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Cellular Reprogramming as a Tool for Harvesting Patient-specific Stem Cells
Pisal, Rishikaysh ; Mokrý, Jaroslav (vedoucí práce) ; Hampl, Aleš (oponent) ; Jendelová, Pavla (oponent)
Cellular reprogramming as a tool for harvesting patient specific stem cells In the year 2006, Dr. Yamanaka surprised the entire field of medicine, by reporting a technique of inducing pluripotency in somatic cells. In his article, he had displayed that fibroblasts could be reprogrammed to pluripotent stem cell state, by ectopic expression of four transcription factors namely OCT4, SOX2, c-MYC and KLF4. His discovery made a paradigm shift in the field of reprogramming because previous methods of reprogramming were dependent on use of human oocytes and this raised ethical concerns. Moreover, his technique of cellular reprograming broadened the spectrum of application of somatic cells in regenerative medicine. Objectives of my research were focused on; development of an optimised protocol for detection of mycoplasma that commonly infects animal tissue culture; detailed characterization of reprogrammed clones; targeted differentiation of iPSC towards myogenic lineage, and construction of an expression vector, optimised for miRNA expression. For detecting mycoplasma infection, we adapted the protocol of Uphoff et al. (2002). By skipping the DNA extraction step (reported in the original protocol) and instead directly using cell culture supernatant and a robust polymerase enzyme for performing PCR, we...
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