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Human Dental Pulp Stem Cells Cultured in Xenogeneic-Free Supplemented Media
Suchánková Kleplová, Tereza ; Jouklová, Nela (advisor) ; Merglová, Vlasta (referee) ; Bartoňová, Marie (referee)
Human dental pulp stem cells cultured in xenogeneic-free supplemented media Summary Introduction: The topic of the study is the cultivation of dental pulp stem cells (hDPSCs) in a xenogeneic-free culture medium. It is not permissible to use cells upon growing under the influence of xenogeneic (extraneous) substances in human clinical practice. The most frequently used in cultivation of hDPSCs is fetal calf serum (FCS/FBS). Unfortunately, these supplements are widespread in hMSCs cultivation, and all gold standard hMSCs properties were postulated in cells cultivated using these supplements. This raises the basic question if and how xenogeneic blood derivatives affect the properties of cells and their growth characteristics. There are two options for replacing these xenogeneic substances in the culture medium: the so-called serum-free media, or human blood supplements, ideally autologous ones. The conducted research was aimed at identifying the effects of xenogeneic and human blood supplements on basic hDPSCs characteristics that are fundamental to introduce the cell therapy into regular medical practice. Method: By culturing 12 hDPSC lines obtained from adult, deciduous, and natal teeth in 12 different culture media, we investigated the effect of FCS, human blood derivatives, i.e., blood plasma (HP), and...
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Mitochondrial transfer and its role in regenerative properties of mesenchymal stem cells
Jaborová, Natálie ; Krulová, Magdaléna (advisor) ; Nahácka, Zuzana (referee)
Mitochondrial transfer represents a form of intercellular communication. In this process, mitochondria are delivered from the donor cell to the recipient cell through several structures. The transfer of mitochondria is observed under pathological and physiological conditions and is accompanied by specific signaling. Mitochondria uptake by injured cells promotes tissue regeneration. This bachelor thesis discusses general knowledge of mitochondrial transfer focusing on mesenchymal stem cells (MSCs) as a donor cell type. Furthermore, the regenerative effect of MSC-derived mitochondria transfer to cells damaged by various pathologies is summarized. In this regard, MSCs have significant therapeutic potential that could be used in future clinical strategies for a number of diseases. Keywords: mitochondria, transfer, mesenchymal stem cells, regeneration
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Immunomodulatory and differentiation properties of MSCs in a mouse model of the injured cornea and retina
Kössl, Jan ; Holáň, Vladimír (advisor) ; Vištejnová, Lucie (referee) ; Heissigerová, Jarmila (referee)
Stem cells, in general, represent the potential for treating many diseases and disorders that are currently difficult to treat or the therapy has many side effects. One of the stem cells widely investigated these days are mesenchymal stem cells (MSCs). MSCs have the considerable immunomodulatory and regenerative potential for treating degenerative disorders and severe damage to various parts of the eye or other organs. Likewise, their application could serve as supportive therapy in corneal transplantation and other eye inflammatory conditions. In this study of immunomodulatory properties of MSCs, we have focused mainly on their ability to differentiate into cells of different tissue types (in our case, corneal epithelium and retina), their production of immunomodulatory molecules in the inflammatory environment, their ability to migrate to the site of the injury, and their local anti-inflammatory, regenerative, and anti-apoptotic effects. In addition, we tested the therapeutic effects of MSCs in a mouse model of ocular surface injury and a model of retinal degeneration. Finally, we investigated the mechanism of this effect in in vitro models with explants of these tissues. Limbal stem cells (LSCs) are already used to treat severe corneal damage as limbal stem cell deficiency. However, this...
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Effect of oxidative stress on the differentiation of mesenchymal stem cells.
Bura, Radek ; Tlapáková, Tereza (advisor) ; Pacherník, Jiří (referee)
Mesenchymal stem cells are capable of forming different types of tissue such as muscle, bone, fat or cartilage tissue, thanks to the ability to divide and transform into another cell type. Mesenchymal stem cells obtained from various tissues are used for cell therapy and regenerative medicine. Knowledge of the influence of various factors on the differentiation of these multipotent cells is important. Currently, little is known about the effect of reactive oxygen species (ROS) on the differentiation potential of mesenchymal stem cells. The aim of this bachelor's thesis is to summarize the knowledge of the effect of oxidative stress on the differentiation of mesenchymal stem cells and the affected signaling pathways.
