Comparative Analysis of the Different Dyes' Potential to Assess Human Normal and Cancer Cell Viability In Vitro under Different D/H Ratios in a Culture Medium.

In this study, using new approach (laser diffraction + biological dyes), we have demonstrated the decrease of cells viability in vitro in the deuterated growth medium, whereas in the deuterium-depleted medium, there was an increase of cell viability. We have also found that not all dyes are equally sensitive to the D/H ratios in the culture medium (system) as well as to the different cell types (cancer vs normal cells).

[Electroconvulsive therapy in treatment of resistant schizophrenia: biological markers of efficacy and safety]. / Primenenie élektrosudorozhnoi terapii pri rezistentnoi shizofrenii: biologicheskie pokazateli éffektivnosti i bezopasnosti.

AIM: To evaluate clinical and biological efficacy and safety of electroconvulsive therapy (ECT) in patients with treatment-resistant paranoid schizophrenia. MATERIAL AND METHODS: Determination of CNS specific biological markers (BDNF, NSE, S100B), together with markers of inflammation and CNS alteration (IL-2, CPK, CPK-MB), and clinical evaluation were performed in two groups of patients: the ECT + antipsychotic treatment group (n=66) and the antipsychotic treatment group (n=32). RESULTS AND CONCLUSION: In the ECT + antipsychotic treatment group, the more pronounced reduction of psychotic symptoms has been revealed compared with subjects on antipsychotic treatment as monotherapy. Patients receiving ECT showed no increase in plasma levels of inflammation and CNS alteration biomarkers (NSE, S100B, CPK, CPK-MB, IL-2). The plasma level of BDNF, capable to characterize both the efficacy and safety of antipsychotic therapy, had a more pronounced upward trend in subjects with combined electroconvulsive and antipsychotic treatment, which may indicate good tolerability and high effectiveness of ECT.

Postnatal extra-embryonic tissues as a source of multiple cell types for regenerative medicine applications.

AIM: We aimed to isolate and characterize the cell types which could be obtained from postnatal extra-embryonic tissues. MATERIALS AND METHODS: Fresh tissues (no more than 12 h after delivery) were used for enzymatic or explants methods of cell isolation. Obtained cultures were further maintained at 5% oxygen. At P3 cell phenotype was assessed by fluorescence-activated cell sorting, population doubling time was calculated and the multilineage differentiation assay was performed. RESULTS: We have isolated multiple cell types from postnatal tissues. Namely, placental mesenchymal stromal cells from placenta chorionic disc, chorionic membrane mesenchymal stromal cells (ChM-MSC) from free chorionic membrane, umbilical cord MSC (UC-MSC) from whole umbilical cord, human umbilical vein endothelial cells (HUVEC) from umbilical vein, amniotic epithelial cells (AEC) and amniotic MSC (AMSC) from amniotic membrane. All isolated cell types displayed high proliferation rate together with the typical MSC phenotype: CD73+CD90+CD105+CD146+CD166+CD34-CD45-HLA-DR-. HUVEC constitutively expressed key markers CD31 and CD309. Most MSC and AEC were capable of osteogenic and adipogenic differentiation. CONCLUSION: We have shown that a wide variety of cell types can be easily isolated from extra-embryonic tissues and expanded ex vivo for regenerative medicine applications. These cells possess typical MSC properties and can be considered an alternative for adult MSC obtained from bone marrow or fat, especially for allogeneic use.

Tissue-engineered bone for treatment of combat related limb injuries.

AIM: Based on our preliminary positive clinical results with use of cultured bone marrow-derived multipotent mesenchymal stem/stromal cells in traumatology, our aim was to develop living three-dimensional tissue-engineered bone equivalent transplantation technology for restoration of critical sized bone defects caused by combat related high energy trauma. MATERIALS AND METHODS: To fabricate bone equivalent we used devitalized allogeneic bone scaffolds (blocks and chips) seeded with cultured autologous cells: bone marrow-derived multipotent mesenchymal stem/stromal cells in mix with periosteal progenitor cells and endothelial progenitor cells. Quality/identity of cell cultures was assured by donor and cell culture infection screening (immunofluorescence assay, polymerase chain reaction), flow cytometry (cell phenotype), karyotyping (GTG banding), functional assays (colony forming units analysis, multilineage differentiation assay). Bone defect treatment with bone equivalent application was fully completed in 39 combat-injured with 42 defects. New bone formation was assessed by the radiographic examination. RESULTS: Casualties were included in a treatment program an average of 10.1 months after injury, provided the ineffectiveness of conventional surgery methods. All cell type cultures had a normal karyotype and appropriate phenotype, differentiation potential and functional properties, ~30% colony forming units frequency and hadn't any signs of cell senescence. The fluorescein diacetate/propidium iodide combined staining and histology analysis of graft samples before transplantation showed their regular seeding with viable cells. Pathomorphological analysis of bone equivalent specimens 3-6 months post-op revealed the active remodeling processes and immature bone tissue formation. Bone defect restoration was observed 5-6 months post-op. CONCLUSION: The developed biotechnology of living three-dimensional tissue-engineered bone equivalent transplantation with overall effectiveness 90.4% allows restoring the bone integrity, forming new bone tissue in a site of bone defect, and significantly reducing the rehabilitation period of a patient.

