[Regulation of Metabolism and the Role of Redox Factors in the Energy Control of Quiescence and Proliferation of Hematopoietic Cells].

One of the key regulators of hematopoietic stem cell (HSC) maintenance is cellular metabolism. Resting HSCs use anaerobic glycolysis as the main source of energy. During expansion and differentiation under conditions of steady state hematopoiesis, the energy needs of activated HSCs increase by many fold. To meet the increased demands, cells switch to mitochondrial oxidative phosphorylation, which is accompanied by an increase in reactive oxygen species (ROS) production. Here, the molecular mechanisms maintaining glycolysis in HSCs, as well as the factors determining the increase in metabolic activity and the transition to mitochondrial biogenesis during HSC activation are discussed. We focus on the role of HIF (hypoxia-inducible factor) proteins as key mediators of the cellular response to hypoxia, and also consider the phenomenon of extraphysiological oxygen shock (EPHOSS), leading to the forced differentiation of HSCs as well as methods of overcoming it. Finally, the role of fatty acid oxidation (FAO) in hematopoiesis is discussed. Understanding the metabolic needs of normal HSCs and precursors is crucial for the development of new treatments for diseases related to the hematopoietic and immune systems.

[Maintenance of Plasmid Expression in vivo Depends Primarily on the CpG Contents of the Vector and Transgene].

Plasmid-mediated gene therapy, being a safe and relatively inexpensive therapeutic strategy, is plagued by a fast silencing of transgene expression. The silencing severely reduces the long-term efficiency of plasmid vectors. We have earlier constructed a low-CpG pMBR2 plasmid vector supporting prolonged expression of transgenes in mesenchymal stem cells in vitro. Long-term expression from the pMBR2 vector was studied for the wild-type mouse secreted alkaline phosphatase gene (mSEAPTwt) and its version devoid of CpGs (mSEAP0) after vector electroporation into mouse hindlimb muscles and hydrodynamic delivery to the liver. The mSEAP levels in the blood were measured over one year. With the pMBR2-mSEAP0 construct, the mSEAP levels in leg muscles increased more than 2.5-fold in the first two months and remained higher than the initial level until the end of the experiment. Far lower expression levels were observed with the control pCDNA3.1-mSEAP0 construct. Expression from pMBR2-mSEAPwt decreased to about 40% after 6 months and remained at similar levels thereafter. In the mouse liver, expression from pMBR2-mSEAP0 was approximately halved within the first 18 weeks and then decrease slowly to the final 17% level. Expression from pMBR2-mSEAPwt initially dropped to 18% and remained at approximately 10% thereafter. In contrast, expression from pCDNA3.1-mSEAP0 sharply dropped to 5% after 2 weeks and remained at nearly zero levels throughout the rest of the experiment. Thus, both vector and transgene should have significantly reduced CpG contents to ensure prolonged plasmid-mediated expression in the liver, while minimizing the vector CpG content is sufficient for expression in skeletal muscles. The results suggested additionally that the localization of S/MAR elements within the transcription unit, in contrast to their outside location, results in significant reduction of the level of secreted, but not cytoplasmic, proteins.

[Homogeneous and heterogeneous 3D melanoma models in vitro].

Growth of malignant tumors occurs in three-dimensional space and depends on a presence of stromal component, which performs critical functions of tumor cell protection and growth support. Therefore, development and analysis of tumor models in 3D cell cultures in vitro, including co-culture systems, presents a significant interest. In this study, the results of 3D culturing of two human melanoma cell lines using the hanging drop method, with or without human mesenchymal stem cells (MSCs), are presented. Melanoma lines were shown to behave differently in 3D cultures; in particular, Mel Cher melanoma cells have the ability to form uniform spheroids within 24 h, whereas MeWo cells under similar conditions failed to form spheroids even after 2 days of culture. However, co-culturing of melanoma cells with MSCs resulted in formation of compact 3D cell spheroids in both cases. Visualization of MeWo cells and MSCs in the mixed spheroids using fluorescent dyes revealed certain clustering of melanoma cells. The observed properties of melanoma cells in homogeneous and heterogeneous spheroids may be used in the complex analysis of results of testing of antimelanoma chemotherapy drugs and evaluation of their therapeutic properties.