Human Umbilical Cord Blood Serum: Effective Substitute of Fetal Bovine Serum for Culturing of Human Multipotent Mesenchymal Stromal Cells.

Optimal conditions for culturing of multipotent mesenchymal stromal cells in the presence of pooled umbilical cord blood serum were determined. It was found that umbilical cord blood serum in a concentration range of 1-10% effectively supported high viability and proliferative activity of cells with unaltered phenotype and preserved multilineage differentiation capacity. The proposed approach allows avoiding the use of xenogenic animal sera for culturing of multipotent mesenchymal stromal cells and creates prerequisites for designing and manufacturing safe cellular and/or acellular products for medical purposes.

Expression of Surface Molecules in Human Mesenchymal Stromal Cells Co-Cultured with Nucleated Umbilical Cord Blood Cells.

We studied the expression of different classes of surface molecules (CD13, CD29, CD40, CD44, CD54, CD71, CD73, CD80, CD86, CD90, CD105, CD106, CD146, HLA-I, and HLA-DR) in mesenchymal stromal cells from human umbilical cord and bone marrow during co-culturing with nucleated umbilical cord blood cells. Expression of the majority of surface markers in both types of mesenchymal stromal cells was stable and did not depend on the presence of the blood cells. Significant differences were found only for cell adhesion molecules CD54 (ICAM-1) and CD106 (VCAM-1) responsible for direct cell-cell contacts with leukocytes and only for bone marrow derived cells.

Human Umbilical Cord Mesenchymal Stromal Cells Support Viability of Umbilical Cord Blood Hematopoietic Stem Cells but not the "Stemness" of Their Progeny in Co-Culture.

Cell-cell interactions and the ability of mesenchymal stromal cells to support the expansion of hematopoietic progenitor cells were studied in co-culture of human umbilical cord tissue-derived mesenchymal stromal cells and nucleated umbilical cord blood cells. It was found that hematopoietic stem cells from the umbilical cord blood are capable to adhere to mesenchymal stromal cells and proliferate during 3-4 weeks in co-culture. However, despite the formation of hematopoietic foci and accumulation of CD34+ and CD133+ cells in the adherent cell fraction, the ability of newly generated blood cells to form colonies in semi-solid culture medium was appreciably reduced. These findings suggest that human umbilical cord tissue-derived mesenchymal stromal cells display a weak capability to support the "stemness" of hematopoietic stem cell progeny despite long-term maintenance of their viability and proliferation.

Changes in Cell Composition of Umbilical Cord Blood and Functional Activity of Hematopoietic Stem Cells during Cryogenic Storage and Repeated Freezing/Thawing Cycles.

We analyzed changes in cell composition of umbilical cord blood and functional activity of hematopoietic stem cells during cryogenic storage and after repeated freezing/thawing cycles. It was found that repeated freezing/thawing cycles performed according to the optimal programmable freezing protocol did not significantly affect viability and functional activity of hematopoietic stem cells. When fast freezing program was used, the cells completely lost their capacity to form colonies in semisolid medium, despite high viability parameters in the test with 7-AAD.

Isolation of Multipotent Mesenchymal Stromal Cells from Cryopreserved Human Umbilical Cord Tissue.

Umbilical cord stroma is an easily available, convenient, and promising source of multipotent mesenchymal stromal cells for regenerative medicine. Cryogenic storage of umbilical cord tissue provides more possibilities for further isolation of multipotent mesenchymal stromal cells for autologous transplantation or scientific purposes. Here we developed a protocol for preparation of the whole umbilical cord tissue for cryogenic storage that in combination with the previously described modified method of isolation of multipotent mesenchymal stromal cells allowed us to isolate cells with high proliferative potential, typical phenotype, and preserved differentiation potencies.

Optimized Protocol for Isolation of Multipotent Mesenchymal Stromal Cells from Human Umbilical Cord.

Extraembryonic tissues, in particular, umbilical cord stroma are promising sources of multipotent mesenchymal stromal cells for regenerative medicine. In recent years, methods for isolation of mesenchymal stromal cells from different compartments of the umbilical cords based on enzymatic disaggregation of the tissue or on tissue explants have been proposed. Here we propose a protocol of isolation of multipotent mesenchymal stromal cells from the whole umbilical cord that combines the advantages of each approach and ensures sufficient cell yield for further experimental and clinical applications. A combination of short-term incubation of tissue fragments on cold collagenase solution followed by their culturing in the form of explants significantly increased the yield of cells with high proliferative activity, typical pluripotent mesenchymal stromal cell phenotype, and preserved differentiation capacity.

[EFFECT OF STROMAL CELLS AND OXYGEN CONCENTRATION ON THE MAINTENANCE OF CORD BLOOD HEMATOPOIETIC PRECURSORS].