Large-scale expansion and characterization of human adult neural crest-derived multipotent stem cells from hair follicle for regenerative medicine applications.

AIM: The purpose of this work was to obtain, multiply and characterize the adult neural crest-derived multipotent stem cells from human hair follicle for their further clinical use. MATERIALS AND METHODS: Adult neural crest-derived multipotent stem cells were obtained from human hair follicle by explant method and were expanded at large-scale up to a clinically significant number. The resulted cell cultures were examined by flow cytometry and immunocytochemical analysis. Their clonogenic potential, ability to self-renewal and directed multilineage differentiation were also investigated. RESULTS: Cell cultures were obtained from explants of adult human hair follicles. Resulted cells according to morphological, phenotypic and functional criteria satisfied the definition of neural crest-derived multipotent stem cells. They had the phenotype Sox2+Sox10+Nestin+CD73+CD90+CD105+CD140a+CD140b+CD146+CD166+CD271+CD349+ CD34-CD45-CD56-HLA-DR-, showed high clonogenic potential, ability to self-renewal and directed differentiation into the main derivatives of the neural crest: neurons, Schwann cells, adipocytes and osteoblasts. CONCLUSION: The possibility of a large-scale expansion of adult neural crest-derived multipotent stem cells up to 40-200·106 cells from minimal number of hair follicles with retention of their phenotype and functional properties are the significant step towards their translation into the clinical practice.Key Words: regenerative medicine, neural crest, hair follicle, neural crest-derived multipotent stem cells, directed differentiation, large-scale expansion.

Endometrial stromal cells: isolation, expansion, morphological and functional properties.

AIM: We aimed to study biological properties of human endometrial stromal cells in vitro. MATERIALS AND METHODS: The endometrium samples (n = 5) were obtained by biopsy at the first phase of the menstrual cycle from women with endometrial hypoplasia. In all cases, a voluntary written informed consent was obtained from the patients. Endometrial fragments were dissociated by enzymatic treatment. The cells were cultured in DMEM/F12 supplemented with 10% FBS, 2 mМ L-glutamine and 1 ng/ml FGF-2 in a multi-gas incubator at 5% CO2 and 5% O2. At P3 the cells were subjected to immunophenotyping, multilineage differentiation, karyotype stability and colony forming efficiency. The cell secretome was assessed by BioRad Multiplex immunoassay kit. RESULTS: Primary population of endometrial cells was heterogeneous and contained cells with fibroblast-like and epithelial-like morphology, but at P3 the majority of cell population had fibroblast-like morphology. The cells possessed typical for MSCs phenotype CD90+CD105+CD73+CD34-CD45-HLA-DR-. The cells also expressed CD140a, CD140b, CD146, and CD166 antigents; and were negative for CD106, CD184, CD271, and CD325. Cell doubling time was 29.6 ± 1.3 h. The cells were capable of directed osteogenic, adipogenic and chondrogenic differentiation. The cells showed 35.7% colony forming efficiency and a tendency to 3D spheroid formation. The GTG-banding assay confirmed the stability of eMSC karyotype during long-term culturing (up to P8). After 48 h incubation period in serum-free medium eMSC secreted anti-inflammatory IL-1ra, as well as IL-6, IL-8 and IFNγ, angiogenic factors VEGF, GM-CSF and FGF-2, chemokines IP-10 and MCP-1. CONCLUSION: Thus, cultured endometrial stromal cells meet minimal ISCT criteria for MSC. Proliferative potential, karyotype stability, multilineage plasticity and secretome profile make eMSC an attractive object for the regenerative medicine use.

Effect of tripeptides on lymphoid and stem cells.

Tripeptides T-36 and, particularly, T-38 in concentrations of 0.1, 1, and 10 ng/ml inhibited proliferation of primary trypsinized embryonic mesenchymal stem cells, rat transplantable KF-1 fibroblasts, and human erythromyelosis K-562 cells. Inhibition of proliferation in embryonic and immortalized cells under the influence of tripeptides probably reflects antitumor activity of these substances. Tripeptides had no effect on lymphocyte survival and their adhesive, cytotoxic, and induced proliferative activities. T-36 did not modulate the proliferative properties of erythromyelosis K-562 cells. Tripeptides did not change engulfment activity and spontaneous and induced bactericidal activities of granulocytes. T-36 in a concentration of 0.1 ng/ml increased spontaneous proliferation of normal lymphocytes. These data suggest that tripeptides stimulate nontumor immune cells in adult people.

