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Differentiation of pancreatic stem cells into insulin producing β-cells.
Leontovyč, Ivan ; Saudek, František (advisor) ; Štechová, Kateřina (referee) ; Holáň, Vladimír (referee)
Diabetes mellitus (DM) is a severe and frequent disease with increasing prevalence. It is not possible to achieve long term cure without late complications. Recent advances in cell fate modifications open a pathway to alternative cell therapies for DM cure. My doctoral thesis "Differentiation of pancreatic stem cells into insulin producing β- cells" is focused on the development of a new source of insulin secreting cells for transplantation. Combinatorial testing of numerous potential transcription factors and epigenetic modifiers resulted in a final protocol for the reprogramming pancreatic of exocrine cells into insulin secreting cells. The key transcriptional factors TF (Pdx1, Ngn3 a MafA) were applied in the form of synthetic mRNA. In four independent experiments we applied transcriptional factors in a specific sequence, thus obtaining 14.3 ± 1.9 % insulin positive cells. When challenged in vitro by the glucose levels of 2.5 and 20 mmol/l glucose, respectively, these cells exhibited glucose-sensitivity of insulin secretion (842 ± 72 and 1 157 ± 58 pg insulin/µg DNA/ml, n=5). They also demonstrated a sensitivity of insulin secretion (863 ± 78 and 1 025 ± 66 pg insulin/µg DNA/ml, n=5) to the concentration of depolarization agent KCl applied at 0 and 30 mmol/l, respectively together with 2.5...
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Specification and differentiation of cells in pancreatic development
Malfatti, Jessica ; Pavlínková, Gabriela (advisor) ; Horáková, Olga (referee)
Pancreas is divided into exocrine and endocrine tissue. The exocrine part contains acinar cells, which produce digestive enzymes, and ductal cells that help with their transportation to the duodenum. The islets of Langerhans form the endocrine part and consist of 5 types of cells; α, β, δ, ε, and PP-cells, producing hormones glucagon, insulin, somatostatin, ghrelin and pancreatic polypeptide, respectively. Pancreas development is divided into primary, secondary and tertiary transition. Many transcription factors participate in the cell specification and differentiation processes. Pancreatic duodenal homeobox 1 specifies the pancreatic domain in primary transition. Pancreas-specific transcription factor 1A is important for the specification and differentiation of acinar cells. All endocrine cell precursors express Neurogenin 3, a key specification factor of endocrine cells. A large number of transcription factors regulate differentiation of endocrine cells as well as their function. Absence or dysfunction of some transcription factors have been associated with pathologies, for example diabetes mellitus, pancreatic carcinoma or pancreatitis. Key words Pancreas, islets of Langerhans, insulin, glucagon, development, differentiation, diabetes
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Targeted differentiation of mesenchymal stem cells and their clinical application
Hámor, Peter ; Kubinová, Šárka (advisor) ; Trošan, Peter (referee)
The goal of this work is to point out possibilities of neuronal differentiation of mesenchymal stem cells and their application for clinical purposes, primary for repairing and regeneration of central nervous system tissues. Because this system works as a control center for functional features of the whole body, and treating this injuries and degenerations often bring many problems and obstacles, the possibility of using autologous cells for a transplantation or inducer of the natural regenerative properties of tissues is worth deeper research. This work progressively focuses on basic characteristics of stem cells and their differentiation potential, characterizing further mesenchymal stem cells together with possibilities of their isolation and cultivation. The main part of the text is formed by studies and methods used for targeted differentiation of mesenchymal stem cells and attempts of their transdifferentiation into neural cell line, together with present and possible future application of these cells in central nervous system therapies.
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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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