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Smart biomaterials for connective tissues regeneration
Hefka Blahnová, Veronika ; Filová, Eva (advisor) ; Maxová, Hana (referee) ; Motlík, Jan (referee)
Connective tissues are characterized by significant volume of extracellular matrix. Their main role is to provide a mechanical support and protection to other body organs. This thesis is focused on regeneration of bone, cartilage and osteochondral defect. In the experimental part we observed viability and differentiation of human mesenchymal stem cells. In vitro we evaluated the potential of PCL scaffold with addition of growth factors, bone xenograft with biomimetic peptides, collagen I composite with bioceramics and a titanium alloy with nanostructured surface. During following in vivo study, we implanted a cell-free scaffold made of PCL, calcium phosphate and IGF-1, bFGF, TGFβ1 and BMP-2 to osteochondral defect. Unfortunately, addition of growth factors resulted in pathological inflammatory process despite clear beneficial effect in vitro. Likewise, the biomimetic peptide sequences promoted osteogenic differentiation of human mesenchymal stem cells. Addition of certain bioceramics influenced the scaffold morphology in the manner of pore size. However, we did not observe any effect of the surface characteristics on cell behavior. The cells were influenced rather by certain material. On the other hand, surface modification of titanium scaffold by anodic oxidation revealed that the most suitable...
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The effect of nanoparticles on functional and regenerative properties of mesenchymal stem cells
Dostálová, Dominika ; Rössner, Pavel (advisor) ; Grobárová, Valéria (referee)
Mesenchymal stem cells (MSCs) represent a type of stem cells, localized in various tissues, where they are involved in the regeneration process. Because of their properties, MSC are tested for cell-based therapy. However, the treatment of injuries is often associated with bacterial infections, which complicate the process of healing. To establish a sterile environment, metallic nanoparticles (NPs) with proven antibacterial properties can be applied, for improving the healing process. Mechanisms of the antibacterial effect of NPs are non-specific and may cause the damage of eukaryotic cells. The aim of this thesis was to evaluate the effect of three antibacterial metallic NPs (silver, copper oxide and titanium dioxide) on the functional and regenerative properties of MSCs. The effect of NPs on the metabolic activity, differentiational potential, expression of genes for immunoregulatory molecules and production of cytokines by MSCs was determined. Additionally, the immunoregulatory properties of MSCs after cultivation with NPs were tested. The results showed that the metabolic activity was reduced in the presence of silver and copper oxide NPs, with the decrease dependent on the type of the tested NPs. The expression of selected immunoregulatory molecules was inhibited and the immunoregulatory...
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Mitochondrial transfer-mediated modulatory action of stem cells on immune cells
Somova, Veronika ; Krulová, Magdaléna (advisor) ; Balounová, Jana (referee)
Stem cells use different mechanisms of intercellular communication to modulate an immune response. Mitochondrial transfer is one of the mechanisms which induce metabolic changes, support cell survival, and change the phenotype of immune cells. Nevertheless, little is known about the mechanism used for transfer of mitochondria between different cell populations and the faith of mitochondria inside the acceptor cell. This thesis aims to describe the mechanism of transfer and the provided modulation. Factors that could affect mitochondrial transfer including reactive oxygen species production, apoptosis and mitochondria function were analyzed. And the impact of mitochondrial transfer on cell survival and mitophagy was described. The next aim was to compare the ability of mesenchymal stem cells (MSC) and Sertoli cells (SC) to transfer mitochondria, with MSC being more productive in the transfer of mitochondria than SC. Significant differences in the presence of mitochondria from donor MSC or SC in individual populations of immune cells were also detected. To explain these findings, the impact of reactive oxygen species on the transfer of mitochondria was analyzed in detail, although it wasn't confirmed. However, it needs to be highlighted that mitophagy plays an important role before and after...
