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Immunomodulatory mechanisms of stem cells and their use for therapy of ocular disorders
Heřmánková, Barbora ; Holáň, Vladimír (advisor) ; Heissigerová, Jarmila (referee) ; Indrová, Marie (referee)
Stem cell-based therapy represents a perspective approach for the treatment of many so far incurable diseases. Mesenchymal stem cells (MSC) are currently the most studied stem cells. They are able to differentiate into different cell types, to produce growth and trophic factors and can suppress the functions of cells of the immune system. During the study of the immunomodulatory properties of MSC, we focused on their effect on B cells. The mechanism of impact of interferon-γ (IFN-γ) on MSC and their effect on the production of interleukin 10 (IL-10) by B cells was analysed. We have demonstrated that MSC-treated with IFN-γ inhibit production of IL-10 by activated B cells via the cyclooxygenase-2 involving pathway. Due to their regenerative and immunomodulatory properties, MSC can be for treatment of many diseases. In this study we focused on the disease and damage of the eye. The limbal stem cells (LSC) are used for the treatment of damaged ocular surface, however their isolation is difficult and they can not be used in all cases of damage. Appropriate candidates in these cases are MSC. Therefore we have decided to compare the therapeutic potential of LSC and MSC isolated from bone marrow or adipose tissue. The study have shown that MSC isolated from bone marrow have a similar regenerative effect on...
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Limbal stem cell transplantation and their utilization for ocular surface reconstruction.
Lenčová, Anna ; Filipec, Martin (advisor) ; Heissigerová, Jarmila (referee) ; Ardan, Taras (referee)
Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...
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Immunomodulatory mechanisms of stem cells and their use for therapy of ocular disorders
Heřmánková, Barbora
Stem cell-based therapy represents a perspective approach for the treatment of many so far incurable diseases. Mesenchymal stem cells (MSC) are currently the most studied stem cells. They are able to differentiate into different cell types, to produce growth and trophic factors and can suppress the functions of cells of the immune system. During the study of the immunomodulatory properties of MSC, we focused on their effect on B cells. The mechanism of impact of interferon-γ (IFN-γ) on MSC and their effect on the production of interleukin 10 (IL-10) by B cells was analysed. We have demonstrated that MSC-treated with IFN-γ inhibit production of IL-10 by activated B cells via the cyclooxygenase-2 involving pathway. Due to their regenerative and immunomodulatory properties, MSC can be for treatment of many diseases. In this study we focused on the disease and damage of the eye. The limbal stem cells (LSC) are used for the treatment of damaged ocular surface, however their isolation is difficult and they can not be used in all cases of damage. Appropriate candidates in these cases are MSC. Therefore we have decided to compare the therapeutic potential of LSC and MSC isolated from bone marrow or adipose tissue. The study have shown that MSC isolated from bone marrow have a similar regenerative effect on...
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Immunomodulatory mechanisms of stem cells and their use for therapy of ocular disorders
Heřmánková, Barbora
Stem cell-based therapy represents a perspective approach for the treatment of many so far incurable diseases. Mesenchymal stem cells (MSC) are currently the most studied stem cells. They are able to differentiate into different cell types, to produce growth and trophic factors and can suppress the functions of cells of the immune system. During the study of the immunomodulatory properties of MSC, we focused on their effect on B cells. The mechanism of impact of interferon-γ (IFN-γ) on MSC and their effect on the production of interleukin 10 (IL-10) by B cells was analysed. We have demonstrated that MSC-treated with IFN-γ inhibit production of IL-10 by activated B cells via the cyclooxygenase-2 involving pathway. Due to their regenerative and immunomodulatory properties, MSC can be for treatment of many diseases. In this study we focused on the disease and damage of the eye. The limbal stem cells (LSC) are used for the treatment of damaged ocular surface, however their isolation is difficult and they can not be used in all cases of damage. Appropriate candidates in these cases are MSC. Therefore we have decided to compare the therapeutic potential of LSC and MSC isolated from bone marrow or adipose tissue. The study have shown that MSC isolated from bone marrow have a similar regenerative effect on...