The paper analyses the morphological and functional features of cord blood cells (CBCs) in the co-culture with multipotent mesenchymal stromal cells (MMSCs) from human adipose tissue under tissue-related oxygen. We have established that MMSCs effectively maintained viability of CBCs at different oxygen concentrations (1, 5 and 20%). According to the data obtained, the oxygen concentration affected the number of colony-forming units (CFUs) formed by CBCs in selective medium. In particular, not co-cultured CBCs the 1 and 5% O2 than at 20% O2. After co-culture with MMSCs, the CFUs numbers were similar at 1 and 20% O2 and increased by 30 at 5% O2. Tissue related O2 concentrations had an impact on the proportion of lineage-resctricted CFCs among CBCs: the number of more committed progenitors--CFU-G and CFU-M, increased, and multi and bipotent--CFU-GEMM and CFU-GM, decreased at low oxygen concentrations. This effect was more pronounced after co-culture compared to that of initial CBCs. Thus, the presence of stromal cells and tissue-related oxygen jointly and severally influenced CBCs in vitro.

Umbilical cord blood for autologous transfusion in the early postnatal ontogeny: analysis of cell composition and viability during long-term culturing.

Changes in cell composition and viability as well as the content and functional activity of hemopoietic progenitor cells were analyzed during long-term (up to 1 month at 4°C) storage of human umbilical cord blood cells. No significant quantitative changes in erythrocytes were found during this period. The total content and viability of leukocytes changed, which resulted in the prevalence of mononuclear cells (lymphocytes and monocytes). Analysis of functional activity of hemopoietic stem cells in semisolid culture revealed a decrease in the relative content of CFU during the first week of storage [corrected] and inability of cells to colony formation after 2 weeks.

Enrichment of umbilical cord blood mononuclears with hemopoietic precursors in co-culture with mesenchymal stromal cells from human adipose tissue.

We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4 ± 9.5 and 24.2 ± 11.2% CD34(+) and CD133(+) cells, respectively, which corresponds to enrichment of precursor cell population by 148 ± 60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine.

[Metabolome profiling in the study of aging processes]. / Metabolomnoe profilirovanie v izuchenii protsessov stareniia.

Aging of a living organism is closely related to systemic metabolic changes. But due to the multilevel and network nature of metabolic pathways, it is difficult to understand these connections. Today, this problem is solved using one of the main approaches of metabolomics - untargeted metabolome profiling. The purpose of this publication is to systematize the results of metabolomic studies based on such profiling, both in animal models and in humans.

[Blood metabolome analysis for creating a digital image of a healthy person]. / Metabolomnyi analiz krovi dlia sozdaniia tsifrovogo obraza zdorovogo cheloveka.

In the frame of the work, data on the implementation of metabolomics tests in medicine have been systematized. Based on the obtained data, a set of protocols was proposed, the sequential realization of which makes it possible to conduct a blood metabolome analysis for medical purposes. Using this analysis and the number of blood samples from healthy volunteers, a prototype of a healthy person's metabolomic image has been developed; it allows visually and digitally to assess the compliance of the human blood metabolome with the norm. At the same time, 99% of the metabolic processes reflected in the blood plasma are estimated. If abnormalities are detected, the metabolomic image allows to get the value of these deviations of metabolic processes in digital terms.

[Ten years of the Russian metabolomics: history of development and achievements]. / Desiat' let rossiiskoi metabolomike: istoriia razvitiia i osnovnye rezul'taty.

Metabolomics is one of the omics sciences, the technologies of which are widely used today in many life sciences. Its application influenced the discovery of new biomarkers of diseases, the description of biochemical processes occurring in many organisms, laid the basis for a new generation of clinical laboratory diagnostics. The purpose of this review is to show how metabolomics is represented in the studies of Russian scientists, to demonstrate the main directions and achievements of the Russian science in this field. The review also highlights the history of metabolomics, existing problems and the place of Russian metabolomics in their solution.

[The pattern of centriole immunostaining for tyrosinated or acetylated alpha-tubulin changes during mitosis in 3T3 (A31) cells but not in SV40-3T3 cells].

Comparative electron microscopic analysis of the patterns of centriolar and cytoplasmic microtubules immunostaining for acetylated or tyrosinated alpha-tubulin was performed during the cell cycles of the mouse diploid 3T3 (A31) and virus-transformed heteroploid SV40-3T3 cell lines. It was shown that the pattern of centriole immunostaining changed during mitosis in control 3T3 (A31) cells, but not in tumorigenic SV40-3T3 cells. A sharp change in the pattern of centriole immunostaining was observed in those cell cycle periods when the protein organization of centrosome underwent changes during interphase/mitosis or mitosis/interphase transitions due to the rearrangement of microtubule system. A high level of centriole immunostaining for tyrosinated and acetylated alpha-tubulin was observed at all cell cycle stages with the exception of entering mitosis for the former and the end of mitosis for the latter.

