56 research outputs found
A nagy (őssejt)kaland; pluripotens őssejtvonalak létrehozása, jellemzése és felhasználási lehetőségeik
Differenciáció és apoptózis indukálta jelpályák kölcsönhatása korai mieloeritroid és eritroid sejtekben = Interaction of differentiation and apoptosis inducing signalling pathways in myeloerythroid and erythroid cells
Munkánk során összefüggést találtunk az eritroid differenciáció és a növekedési útvonalak, elsősorban az ERK MAPK, aktivitásának csökkenésében. Igazoltuk, hogy az ERK aktivitás csökkenése az eritroid differenciáció kezdeti szakaszában játszik szerepet, de nem elegendő a terminális differenciációhoz. ELM1 és TF1 sejteken kimutattuk, hogy a sejt ugyanarra stimulusra adott válaszát az ERK aktivitás időbeli lefutása döntően befolyásolhatja. K562 sejteken igazoltuk, hogy a Bcr/Abl jelpálya gátlása a sejtek hemoglobin termelésének növekedéséhez vezet. K562 és leukémiás betegekből származó sejteken bizonyítottuk, hogy a növekedési jelpálya egyidejű, több irányból történő támadása kedvező proapoptótikus hatást eredményez. Ezek az eredmények hozzájárulhatnak egyedi terápiák kidolgozásához. A431 sejteken kimutattuk, hogy az ABCG2 multidrog rezisztencia fehérje kifejeződése jelentős mértékben csökkenti a klinikumban alkalmazott TK gátló Iressa hatását. Összefüggést találtunk az ERK, p38 kinázok aktivitásainak mértéke, időbeli lefutása és az isoproterenol stimulus immunmoduláló hatása között. Beállítottunk egy új vizsgálati rendszert, mely fontos információkkal szolgálhat az új TK gátlók anti-tumor hatásának megítélésében. Résztvettünk új analitikai módszerek kidolgozásában. A külső sejt kontrollon alapuló mennyiségi mRNS meghatározási módszer szabadalmi bejelentéshez vezetett. Az új, nagy érzékenységű HPLC-MS módszer lehetővé teszi a sejten belüli TK gátlószerek direkt meghatározását. | We found a correlation between erythroid differentiation and a decrease in the activity of mitogen, especially of the ERK MAPK, pathways. We showed that a decrease in ERK activity was connected with the initiation steps of erythroid differentiation, but was not sufficient to induce terminal differentiation. We showed that the response of the cells was highly influenced by the kinetic of ERK activation. In K562 cells we found that the decrease of the Bcr/Abl kinase activity induced haemoglobin production. In K562 and in patient-originated primer cells, we demonstrated that the simultaneous action of different drugs along the mitogen pathway can have more favorable cytotoxic properties than those of individual drugs. All these results may serve as background for individual therapies. Strongly decreased cytotoxic efficiency could be observed in ABCG2 expressing A431 cells treated with Iressa. A strong correlation was found between the kinetic of ERK and p38 MAPK-s activation and the immuno-stimulating effect of beta-receptor activation by isoproterenol. We also participated in developing new analytical methods. A patent application has been filed covering our external control based quantitative mRNS detection. A new HPLC-MS based quantitative method has been developed for the detection of TKIs in the cells. Moreover we introduced a proper combination of in vitro assays that may provide valuable preclinical information for the targeted anticancer applicability of novel TKIs
Neurológiai és pszichiátriai betegségek in vitro modellezése indukált pluripotens őssejtek felhasználásával: fókuszban az Alzheimer-kór és a szkizofrénia
Over the past decade we witnessed the birth of a new scientific area that lies at the borders of developmental biology, stem cell biology, basic and clinical neuroscience. In vitro disease modeling refers to the approach that exploits the capacity of stem cells for self-renewal and pluripotency by generating specific cell types that are relevant for a given disorder. Based on this method, neurological and psychiatric disorders can be investigated by differentiating stem cells into neurons in a dish, and studying the relevant neuronal populations affected in the pathophysiology of the disorder in terms of specific cellular phenotypes. The advent of induced pluripotent stem cells (IPSCs) has made it possible to reprogram IPSCs from somatic cells of patients carrying specific genetic risk variants, and to analyze the in vitro cellular findings in the context of the clinical picture. Pluripotent stem cell based disease modeling offers an alternative solution for invasive and mostly not performable central nervous system biopsies in neuropsychiatric disorders, and is an appealing laboratory method for studying biomarkers of these disorders and for future drug development. This review summarizes the pluripotent stem cell based disease modeling literature in two important neuropsychiatric disorders, Alzheimer's disease and schizophrenia