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The culture of limbal and mesenchymal cells on various feeders for their use in ophthalmology.
Trošan, Peter ; Jirsová, Kateřina (advisor) ; Heissigerová, Jarmila (referee) ; Netuková, Magdaléna (referee)
P.Trošan Ph.D. Thesis Abstract Limbal stem cell deficiency (LSCD) is a disease characterized by the deficiency of stem cells in the limbus, which are responsible for the homeostasis and renewal of the corneal epithelium. This disorder results in corneal neovascularization, chronical inflammation and opacification, which may lead to loss of vision. The most successful treatment is the transplantation of limbal tissue or cultured limbal epithelial cells (LECs) onto the damaged ocular surface. The human amniotic membrane (HAM) is used as the feeder of the LECs culture, as well as for the LSCD treatment. HAM is also widely used in clinical practice, particularly for the treatment of chronic wounds. This dissertation is particularly concerned on cell therapy for LSCD, on preparation of cells suitable for grafting onto the ocular surface, on the improvement of the LECs culture conditions, and on the preparation of appropriate carrier for the transfer of cells onto the damaged cornea. During my work I have used a wide spectrum of methods, e.g. cell cultures (LECs, mesenchymal stem, amniotic epithelial, conjunctival epithelial, goblet and 3T3 cells), immunohisto- and immunocytochemistry, microscopy, proliferation and colony forming assays, reverse transcription and quantitative real-time PCRs and statistical...
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Immunomodulatory mechanisms of stem cells and their use for therapy of ocular disorders
Heřmánková, Barbora ; Holáň, Vladimír (advisor) ; Heissigerová, Jarmila (referee) ; Indrová, Marie (referee)
Stem cell-based therapy represents a perspective approach for the treatment of many so far incurable diseases. Mesenchymal stem cells (MSC) are currently the most studied stem cells. They are able to differentiate into different cell types, to produce growth and trophic factors and can suppress the functions of cells of the immune system. During the study of the immunomodulatory properties of MSC, we focused on their effect on B cells. The mechanism of impact of interferon-γ (IFN-γ) on MSC and their effect on the production of interleukin 10 (IL-10) by B cells was analysed. We have demonstrated that MSC-treated with IFN-γ inhibit production of IL-10 by activated B cells via the cyclooxygenase-2 involving pathway. Due to their regenerative and immunomodulatory properties, MSC can be for treatment of many diseases. In this study we focused on the disease and damage of the eye. The limbal stem cells (LSC) are used for the treatment of damaged ocular surface, however their isolation is difficult and they can not be used in all cases of damage. Appropriate candidates in these cases are MSC. Therefore we have decided to compare the therapeutic potential of LSC and MSC isolated from bone marrow or adipose tissue. The study have shown that MSC isolated from bone marrow have a similar regenerative effect on...
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Limbal stem cell transplantation and their utilization for ocular surface reconstruction.
Lenčová, Anna ; Filipec, Martin (advisor) ; Heissigerová, Jarmila (referee) ; Ardan, Taras (referee)
Aims: Limbal stem cell (LSC) deficiency is one of the most challenging ocular surface diseases. The aim of this thesis was to study damaged ocular surface reconstruction. Therefore, a mouse model of limbal transplantation was estab- lished. Furthermore, LSC isolation, transfer of LSCs and bone marrow-derived mesenchymal stem cells (MSCs) on nanofiber scaffolds were studied. Material and methods: Syngeneic, allogeneic and xenogeneic (rat) limbal grafts were transplanted orthotopically into BALB/c mice. Graft survival, immune re- sponse and the effect of monoclonal antibodies (mAb) (anti-CD4 and anti-CD8 cells) were analyzed. Mouse LSCs were separated by Percoll gradient; subse- quently, they were analyzed for the presence of LSC and differentiation corneal epithelial cell markers and characteristics using real-time PCR and flow cytom- etry. Nanofiber scaffolds seeded with LSCs and MSCs were transferred onto the damaged ocular surface in mouse and rabbit models. Cell growth on scaffolds, post-operative inflammatory response and survival of transferred cell were ana- lyzed. Results: Limbal allografts were rejected promptly by the Th1-type of immune response (IL-2, IFN-γ) involving CD4+ cells and nitric oxide produced by macro- phages, contrary to the prevailing Th1 and Th2 immune responses (IL-4, IL-10) in...