[The organization of the mitotic apparatus poles in etoposide-treated CHO-K1 cells].

In this study, we have examined the organization of the mitotic spindle poles in CHO-K1 cells dividing after treatment with the etoposide (1 h, 25 microM). We studied at various periods after the treatment: 1) the distribution of gamma-tubulin in mitotic cells by immunofluorescent staining; 2) the level of posttranslational modification of a-tubulin in the spindle microtubules by immunoelectron microscopy; 3) the ultrastructure of the mitotic apparatus poles by standard electron microscopy. In 48 h after the addition of the agent we identified considerable changes in the ultrastructure of poles in etoposide-treated CHO-K1 cells with bipolar and multipolar spindles. The number of centrioles increased. The centrioles were unevenly distributed among the poles, and some centrioles were not explicitly involved in the organization of mitotic spindle, furthermore they can differ in the number of outgrowing microtubules. Most centrioles were without fibrillar halo. In 48 h after the addition of etoposide, electron microscopy of cells after immunoperoxidase staining with antibodies to acetylated and tyrosinated alpha-tubulin has shown that different poles of a multipolar spindle within the same cell are stained differently for tyr-tubulin but not for acet-tubulin. Immunofluorescence staining for gamma-tubulin also points to different organization of poles in the same spindle. Our findings provide the first evidence that the pattern of immunostaning and the ultrastructure of mitotic apparatus poles differ in the cells dividing at various periods after etoposide treatment.

[The reorganization of the mitotic apparatus in etoposide-treated CHO-K1 cells precedes accumulation of the apoptotic cells].

Etoposide (1 h, 25 microM) causes interphase arrest in CHO-K1 after which cells resume mitotic division and die due to apoptosis after a certain time period. Accumulation of apoptotically dying cells in the culture follows a gradual increase in the number ofmultipolar mitoses. Our findings provide the first evidence that the pattern of immunostaning for alpha-tubulin, acetylated alpha-tubulin and tyrosinated alpha-tubulin in cells dividing at various periods after etoposide treatment. Moreover, some parts of the multipolar mitotic spindle differ in the presence of antigenic determinants accessible to anti-tyrosinated alpha-tubulin antibodies. It is noteworthy that these abnormalities are aggravated just before the increase in the number of apoptotically died cells. Our findings also suggest that some cells pass through at least two mitotic cycles prior to a sharp increase in the number of apoptotically died cells in the cell culture.

[Pharmacometabonomics - the novel way to personalized drug therapy]. / Farmakometabonomika ­ novyi podkhod k personalizatsii lekarstvennoi terapii.

The review is devoted to pharmacometabonomics - a new branch of science focused on personalization of drug therapy through the comprehensive analysis of metabolites of patient's biological fluids. It considers the history of pharmacometabonomic, positioning to other "-omic" sciences, and system approach, realized by this science, in determination of individual therapeutic dose of the drugs and also a technical implementation of pharmacometabonomic based on direct mass spectrometry of blood plasma metabolites. Special attention is paid to a comparative analysis of pharmacometabonomics and other main approaches to personalized therapy in the clinic, such as pharmacogenetics and therapeutic drug monitoring. Finally, prospects of pharmacometabonomics applications in clinical practice were also discussed.

[Mass spectrometry analysis of blood plasma lipidome as method of disease diagnostics, evuation of effectiveness and optimization of drug therapy].

A new method for the analysis of blood lipid based on direct mass spectrometry of lipophilic low molecular weight fraction of blood plasma has been considered. Such technique allows quantification of hundreds of various types of lipids and this changes existing concepts on diagnostics of lipid disorders and related diseases. The versatility and quickness of the method significantly simplify its wide use. This method is applicable for diagnostics of atherosclerosis, diabetes, cancer and other diseases. Detalization of plasma lipid composition at the molecular level by means of mass spectrometry allows to assess the effectiveness of therapy and to optimize the drug treatment of cardiovascular diseases by phospholipid preparations.

[Mass spectrometry of blood low-molecular fraction as a method for unification of therapeutic drug monitoring].

The article describes a new therapeutic drug monitoring (TDM) method based on direct infusion of low-molecular fraction of blood into electrospray ionization source of mass spectrometer. This technique allows performing TDM of almost all drugs used in clinic. In article, the universality and high-throughput of the method, that significantly simplifies its wide application, have been shown. Moreover, the possibility of method application in most cases of drug therapy has been argued as a tool of control of drug doses, rationality of drug therapy, and the quality of the drugs themselves. In conclusion, the prospects for application of the method as primary means of improving the quality and personalization of drug therapy have been discussed.