of X. tropicalis immature Sertoli cells (XtiSCs) derived from juvenile testes of X. tropicalis. The objective of this thesis was to induce the differentiation process of XtiSCs into mature Sertoli cells. In vitro experiments using several factors or primary culture from adult male X. tropicalis did not show any mature markers in differentiated XtiSCs. Another experiment using cell culture derived from pubertal mice was partially successful in the induction of the differentiation process. These results indicate that XtiSCs do have some differentiation potential into mature Sertoli cells. Part of this work was to test the ability of testicular cells isolated from juvenile males of X. tropicalis to form de novo organoids. In vitro experiments were successful when these cells were cultured in a three-layer matrigel.

Sertoli cells (SCs) are somatic cells located in the testes. They are the only cells in direct contact with germ cells and play a key role in process of spermatogenesis. New insights in the biology of SCs are highlighting the immunological function of these cells: germ cells protection by maintaining the immunoprotective niche, creating the blood- testis barrier and local modulation of the immune response to spermatic cells. Immunomodulatory activity of SCs is preserved after their allo- and xenogeneic transplantation, and thus SCs prolongs survival not only of themselves but also of cells transplanted with them. The aim of this thesis was to study the survival and migration of SCs precursors (TSC) in mice recipients. The project is employing the neonatal tolerance phenomenon and evolutionary distinct donor organism, Xenopus tropicalis, to monitor conserved mechanisms of immune system (IS) modulation using SCs. SCs were detectable in the lungs and thymus 7 days after transplantation. The phenotype of immune cells was not altered 30 days after transplantation, however we detected changes in cytokine environment, namely increased levels of cytokines typical for Th2 and Treg immune responses. In vitro experiments further confirmed IS modulation by SCs - changing the phenotype of macrophages to alternatively...

African clawed frogs (Xenopus) represent an ideal model organism for study of regeneration mechanisms. In frogs, complete regeneration occurs in the tadpole stage. In later stages the regeneration capacity is lost. The Laboratory of Developmental biology was successful in establishment of cell culture called Xenopus tropicalis immature Sertoli cells (XtiSCs) derived from X. tropicalis testes. These cells are common progenitors of Sertoli cells and peritubular myoid cells. XtiSCs show similar characteristics as mesenchymal stem cells. MSCs hold interest of scientists for their immunomodulatory properties and multipotent differential and regeneration potential. In this thesis, we studied regeneration and migration potential of XtiSCs after X. tropicalis tadpole's tail amputation in developmental stage 47 - 50. Transgenic XtiSCs culture expressing RFP was prepared to facilitate transplantation experiments. Transplantation experiments showed preferential migration of XtiSCs into the site of injury. XtiSCs transplantations in X. laevis tadpoles with downregulated NO synthases eNOS and nNOS revealed their migratory dependence on nitric oxide signalization. Imunocytochemical staining of XtiSCs in vitro showed positive iNOS, nNOS and Pax7 expression. Imunohistochemical staining of tadpole's tail vibratome...

Cullin-dependent ubiquitin ligases are responsible for the regulation of most cellular processes. Despite their mutated forms being the cause of many human diseases, their physiological roles are not sufficiently described. In the presented results, we focused on the physiological role of ubiquitin ligase SCFFBXO38 (SKP1-CULLIN1-FBXO38), whose mutated forms are responsible for the progression of distal neuropathy. Preparation of mouse model deficient in FBXO38 revealed that homozygous pups were born in a lower than expected ratio. Animals were growth-retarded, both at the level of the whole organism and individual organs, especially the liver and testes. Males with a deletion in the Fbxo38 gene had significantly lower reproductive capacity, which was associated with lower production of mature sperm and pathological changes in the structure of seminiferous tubules. We found that the FBXO38 protein is functionally expressed in Sertoli cells responsible for regulating spermatogenesis and seminiferous tubules integrity. Detailed analysis of spermatogenic populations revealed a defect at the level of spermatocyte differentiation. The dynamics of this differentiation depend on the hematotesticular barrier functional integrity formed by the intercellular junctions of Sertoli cells. We confirmed that the...

Sertoli cells (SCs) are somatic cells of testicular tissue that are involved in spermatogenesis and maturation of germ cells. They are currently being extensively studied for their immunomodulatory abilities, and recent studies have shown that they share some properties with mesenchymal stromal cells (MSCs). Detailed characterization of SCs and clarification of their role in testicular tissue is crucial for potential use of SCs as a therapeutic tool in regenerative medicine. Cell culture of Xenopus tropicalis immature Sertoli cells (XtiSCs) and Mus musculus (mSCs) Sertoli cells were established in the Laboratories of Developmental Biology and Immunoregulations, Faculty of Science, Charles University. Previous research has characterized XtiSCs and demonstrated their multipotent potential by in vitro differentiation into a mesodermal line. Following this research, one of the goals of the diploma project was the induction of in vitro differentiation of XtiSC into other cell types, which would verify the differentiation potential of XtiSCs. The mSC expression profile confirmed the somatic origin of this culture as well as the transcription of Sertoli cell gene markers. Differentiation of mSCs along the mesodermal line into osteoblasts, chondrocytes and adipocytes has been successfully induced in vitro....