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Local production of cytokines after treatment with stem cells of damaged ocular surface
Kössl, Jan ; Holáň, Vladimír (advisor) ; Kovář, Marek (referee)
The damage of ocular surface represents one of the most common causes of decreased quality of vision or even blindness. If the injury is extensive and includes the region of limbus, niche of limbal stem cells (LSC), LSC deficiency occurs and the natural corneal regeneration is stopped. Conjunctival epithelium migrates into the injured area. Neovascularization, local inflammation and corneal opacity occur. Corneal transplantation is an insufficient treatment in such case. If the injury is bilateral, the allogenic limbal graft or LSC transplantation is required. In such cases systemic immunosuppressive drugs with many negative side-effects must be administered. The search for an adequate autologous substitution is important for avoid immunosuppressive medication. Mesenchymal stem cells (MSC) represent a perspective substitution for the reason of their immunomodulatory properties and the capability to differentiate in many cell types. There is possibility to isolate sufficient number of these cells from adipose tissue or bone marrow which are relatively easily accessible. Our goal was to observe local production of cytokines and other molecules which are present in inflammatory reaction after the chemical burn of the murine cornea and after the treatment with stem cells growing on nanofiber scaffold....
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Possibilities of using stem cells for treatment of ocular surface injuries
Kössl, Jan ; Holáň, Vladimír (advisor) ; Drbal, Karel (referee)
Ocular surface defects are one of the most common causes of impaired vision or even blindness. Corneal transplantation is the first choice of the treatment of these defects. If the damage is extensive and includes the limbus, niche of limbal stem cells (LSCs), LSC deficiency (LSCD) occurs and reparation with regeneration of cornea is impaired. The only way to treat LSCD is limbal transplantation or transplantation of autologous LSCs from the healthy eye. In cases of bilateral LSCD there are no autologous LSCs available. The use of allogeneic LSCs is associated with the requirement of systemic or local administration of immunosuppressive drugs which have often negative side-effects and outcomes remain uncertain. The alternative for treatment of ocular surface defects and LSCD is finding a new source of an appropriate autologous stem cell substitute which are e.g. mesenchymal stem cells (MSCs). These cells can be obtained from bone marrow or adipose tissue of the particular patient. MSCs can be easily cultivated ex vivo and can be transferred onto the damaged ocular surface using appropriate scaffold. Here they can differentiate to corneal epithelial cells, have immunomodulatory properties and produce numerous trophic and growth factors. The experiments with MSCs on animal models of mechanically or...
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Targeted differenciation of limbal and mesenchymal stem cells and their therapeutic application
Kuthanová, Hana ; Holáň, Vladimír (advisor) ; Indrová, Marie (referee)
The research of stem cells slowly transfers from the experimental to the preclinical and clinical level. They are in the centre of interest thanks to their potential to treat many of severe injuries and genetically determined diseases. However, the clinical application of these cells has to be based on a basic research of their characteristics and differential potential. Adult stem cells are in organism in minor populations in unique niches. In comparison with embryonic and induced pluripotent stem cells, the adult stem cells have lower differential potential but they also tend less to making teratomas. The therapeutic use of differential and transdifferential potential of limbal and mesenchymal stem cells is described here in more detail with focus on their use in damaged ocular surface treatment. Limbal stem cells are the only source of stem cells for corneal epithelium regeneration in most organisms. Deficiency of these stem cells leads to severe eye disorders even to blindness. Nowadays, a transplantation of allogeneic limbal stem cells or allogeneic limbus is the only chance for patients with total limbal stem cell deficiency. In clinical trials with patients with particular limbal stem cell deficiency, autologous limbal stem cells were successfully transplanted. Mesenchymal stem cells derived...
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