E. S. Manuilova

Trim14 overexpression causes the same transcriptional changes in mouse embryonic stem cells and human HEK293 cells

The trim14 (pub, KIAA0129) gene encodes the TRIM14 protein which is a member of the tripartite motif (TRIM) family. Previously, we revealed high expression levels of trim14 in HIV- or SIV-associated lymphomas and demonstrated the influence of trim14 on mesodermal differentiation of mouse embryonic stem cells (mESC). In the present work, to elucidate the role of trim14 in normal and pathological processes in the cell, we used two different types of cells transfected with trim14: mESC and human HEK293. Using subtractive hybridization and real-time PCR, we found a number of genes which expression was elevated in trim14-transfected mESC: hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), hlx1, hbp1, junb, and pdgfrb. A further analysis of the trim14-transfected mESC at the initial stage of differentiation (embryoid bodies (EB) formation) showed essential changes in the expression of these upregulated genes. The transfection of trim14 into HEK293 also induced an enhanced expression of the several genes upregulated in trim14-transfected mESC (hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), and hlx1). Summarizing, we found similar genes that participated in trim14-directed processes both in mESC and in HEK293. These results demonstrate the presence of the similar mechanism of trim14 gene action in different types of mammalian cells.

Expression of Regulatory GenesOct-4, Pax-6, Prox-1, and Ptx-2 at the Initial Stages of Differentiation of Embryonic Stem Cells in vitro

The expression of regulatory genes of the POU, Pax, Prox, and Ptx gene families was studied at the initial stages of differentiation of murine embryonic stem cells of R1 line. mRNAs were isolated from undifferentiated embryonic stem cells and embryoid bodies formed at the early stages of in vitro differentiation and cDNA sequences were synthesized for comparative PCR analysis of the expression of studied genes. The levels of expression of the gene Oct-4involved in maintenance of the pluripotent status of embryonic stem cells proved to be practically indistinguishable in undifferentiated cells and embryoid bodies, while the expression of Pax-6 markedly increased in the latter. The levels and patterns of expression of the homeobox transcription factors Prox-1 and Ptx-2 were compared on this cell model for the first time. A probable role of these genes in differentiation of the murine embryonic stem cells is discussed.

Differentiation of Pluripotent Embryonic Stem Cells in the Peritoneal Cavity of Irradiated Mice

We studied the behavior and differentiation of pluripotent embryonic stem cells of R1 mice in vivo. Undifferentiated embryonic stem cells and differentiating embryoid bodies implanted in the abdominal cavity of irradiated mice were shown to form tumors containing the derivatives of all germ layers. Cells of the embryoid bodies form tumors two weeks after implantation, while undifferentiated embryonic stem cells form tumors only by week three.

Effect of Nerve Growth Factor on Neural Differentiation of Mouse Embryonic Stem Cells

The cholinergic, GABAergic, and catecholaminergic neurons derived from mouse embryonic stem cells in a culture medium supplemented with recombinant NGF were phenotyped and scored. NGF stimulated generation of neurons, but not neuronal progenitors from embryonic stem cells and affected the proportion of specific types of neurons in cultures of differentiating embryonic stem cells. These findings open vista to employ NGF for generation of specific neuron types from embryonic stem cells for fundamental and toxicological studies.

Investigation of the effect of α-melanocyte-stimulating hormone on proliferation and early stages of differentiation of human induced pluripotent stem cells

We have studied the influence of α-melanocyte-stimulating hormone (α-MSH) on proliferation and early stages of differentiation of human induced pluripotent stem cells (iPSc). We have demonstrated that α-MSH receptor genes are expressed in undifferentiated iPSc. The expression levels of MCR1, MCR2, and MCR3 increased at the embryoid body (EB) formation stage. The formation of neural progenitors was accompanied by elevation of MCR2, MCR3, and MCR4 expression. α-MSH had no effect on EB generation and iPSc proliferation at concentrations ranging from 1 nM to 10 μM. At the same time, α-MSH increased the generation of neural rosettes in human iPSc cultures more than twice.

The effect of α-melanocyte-stimulating hormone on early stages of differentiation of mouse embryonic stem cells.