Use of Transposon-Transposase Systems for Stable Genetic Modification of Embryonic Stem Cells
Expression of Tight Junction Components in Hepatocyte-Like Cells Differentiated from Human Embryonic Stem Cells
Human embryonic stem cells can be differentiated in vitro into a wide variety of progeny cells by addition of different morphogens and growth factors. Our aim was to monitor the expression pattern of tight junction (TJ) components and various cellular markers during differentiation of stem cell lines toward the hepatic lineage. Human embryonic stem cell lines (HUES1, HUES9) were differentiated into endoderm-like cells, and further differentiated to hepatocyte-like cells. Gene expressions of Oct3/4, Nanog, alpha-fetoprotein, albumin, cytokeratins (CK-7, CK-8, CK-18, CK-19), ATP-binding cassette (ABC) transporters (ABCC2, ABCC7, ABCG2), and various TJ components, including claudin-1, claudin-4, claudin-5, claudin-7, and tricellulin, as well as an extracellular matrix component, agrin were monitored during hepatic differentiation by real-time quantitative PCR. The differentiated cells exhibit epithelial morphology and functional assessments similar to that of hepatocytes. The expression level of stem cell marker genes (Oct3/4 and Nanog) significantly and gradually decreased, while liver-associated genes (alpha-fetoprotein, albumin) reached their highest expression at the end of the differentiation. The endoderm-like cells expressed claudin-1, which declined eventually. The expression levels of cholangiocyte markers including claudin-4, CK-7, CK-19, and agrin gradually increased and reached their highest level at the final stage of differentiation. In contrast, these cells did not express notable level of claudin-7, CK-8 and tricellulin. The marker set used for monitoring differentiation revealed both hepatocyte and cholangiocyte characteristics of the differentiated cells at the final stage. This is the first report describing the expression level changes of various TJ components, and underlining their importance in hepatic differentiation. © 2015 Arányi Lajos Foundatio
Excision efficiency is not strongly coupled to transgenic rate: cell type dependent transposition efficiency of Sleeping Beauty and piggyBac DNA transposons
The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, “overproduction inhibition,” a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own “in-house” platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2–3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future
Investigation of the effects of acute nicotine exposure and long term smoking on human induced pluripotent stem cell-derived endothelial cells from identical twins with DM. Investigation of the effects of acute nicotine exposure and long term smoking on human induced pluripotent stem cell-derived endothelial cells from identical twins with DM. : [abstract]
Mesenchymal stromal cell-like cells set the balance of stimulatory and inhibitory signals in monocyte-derived dendritic cells
Ct shift: A novel and accurate real-time PCR quantification model for direct comparison of different nucleic acid sequences and its application for transposon quantifications
There are numerous applications of quantitative PCR for both diagnostic and basic research. As in many other techniques the basis of quantification is that comparisons are made between different (unknown and known or reference) specimens of the same entity. When the aim is to compare real quantities of different species in samples, one cannot escape their separate precise absolute quantification. We have established a simple and reliable method for this purpose (Ct shift method) which combines the absolute and the relative approach. It requires a plasmid standard containing both sequences of amplicons to be compared (e.g. the target of interest and the endogenous control). It can serve as a reference sample with equal copies of templates for both targets. Using the DeltaDeltaCt formula we can quantify the exact ratio of the two templates in each unknown sample. The Ct shift method has been successfully applied for transposon gene copy measurements, as well as for comparison of different mRNAs in cDNA samples. This study provides the proof of concept and introduces some potential applications of the method; the absolute nature of results even without the need for real reference samples can contribute to the universality of the method and comparability of different studies
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