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Importance of glycolysis and oxidative phosphorylation in the metabolism of mesenchymal stem cells
Fráňová, Markéta ; Krulová, Magdaléna (advisor) ; Rohlenová, Kateřina (referee)
Mesenchymal stem cells (MSCs) are classified as multipotent stem cells. They possess the ability to differentiate into many cell types, promote angiogenesis, increase cell survival in damaged tissue and modulate the immune response. These functions of MSCs are used in the treatment of various injuries and some diseases. This work characterizes MSCs, with a focus on their energy metabolism, specifically on the switch in their metabolic phenotype between glycolysis and oxidative phosphorylation in different states of MSCs, during cell culture and after transplantation. Finally, two modulations of MSC metabolism are presented, including cultivation in a hypoxic environment and quiescence induced by serum deprivation, which increase cell survival under the ischemic conditions that MSCs enter after transplantation. Key words: mesenchymal stem cells, metabolism, glycolysis, oxidative phosphorylation
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Fluorescent Methods in Research of Eukaryotic Cells
Chmelíková, Larisa ; Babula, Petr (referee) ; Pešl,, Martin (referee) ; Provazník, Ivo (advisor)
Tato práce zkoumá aplikaci fluorescenčních metod používaných v in vitro studiích v oblasti regenerace srdeční tkáně. Konfokální fluorescenční mikroskopie je vhodnou mikroskopickou technikou pro výzkum v této oblasti, protože umožňuje vizualizaci 3D struktur a distribuce buněk ve 3D modelech. Používané fluorescenční markery by měly být dlouhodobě stabilní, biokompatibilní a netoxické pro živé buňky. V současné době je použití nanočástic jako superparamagnetické nanočástice oxidu železa (SPION) velmi populární; velké množství studií ukazuje, že jsou vhodné pro dlouhodobé experimenty. Tento výzkum využívá superparamagnetické maghemitové nanočástice svázaným rhodaminem na jejich povrchu (SAMN-R) a popisuje jejích excitační a emisní spektrum, velikost a lokalizaci vbuňkách. Stanovení toxicity bylo provedeno měřením reaktivních forem kyslíku (ROS) a nekvantitativním měřením pomocí fluorescenční mikroskopie bylo zjištěno, že hodnota dávky 20 µg·cm-2 je optimální pro aplikaci na živé buňky. Dále byl zkoumán vliv aplikace SAMN-R na buněčnou proliferaci a motilitu, kdy ve studii buněčné proliferace a scratch assay byla použita buněčná linie fibroblastů 3T3. Poté byla studována migrace jednotlivých buněk s použitím mezenchymálních kmenových buněk (MSCs), izolovaných zlidské tukové tkáně. Následná statistická analýza nepotvrdila, že by aplikace SAMN-R měla významný vliv na buněčnou proliferaci, kolektivní migraci nebo na migraci jednotlivých buněk. Lze tedy předpokládat, že SAMN-R jsou vhodným fluorescenčním markerem pro výzkum živých buněk, včetně experimentů voblasti regenerace tkáně. MSC buňky izolované z tukové tkáně mají velký potenciál v regeneraci srdeční tkáně. Jejich interakce s buněčnou linií srdečních svalových buněk HL-1 byly studovány pomocí scratch assay, kdy se tento model jeví jako nadějný a vhodný pro studium buněčných kontaktů a jejich roli přiregeneraci buněk.
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Immunomodulatory properties of mesenchymal stem cells - use in therapy
Pavlíková, Michaela ; Krulová, Magdaléna (advisor) ; Stříž, Ilja (referee)
Mesenchymal stem cells (MSC) are extensively studied mainly due to their feasible clinical application. Therapeutic potential of MSC consists not only of the ability to differentiate into mesenchymal cells, ectodermal and endodermal cell lines, but primarily in their immunomodulatory functions. Due to their effect on immune cells, MSC promote the shift of the inflammatory immune response to antiinflammatory. The ability to suppress inflammation, together with their differentiation potential and antiapoptotic potential on the surrounding cells makes MSC a promising tool for treating serious diseases. This work discusses the effect of MSC on the individual cells of the immune system. It focuses on the description of the effect of MSC in four model cases. These are an experimental autoimmune encephalomyelitis, myocardial infarction, diabetes mellitus and skin graft transplantation. The knowledge of the mechanisms of the interactions between MSC and the cells of the immune system, together with the understanding the effect of specific conditions on MSC is essential for their use in clinical therapy. Keywords: mesenchymal stem cells, immunomodulation, autoimmune diseases, transplantation
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