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.

Neuroprotective Properties of Endocannabinoids N-Arachidonoyl Dopamine and N-Docosahexaenoyl Dopamine Examined in Neuronal Precursors Derived from Human Pluripotent Stem Cells.

Neuroprotective properties of endocannabinoids N-arachidonoyl dopamine (NADA) and N-docosahexaenoyl dopamine (DHDA) were examined in neuronal precursor cells differentiated from human induced pluripotent stem cells and subjected to oxidative stress. Both compounds exerted neuroprotective activity, which was enhanced by elevating the concentration of the endocannabinoids within the 0.1-10 µM range. However, both agents at 10 µM concentration showed a marked toxic effect resulting in death of ~30% of the cells. Finally, antagonists of cannabinoid receptors as well as the receptor of the TRPV1 endovanilloid system did not hamper the neuroprotective effects of these endocannabinoids.

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 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.

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.

[Clinostatting effects on neuronal differentiation of embryonic stem cells from mice strain R1].

The model of embryonic stem cells from R1 mice at the stage of embryoid bodies was used to study effects of slow clinostatting on neuronal differentiation with the help of two markers--beta-III tubulin (early differentiation) and MAP2 (late differentiation). As compared with the control, the number of beta-III tubulin-positive neurons was found increased and of MAP2-positive neurons--decreased. As regards MAP2- positive neurons, it is concluded that the gravity factors have a specific effect on EB. The beta-III tubulin staining makes possible determination of the total number of neuronal cells at different stages of development. The observed increase in the number of beta-III tubulin-positive neurons may evidence a nonspecific mechanic effect of clinostatting at the EB stage. It was shown that EB cells are particularly sensitive to clinostatting.

The Production and Characteristics of a Mouse's Embryonic Stem Cell Lineage, Transfected by the Glia Neurotrophic Factor and Gene Fused with the Green Fluorescent Protein Gene.

The influence that the expression of the human (glial-derived neurotrophic factor (GDNF)) neurotrophic factor has on the morphology and proliferative activity of embryonic stem cells (SC) of a mouse with R1 lineage, as well as their ability to form embroid bodies (EB), has been studied. Before that, using a PCR (polymerase chain reaction) coupled with reverse transcription, it was shown that, in this very lineage of the embryonic SC, the expression of the receptors' genes is being fulfilled for the neurotropfic RET and GFRα1 glia factor. The mouse's embryonic SC lineage has been obtained, transfected by the human GDNF gene, and has been fused with the "green" fluorescent protein (GFP) gene. The presence of the expression of the human GDNF gene in the cells was shown by northern hybridization and the synthesis of its albuminous product by immunocitochemical coloration with the use of specific antibodies. The reliable slowing-down of the embriod-body formation by the embryonic SC transfected by the GDNF gene has been shown. No significant influence of the expression of the GDNF gene on the morphology and the proliferative activity of the transfected embryonic SCs has been found when compared with the control ones.

Influence of pub Gene Expression on Differentiation of Mouse Embryonic Stem Cells into Derivatives of Ecto-, Meso-, and Endoderm in vitro.

The influence of low and high pub gene expression on the initial stages of the differentiation of mouse embryonic stem cells into derivatives of ecto-, meso-, and endoderm in vitro was investigated. As follows from the results of a RT -PCR analysis, the expression of the vimentin, somatostatin, GATA 4, and GATA 6 genes, being the markers of endodermal differentiation, does not vary in both the cells with high pub gene expression and the cells with low pub gene expression, as well as in the corresponding control lines. The cells with high pub gene expression are characterized by an increase in the expression of mesodermal differentiation gene-markers (trI card, trI skel, c-kit, and IL-7), whereas the cells with low pub gene expression are specified by a decrease in their expression. According to the analyses carried out, the reverse is characteristic of the expression of ectodermal differentiation gene-markers (nestin, ≤-III tubulin, gfap, and th). Expression of these genes decreases in cell lines with high pub gene expression, whereas their expression increases with the decrease in pub gene expression. Hence, it is suggested that the variations in the pub gene expression in the embryonic stem cells influence significantly the mesodermal and ectodermal differentiation of these cells.

Different Effects of Regulatory Genes (tat, nef) of Human Immunodeficiency Virus Type 1 (HIV-1) on the Proliferation and Differentiation of Mouse Embryonic Stem Cells in vitro.

To examine the effects of the tat and nef regulatory genes of human immunodeficiency virus (HIV-1) on cell differentiation we used the mouse embryonic stem cells (ESC) as a model. Proliferation, embryoid bodies (EB) formation and subsequent differentiation into cardiomyocytes, glial and neuronal cells were investigated in ESC lines transfected with these genes. It has been shown that the transfection of ESC by the tat gene increased their proliferating activity, whereas the nef gene transfected ESC showed its decrease. The number of embryoid bodies formed was higher in the cultures of ESC transfected by the nef and lower in the cells transfected by the tat in comparison with controls. The percentage of embryoid bodies with contracting cardiomyocytes was higher against control in the nef transfected cells and lower in ESC transfected with the tat. There were no reliable differences in the appearance of glial cells between control and the nef and tat transfected cell lines. Spontaneous differentiation of ESC into neuronal cells was almost not observed in the nef transfected cells, in contrast to control and the tat transfected cells. However, addition of retinoic acid (RA) to the nef transfected cells caused even a slight increase in neuron formation as compared to control ESC treated with RA. Thus, for the first time we have shown that the tat and nef regulatory genes of HIV-1 had a visible effect on proliferation of ESC and some first steps of their differentiation. In general, the reverse correlation between the effects of these two viral genes on ESC proliferation and differentiation were observed.

