Cell and Tissue Biology

Mechanisms of Dedifferentiation of Adult Human Retinal Pigment Epithelial Cells in vitro. Morphological and Molecular Genetic Analysis Abstract Changes in morphology and molecular genetic profile of human retinal pigment epithelial (RPE) cells in vitro were studied when exposed to the basic fibroblast growth factor (bFGF). The cell morphology was estimated by cell area and perimeter, spreading and polarization coefficients. It was demonstrated that the number of elongated (fibroblast-like) cells and the number of cells, which size was less than in the control increased in 48 h after adding the factor to the culture. At the same time, the cell proliferative activity decreased (according to MTT test). Immunocytochemical analysis demonstrated a decrease in the staining for connexin Cx43 and an increase in the intensity of staining for protein the Otx2 neuroepithelium protein. Simultaneously, the number of nestin-positive cells and βIII-tubulin positive cells increased. Using quantitative real-time PCR method, an increase in mRNA expression of the KLF4, OCT4, NANOG, OTX2, and NES and decrease in mRNA expression of the MITF and KRT18 were detected in RPE cells treated with bFGF; this indicates enhancement of cell dedifferentiation. These data are confirmed by a decrease in the COL1A1 mRNA expression indicating a decrease in synthetic cell activity. The results indicate that single (short-time) effect by bFGF is sufficient to activate the mechanism, which decreases the cell differentiation level toward neuroepithelium.

The Influence of an Electret-Generated Electric Field Based on a Tantalum Oxide Anode on Differentiation Properties of Bone Marrow Stromal Cells from Patients with Osteoarthritis Abstract The ability of the bone marrow multipotent mesenchymal stromal cells (MMSCs) of a patient with osteoarthritis to differentiate into the osteogenic and chondrogenic lineages under the influence of an electret-generated electric field created on the surface of anodic tantalum oxide was investigated. It has been shown that the synthesis of protein-differentiation markers in MMSCs is intensified in the presence of samples with anodic oxide in the electret state. The effect of electrets with uniform charge distribution over the surface was compared with linear charge distribution, MMSCs osteogenic differentiation was accompanied with enhanced expression of osteocalcin and type I collagen. These features were exhibited much stronger in cells exposed to electret specimens with a charge distribution on the surface close to linear.

Dopamine System Components in Neuroendocrine Complexes in Snail Atrium Abstract Catecholamine dopamine (DA) is an important neurotransmitter and hormone involved in many physiological processes and stress reactions in vertebrates and invertebrates. The aim of the work was to clarify the presence and localization of dopamine system elements (tyrosine hydrokinase (TH), dopamine beta-hydrokinase (DAbetaHK), and dopamine type 1 receptors (DA-R1)) in the Achatina achatina gastropod atrial neuroendocrine complex (NEC) cells. The snail atrial NEC cells are generated by large granular cells (GCs) and nerve fibers that are in close contact with them. The methods of histochemistry, immunofluorescence staining, and immunoelectron microscopy were used. Glyoxylic acid-induced fluorescence demonstrated the presence of catecholamines in the nerve fibers and GC. TH-like and DAbetaH-like immunoreactive material was found both in nerve fibers and in GC granules. DA-R1-positive material was detected only in nerve fibers. In addition, it was demonstrated that exogenous DA induces an enhanced degranulation of GC in vivo. Data obtained indicate the involvement of dopamine system in the functioning of snail NEC.

Ion Homeostasis during the Growth of Human Mesenchymal Stem Culture. I. Density-Dependent Changes of Intracellular K + and Na + Content and K + Influxes Abstract— In this study, we reported proliferation-related changes in cell K+ and Na+ content and K+ influxes in cultivated human mesenchymal stem cells (MSCs). The intracellular K+ content calculated per cell protein amount was found to decrease in the growing culture, whereas intracellular Na+ content was not significantly changed. It was also revealed that, at higher densities of an hMSC monolayer, the ouabain-sensitive K+ influx was decreased thus indicating a decline in Na+, K+ pump-mediated transport. We analyzed the cell cycle profiles of hMSC cultures and found that, under optimal culture conditions in high-density cultures, the decline in K+ content per cell protein was correlated with accumulation of G1 cells and slowing down cell proliferation in the population. It is concluded that cell K+ content per cell protein is an informative test for assessing the functional status of stem cells in vitro.

Human Peripheral Blood Macrophages As a Model for Studying Glucocerebrosidase Dysfunction Abstract— Decreased activity of glucocerebrosidase (GCase) as a result of mutations in the GBA gene causes Gaucher’s disease (GD), which belongs to the group of lysosomal storage disorders. The risk of Parkinson’s disease in homo- and heterozygous carriers of GBA mutations is elevated seven- to eightfold. Screening of novel compounds designed to enhance GCase activity requires development of in vitro models based on primary cell cultures obtained from patients carrying GBA mutations. In this work, the efficiency of different methods used to culture peripheral blood macrophages of GD patients and control subjects was compared, and GCase activity and lysosphingolipid concentrations were evaluated using tandem mass spectrometry (HPLC‒MS/MS) in dried cell spots. For the first time, the efficacy of restoring the activity of mutant GCase has been assessed in primary macrophages of GD patients cultured in the presence of pharmacological GCase chaperones isofagomine and ambroxol. Based on these results, a convenient method of in vitro screening of candidate pharmacological agents designed to increase GCase activity can be proposed.