[Immunoregulatory role of mesenchymal stem cells in bone reparation processes].

Bone marrow contains mesenchymal stem cells (MSC) including osteoblast progenitor cells. When culturedunder conditions promoting an osteoblastic phenotype,MSC proliferate to form colonies that produce alkaline phosphatase and, subsequently, a mature osteoblastic phenotype. Transplantation of cultured autologous MSC to patients with non-healing bone fractures gives a good result leading to complete bone fracture consolidation. The aim of the study is to determine a quantitative production of IL-1beta, IL-2, IL-4, IL-6, IL-8 and TNF-alpha by cultured uncommitted and committed osteogenic MSC. The results showed that the cytokine profile consisting of IL-1beta, IL-2, IL-4, IL-6, IL-8 and TNF-alpha is secreted by cultured MSC. The secretion of IL-1beta and IL-2 by cultured MSC together with hyper production of IL-6 (up to 276.5 pg/ml, p<0.05) and IL-8 (up to 106.6 ng/ml, p<0.05) by osteoinducted MSC are firstly shown. The immunoregulatory role of transplanted autologous cells in inflammation and own bone reparation processes during posttraumatic bone fracture healing is highlighted. In conclusion, the data obtained allow examining of cultured autologous MSC as effective activators of bone resorption, inflammation and some immunological reactions in the process of altered osteoreparation.

[Role of polypeptide growth factors in the regulation of epidermal keratinocyte proliferation]. / Rol' polipeptidnykh faktorov rosta v reguliatsii proliferatsii keratinotsitov epidermisa.

The growth factors, cytokines, adhesive molecules and extracellular matrix components play the leading role in the processes of intercellular interactions. Literary data on structure and mechanisms of functioning of the growth factors and their receptors are summarised in the present review. Some aspects of regulatory functions of such growth factors as EGF, TGFalpha, TGFbeta, FGF, KGF, AR, and HGF in these processes in epidermis keratinocytes both in vivo and in vitro as example were also considered.

Genomic organization and promoter characterization of the gene encoding the human telomerase reverse transcriptase (hTERT).

The enzyme telomerase plays a crucial role in cellular proliferation and tumorigenesis. By adding hexameric repeats to chromosome ends, it prevents telomeric loss and, thus, entry into senescence. Recent data suggest that expression of the human telomerase reverse transcriptase subunit (hTERT) represents the limiting factor for telomerase activity. To gain an insight into the mechanisms regulating hTERT expression, we have determined the complete genomic organization of the hTERT gene and isolated the 5'- and 3'- flanking region. The hTERT gene encompasses more than 37kb and consists of 16 exons. We show that all hTERT insertion and deletion variants described so far most likely result from the usage of alternative splice consensus sequences in intron or exon regions. Furthermore, we identified a new hTERT splice variant. Analysis of the DNA sequence surrounding the putative transcriptional start region revealed a TATA-less promoter located in a CpG island. A promoter fragment spanning the first 1100bp upstream of the initiating ATG start codon exhibited high-level activity in HEK-293 cells. Several consensus binding sites for the transcription factor Sp1 as well as a c-Myc binding site were identified in this promoter region. Altogether, these results provide the basis for more detailed studies on the regulation of telomerase activity in normal and cancer cells, and may lead to the development of new cancer therapies.

A convenient approach to the synthesis of trinucleotide phosphoramidites--synthons for the generation of oligonucleotide/peptide libraries.

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites--trinucleotide phosphotriesters--have been prepared using the phosphotriester approach without protection of the 3'-hydroxyl function. The structures of trinucleotide phosphotriesters and intermediates were confirmed by 1H- and 31P-NMR spectra, mass-spectra and by analysis of SPDE-hydrolysates of deprotected preparations. Purity of the target products has been confirmed by test reactions. The synthons have been used for automated synthesis of oligonucleotides and corresponding libraries by a phosphite-triester approach. A 54mer, containing 12 randomized internal bases, and a 72mer with 24 internal randomized bases have been synthesized.

[Detection of expression of a membrane form of the guanylate cyclase type of GC-B in cattle retina]. / Obnaruzhenie ékspressii membrannoi formy guanilattsiklazy tipa GC-B v setchatke byka.

cDNA clones encoding the central and C-terminal parts of a membrane-bound guanylate cyclase (GC) were isolated from the lambda ZAP bovine retinal library. All of the analysed recombinants appeared to carry inserts encoding the guanylate cyclase GC-B. Analysis of the determined nucleotide and deduced amino acid sequences showed extremely high level of homology to the sequences of known GC-B. The results indicate that a mRNA for GC-B is expressed in the bovine retina.