Browsing by Author "Kozlovska, Tatjana"

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    Characterization of senescence of culture-expanded human adipose-derived mesenchymal stem cells
    (2016) Legzdina, Diana; Romanauska, Anete; Nikulshin, Sergey; Kozlovska, Tatjana; Berzins, Uldis
    Background and Objectives: Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates in regenerative medicine. The need for in vitro propagation to obtain therapeutic quantities of the cells imposes a risk of impaired functionality due to cellular senescence. The aim of the study was to analyze in vitro senescence of previously cryopreserved human ADSCs subjected to serial passages in cell culture. Methods and Results: ADSC cultures from 8 donors were cultivated until proliferation arrest was reached. A gradual decline of ADSC fitness was observed by altered cell morphology, loss of proliferative, clonogenic and differentiation abilities and increased β-galactosidase expression all of which occurred in a donor-specific manner. Relative telomere length (RTL) analysis revealed that only three tested cultures encountered replicative senescence. The presence of two ADSC subsets with significantly different RTL and cell size was discovered. The heterogeneity of ADSC cultures was supported by the intermittent nature of aging seen in tested samples. Conclusions: We conclude that the onset of in vitro senescence of ADSCs is a strongly donor-specific process which is complicated by the intricate dynamics of cell subsets present in ADSC population. This complexity needs to be carefully considered when elaborating protocols for personalized cellular therapy.
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    High efficiency of alphaviral gene transfer in combination with 5-fluorouracil in a mouse mammary tumor model
    (2014-06-20) Zajakina, Anna; Vasilevska, Jelena; Zhulenkovs, Dmitry; Skrastina, Dace; Spaks, Artjoms; Plotniece, Aiva; Kozlovska, Tatjana
    Background: The combination of virotherapy and chemotherapy may enable efficient tumor regression that would be unachievable using either therapy alone. In this study, we investigated the efficiency of transgene delivery and the cytotoxic effects of alphaviral vector in combination with 5-fluorouracil (5-FU) in a mouse mammary tumor model (4 T1).Methods: Replication-deficient Semliki Forest virus (SFV) vectors carrying genes encoding fluorescent proteins were used to infect 4 T1 cell cultures treated with different doses of 5-FU. The efficiency of infection was monitored via fluorescence microscopy and quantified by fluorometry. The cytotoxicity of the combined treatment with 5-FU and alphaviral vector was measured using an MTT-based cell viability assay. In vivo experiments were performed in a subcutaneous 4 T1 mouse mammary tumor model with different 5-FU doses and an SFV vector encoding firefly luciferase.Results: Infection of 4 T1 cells with SFV prior to 5-FU treatment did not produce a synergistic anti-proliferative effect. An alternative treatment strategy, in which 5-FU was used prior to virus infection, strongly inhibited SFV expression. Nevertheless, in vivo experiments showed a significant enhancement in SFV-driven transgene (luciferase) expression upon intratumoral and intraperitoneal vector administration in 4 T1 tumor-bearing mice pretreated with 5-FU: here, we observed a positive correlation between 5-FU dose and the level of luciferase expression.Conclusions: Although 5-FU inhibited SFV-mediated transgene expression in 4 T1 cells in vitro, application of the drug in a mouse model revealed a significant enhancement of intratumoral transgene synthesis compared with 5-FU untreated mice. These results may have implications for efficient transgene delivery and the development of potent cancer treatment strategies using alphaviral vectors and 5-FU.
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    Synthesis and comparative evaluation of novel cationic amphiphile C12-Man-Q as an efficient DNA delivery agent in vitro
    (2018) Apsite, Gunita; Timofejeva, Irena; Vezane, Aleksandra; Vigante, Brigita; Rucins, Martins; Sobolev, Arkadij; Plotniece, Mara; Pajuste, Karlis; Kozlovska, Tatjana; Plotniece, Aiva; Rīga Stradiņš University
    New amphiphilic 1,4-DHP derivative C12-Man-Q with remoted cationic moieties at positions 2 and 6 was synthesised to study DNA delivery activity. The results were compared with data obtained for cationic 1,4-DHP derivative D19, which is known to be the most efficient one among the previously tested 1,4-DHP amphiphiles. We analysed the effects of C12-Man-Q concentration, complexation media, and complex/cell contact time on the gene delivery effectiveness and cell viability. Transmission electron microscopy data confirms that lipoplexes formed by the compound C12-Man-Q were quite uniform, vesicular-like structures with sizes of about 50 nm, and lipoplexes produced by compound D19 were of irregular shapes, varied in size in the range of 25–80 nm. Additionally, confocal microscopy results revealed that both amphiphiles effectively delivered green fluorescent protein expression plasmid into BHK-21 cells and produced a fluorescent signal with satisfactory efficiency, although compound C12-Man-Q was more cytotoxic to the BHK-21 cells with an increase of concentration. It can be concluded that optimal conditions for C12-Man-Q lipoplexes delivery in BHK-21 cells were the serum free media without 0.15 M NaCl, at an N/P ratio of 0.9. Compound D19 showed higher transfection efficiency to transfect BHK-21 and Cos-7 cell lines, when transfecting active proliferating cells. Although D19 was not able to transfect all studied cell lines we propose that it could be cell type specific. The compound C12-Man-Q showed modest delivery activity in all used cell lines, and higher activity was obtained in the case of H2-35 and B16 cells. The transfection efficiency in cell lines MCF-7, HeLa, and Huh-7 appears to be comparable to the reference compound D19 and minimal in the HepG2 cell line.
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    Synthesis and comparative evaluation of novel cationic amphiphile C12-Man-Q as an efficient DNA delivery agent in vitro
    (2018) Apsite, Gunita; Timofejeva, Irena; Vezane, Aleksandra; Vigante, Brigita; Rucins, Martins; Sobolev, Arkadij; Plotniece, Mara; Pajuste, Karlis; Kozlovska, Tatjana; Plotniece, Aiva; Rīga Stradiņš University
    New amphiphilic 1,4-DHP derivative C12-Man-Q with remoted cationic moieties at positions 2 and 6 was synthesised to study DNA delivery activity. The results were compared with data obtained for cationic 1,4-DHP derivative D19, which is known to be the most efficient one among the previously tested 1,4-DHP amphiphiles. We analysed the effects of C12-Man-Q concentration, complexation media, and complex/cell contact time on the gene delivery effectiveness and cell viability. Transmission electron microscopy data confirms that lipoplexes formed by the compound C12-Man-Q were quite uniform, vesicular-like structures with sizes of about 50 nm, and lipoplexes produced by compound D19 were of irregular shapes, varied in size in the range of 25–80 nm. Additionally, confocal microscopy results revealed that both amphiphiles effectively delivered green fluorescent protein expression plasmid into BHK-21 cells and produced a fluorescent signal with satisfactory efficiency, although compound C12-Man-Q was more cytotoxic to the BHK-21 cells with an increase of concentration. It can be concluded that optimal conditions for C12-Man-Q lipoplexes delivery in BHK-21 cells were the serum free media without 0.15 M NaCl, at an N/P ratio of 0.9. Compound D19 showed higher transfection efficiency to transfect BHK-21 and Cos-7 cell lines, when transfecting active proliferating cells. Although D19 was not able to transfect all studied cell lines we propose that it could be cell type specific. The compound C12-Man-Q showed modest delivery activity in all used cell lines, and higher activity was obtained in the case of H2-35 and B16 cells. The transfection efficiency in cell lines MCF-7, HeLa, and Huh-7 appears to be comparable to the reference compound D19 and minimal in the HepG2 cell line.