387 αMelanocyteestimulating hormone (αMSH) belongs to the melanocortin family, which includes adrenocorticotropic hormone (ACTH); α, β, and γMSHs, and several shorter peptides [1]. It is known that melanocortins and their fragments have nootropic, neurotrophic, and neuroprotective activities [2, 3]. The positive melanocortin effect that was observed in cull tured rat neurons was reflected in an increased neuu ronal survival, an enhanced growth of neuronal pro cesses, and expression of the neuronnspecific protein B50 [4]. αMSH stimulated process growth in Neuro 2A neuroblastoma cells, with the introduction of melanoo cortin receptor antagonist (MCR4) DDArg8 ACTH 4–10 inhibiting the positive effect of the peptide [5]. Brannvall [6] studied the effect of αMSH on the proliferation of cells derived from embryonic rat brain. It has been shown that the introduction of αMSH doubled the proliferative activity and formation of secc ondary neurospheres in comparison with the control, indicating the stimulatory effect of αMSH on the ability of cells to selffrenew. Previously, it was found that Semax (Met–Glu–His–Phe–Pro–Gly–Pro), which is a synthetic analogue of the fragment ACTH 4–10 , resulted in a several times higher survival rate of nerve cells in cultures of the embryonic rat brain [7]. The introduction of αMSH and Semax into primary neuu ronal cultures and astrocytes of the rat hippocampus resulted in increased levels of expression of brainn derived neurotrophic factor (BDNF) in these cells [8]. These data suggest that the action of melanocortins may be mediated by the regulation of the expression level of a number of known neurotrophic factors, such as NGF, BDNF, neurotrophinss3, 4/5, and others [7, 8]. The experiments carried out on animals have shown that melanocortins can accelerate the regenerr ation of axons after damage of the peripheral nerve fibers and stimulate neuron growth in the central nerr vous system [4]. Pluripotent stem cells, both of embryy onic and of genetically reprogrammed origin, are widely used as an experimental model for studying the early stages of differentiation of embryonic stem cells (ESCs) in tissues originating from different germ layy ers [9]. ESCs were used to study the effects of pituitary adenylate cyclase activating peptide (PACAP) and vasoactive intestinal peptide (VIP). Both peptides are neurotrophic factors in the embryogenesis of the brain. PACAP is expressed at the mRNA and protein levv els in rat embryos and in the neural tube in mice. VIP was also identified in the brain of mouse embryos. The presence of …

Influence of Regulatory Genes of Type 1 Human Immunodeficiency Virus on Proliferation and Differentiation of Murine Embryonic Stem Cells

Spontaneous formation of embryoid bodies and subsequent differentiation of some cells into cardiomyocytes were demonstrated on murine embryonic stem cells of R1 line. The lines of embryonic stem cells were obtained that had been transfected with genetic constructs carrying expressing regulatory genes of the human immunodeficiency virus tat and nef and green protein gene (GFP). The transfection of embryonic stem cells with the gene tat stimulated their proliferative activity, while this activity decreased in the cells transfected with the gene nef. The time necessary for the formation of embryoid bodies by all lines of transfected cells was similar to that in the control cells. In the cultures of cells transfected with nef and tat, the number of embryoid bodies and the percentage of embryoid bodies with contracting cardiomyocytes were higher and lower than in the control, respectively. Thus, an inverse correlation was observed between the effects of regulatory genes of the human immunodeficiency virus on proliferation and differentiation embryonic stem cells.

Cytogenetic Analysis of the Results of Genome Editing on the Cell Model of Parkinson’s Disease

We performed a cytogenetic analysis of the results of CRISPR/Cas9-correction of G2019S mutation in LRRK2 gene associated with Parkinson’s disease. Genome editing was performed on induced pluripotent stem cells derived from fibroblasts of a patient carrying this mutation. A mosaic variant of tetraploidy 92 XXYY/46,XY (24-43% cells from various clones) was found in neuronal precursors differentiated from the induced pluripotent stem cells after gene editing procedure. Solitary cases of translocations and chromosome breaks were observed. These data confirm the importance of the development of new approaches ensuring genome stability in CRISPR/Cas9-edited cultures.

Investigation of the effects of GABA receptor agonists in the differentiation of human induced pluripotent stem cells into dopaminergic neurons

The influence of GABA receptor agonists on the terminal differentiation in vitro of dopaminergic (DA) neurons derived from IPS cells was investigated. GABA-A agonist muscimol induced transient elevation of intracellular Ca2+ level ([Ca2+]i) in the investigated cells at days 5 to 21 of differentiation. Differentiation of cells in the presence of muscimol reduced tyrosine hydroxylase expression. Thus, the presence of active GABA-A receptors, associated with phenotype determination via Ca2+-signalling was demonstrated in differentiating human DA neurons.

Fibroblast-like cells as an effective feeder for the cultivation and derivation of new lines of human induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) can be a highly informative model of hereditary and sporadic human diseases. In the future, such cells can be used in substitution and regenerative therapy of a wide range of diseases and for the treatment of injuries and burns. The ability of iPSCs derived from patients with Parkinson’s disease to differentiate into fibroblast-like cells (derivatives) was studied. It was found that these cells can serve as an effective feeder layer not only to maintain the pluripotency of allogenic and autologous iPSCs but also to derive new iPSC lines.