[Studies of the primary effects of chronic clinostatting of embryonal mice stem cells in vitro].

Effects of randomized gravity vector (clinostatting) on embryonal stem cells (ESC) of mice were evaluated in vitro with respect to proliferation, proliferative potential, and differentiability. Colony formation remained normal up to hour 72 of clinostatting; however, further exposure led to fusion of the ESC colonies. No reliable shifts in the proliferative activity were found, whereas morphometric analysis showed different dynamics of the ESC colonies size in specific periods of the experiment comparing with the control. Evaluation of the ESC proliferative potential after the experiment revealed a trend upward in the number of colonies when compared with the dynamic and static controls. However, the number of resulted EBs in the control tended upward contrary to EBs formed under the conditions of clinostatting and continuous agitation pointing to the importance of local medium conditioning at the beginning of ESC differentiation.

Different effects of enhanced and reduced expression of pub gene on the formation of embryoid bodies by cultured embryonic mouse stem cell.

The effects of pub gene on proliferation and initial stages of differentiation of embryonic mouse stem cells were studied in vitro. To this end we used enhanced expression of human pub gene (hpub) and suppression of expression of mouse endogenous pub gene with RNA-interference in embryonic stem cells. Proliferative activity of genetically modified polyclonal lines of the embryonic stem cells transfected with plasmids carrying expressing hpub gene or plasmids generating small interference RNA to this gene did not differ from that of the control cells. Inhibition of expression of endogenous pub gene in embryonic stem cells using small interference RNA 2-fold decreased the formation of embryoid bodies, at the same time additional expression of exogenous hpub gene almost 2-fold increased their number in comparison with the control. It was hypothesized that pub gene participates in early stages of differentiation of embryonic stem cells leading to the formation of embryoid bodies.

BFP protein expression in transfected embryonic stem cells.

Cultured mouse embryonic stem cells can be transfected with a reporter gene encoding blue fluorescent protein BFP and regulated by drosophila heat shock protein 70 promoter. This gene is activated after heating and synthesizes matrix RNA. Blue protein is synthesized under these conditions. The system for transfection of stem cells allows us to activate automatically the corresponding transgenes.

[Analysis of expression of a series of lymphoma-specific genes in human fibroblasts immortalized by SV40 virus]. / Analiz ékspressii riada limfomospetsificheskikh genov v immortalizovannykh virusom SV40 fibroblastakh chelovaka.

Subtraction hybridization was earlier used to obtain cDNA clones corresponding to human genes upregulated in HIV-associated centroblast lymphoma (CL) as compared with HIV-associated immunoblast lymphoma (IL). With inverse subtraction hybridization, clones were isolated that correspond to genes upregulated in IL compared with CL. In addition to cDNAs characterized earlier, the resulting clones contained several (seven CL-specific and three IL-specific) sequences with unknown functions. To identify the lymphoma-specific genes that are overexpressed in early carcinogenesis, Northern blotting was used to assess the level of gene transcription in two human fibroblast lines and in their derivatives immortalized with either a temperature-sensitive mutant of SV-40 or with pSV3neo carrying the SV-40 A gene, considering the latter as a model of early cell malignant transformation. Increased expression in at least one immortalized line compared with normal fibroblasts was observed for set, a-myb, ND1, ND2, ND4 (NADH dehydrogenase subunits 1, 2, and 4), COX2, COX3 (cytochrome-c-oxidase subunits 2 and 3), KIAA0129, and the gene corresponding to cDNA hss2-1-7-10. High expression of these genes was assumed to be associated not only with lymphomogenesis, but also with early transformation (immortalization) of other, nonlymphoid cells. Expression of the calpain gene and the gene corresponding to cDNA hss2-2-9-5 proved to be lower in immortalized than in normal fibroblasts. This was considered indicative of an alternative mechanism of fibroblast transformation or of different processes regulating the expression of these genes in early and late carcinogenesis.

[Expression of regulatory genes Oct-4, Pax-6, Prox-1, Ptx-2 at the initial stages of differentiation of embryonic stem cells in vitro]. / Ekspressiia reguliatornykh genov Oct-4, Pax-6, Prox-1, Ptx-2 na nachal'nykh stadiiakh differentsirovki émbrional'nykh stvolovykh kletok 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-4 involved 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.

[Influence of regulatory genes of type 1 human immunodeficiency virus on proliferation and differentiation of murine embryonic stem cells]. / Vliianie reguliatornykh genov virusa immunodefitsita cheloveka tipa 1 na proliferatsiiu i differentsirovku émbrional'nykh stvolovykh kletok myshi.

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.

[Differentiation of pluripotent embryonic stem cells in peritoneal cavity of irradiated mice]. / Izuchenie differentsirovki pliuripotentnykh émbrional'nykh stvolovykh kletok v peritoneal'noi polosti obluchennykh myshei.

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