The Effect of Histone Deacetylase Inhibitor on the Expression Level of Glucococrticoid Receptor in Rat Forebrain under Hypoxia Abstract The activity of the gene transcription depends to a large extent on the histone acetylation status and can be affected by different stimuli such as hypoxia. Histone deacetylase inhibitors promote gene transcription by facilitating histone acetylation and are thus considered as candidates for target therapy of posthypoxic states. In the present study, using immunohistochemistry, the effect of the histone deacetylase inhibitor trihostatin A (TSA) on the expression level of glucocorticoid receptor (GR) have been analyzed in the neocortex and hippocampus of rats in an original model of severe hypobaric hypoxia (SHH). It has been found that injections of TSA in rats facilitated GR expression after SHH in the neocortex and CA1 field but not in the dentate girus of hippocampus. GR overexpression enhances the toxicity of circulating glucocorticoid hormones on the hippocampal neurons. Stimulation of GR expression by TSA injections in the CA1 field of the hippocampus may lead to maladaptation, making possible damaging effects of unfavorable factors, such as hypoxia. The data obtained indicate that therapy using histone deacetylase inhibitors may have adverse side effects for vulnerable brain neurons.

Analysis of Gene Expression in Hondroblasts of Vertebral Body Growth Plates in Patients with Grade III–IV Idiopathic Scoliosis Abstract Idiopathic scoliosis (IS) has been known since ancient times, but there is still no unified conception of the etiology and pathogenesis of this disease. The genetic theory of the scoliosis development is predominant. The search for etiological factors in most studies is carried out using the blood of patients with IS. The aim of the study was to analyze the expression of the genes regulating the differentiation and functioning of the chondroblasts of the growth plates (GP), the synthesis and formation of extracellular matrix components of the GP of vertebral bodies in the pathology localization area in patients with IS of Grade III–IV. As a result of the study, the following profile of gene expression peculiar for chondrocytes of vertebral body GP at Grade III–IV IS was revealed: the dis balance of chondrogenic differentiation genes (PAX1, PAX9, IHH), of receptors of the growth and transcription growth factors (SOX9, TGFR1, GHR), and of genes participating in the sulfation of proteoglycans (SLC26A2, CHST3).The data obtained are consistent with morphological and biochemical results, and may be a marker of pathology.

Infrared Spectroscopy of Blood Serum from Patients with Multiple Myeloma Abstract A combination of high-resolution agarose gel protein electrophoresis, refractometric analysis, and infrared spectroscopy was applied to investigate the blood serum in patients with multiple myeloma (MM). Analysis of the FTIR spectra of the serum obtained from MM patients and healthy donors revealed a decreased amount of α-helixes and increased content of β-sheets in serum proteins from MM patients compared to healthy donors. These findings suggest an interaction between serum proteins and stabilization of the resulting complexes, accompanied by the formation of associated β-sheets.

Obtaining and Characterization of Cells from Native Lung and Diaphragm Rat Tissues for Recellularization of Tissue Engineered Constructs Abstract— The choice of cellular resourses for recellularization of biological scaffolds and synthetic matrices remains a topical issue in regenerative medicine. Isolation of stromal cells from native organs and their utilization for recellularization of biological matrices appears to be a promising alternative for the use of mesenchymal stromal cells. The present work presents modified protocols for obtaining stromal cells from homogenized rat lung and diaphragm tissues. Cell culture typing and cell viability evaluation on the scaffolds was followed by the orthotopic transplantation of a tissue engineered diaphragm construct in rats with histological characteristics of explanted tissue after 24 and 45 days. Recellularized stromal cell scaffolds from native tissues with otherwise equal properties caused less noticeable inflammation reaction compared with recellularized mesenchymal stromal cells (MSCs). The intensity of fibrosis was also lower then when using MSCs for scaffold repopulation.

Activation of Autophagy and Nrf2 Signaling in Human Breast Adenocarcinoma MCF-7 Cells by Novel Monophenolic Antioxidants Abstract The effect of novel water-soluble structurally related monophenolic compounds on the activity of two most important mechanisms of maintaining intracellular homeostasis, autophagy and the redox-sensitive signal system Keap1/Nrf2/ARE, has been studied in human breast adenocarcinoma cell line MCF-7 using confocal microscopy. Autophagy processes were analyzed on the basis of the amount of intracellular vesicles that were positive for the autophagy marker (LC3B). The activation of the Keap1/Nrf2/ARE system was determined by the translocation of the transcription factor Nrf2 into the nucleus. It was found that the effect of the tested compounds depended on their structure and concentration. When the inhibitor of autophagosome–lysosome fusion chloroquine was added to the culture medium (20 μM), the asymmetrically hindered by the tert-butyl group phenols with thiosulfonate (TS-13) and sulfonate group in the para-propyl substituent increased the rate of autophagosome elimination in MCF-7 cells. Shortening of the para-alkyl substituent by one methylene unit abolished the effect. The addition of the second ortho-tert-butyl substituent had the reverse result. Both tested compounds enhanced the translocation of the transcription factor Nrf2 into the nucleus of MCF-7 cells (which is a critical step in Keap1/Nrf2/ARE activation). It was observed after incubation with asymmetrically hindered by the tert-butyl group phenol with selenosulfonate group in para-propyl substituent (5−100 μM) for 4 h and with TS-13 (5−100 μM) for 24 h. Taking into account our previous findings on the toxicity of this group of compounds for MCF-7 cells we can conclude that these compounds exert different effect on autophagy and activation of the antioxidant response element signaling system Keap1/Nrf2/ARE.