[The Efficiency of Gene Activation Using CRISPR/dCas9-Based Transactivation Systems Depends on the System Run Time].

Transactivation systems are a promising application based on the CRISPR/Cas9 system and allow targeted control of gene expression levels in cell culture. However, their performance has been reported to vary considerably depending on the cell type and the activator system. Three activator systems (dCas9-VP160, dCas9-SunTag, and dCas9-VPR) were compared for the efficiency of activating expression of OCT4, NANOG, PDX1, FOXA2, NKX2-2, and NKX6-1 in an immortalized human skin fibroblast line. The activation efficiency was found to depend on the activation system type; the extent of activation depended on the system run time.

[Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures].

In an experimental study using the CRISPR/Cas9 system, "enhanced" NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out ß2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1^(+) overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH^(-/-) or B2M^(-/-) knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH^(-/-) has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1^(+)-CISH^(-/-) and YT-VAV1^(+)B2M^(-/-) lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1^(+) line. The lines of NK-like cells obtained may serve as a prototype for the creation of "enhanced" allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.

IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals.

The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON-NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

NANOS3 downregulation in Down syndrome hiPSCs during primordial germ cell-like cell differentiation.

Human infertility is a complex disorder at the genetic, molecular, cellular, organ, and hormonal levels. New developing technology based on the generation of human primordial germ cell-like cells (hPGCLCs) from induced pluripotent stem cells (hiPSCs) might improve understanding of early germ cell development (specification, migration, gametogenesis, and epigenetic reconstitutions), as well as offering a solution for infertility and hereditary disorders. In this study, we differentiated hiPSCs with trisomy 21 into hPGCLCs. In vitro-derived germ cells from hiPSCs with Down syndrome (DS) express hPGCLC core circuitry, EOMES, SOX17, and PRDM14 at relatively low levels. TFAP2C and PRDM1 were expressed and remained elevated, whereas NANOS3 and NANOG were downregulated in BMP4-induced hiPSCs with DS. The low level of NANOG and NANOS3 expression might negatively influence hPGCLC generation in DS hiPSCs. We suggest that DS hPGCLCs could be a suitable model for studying human early germ cell development, the epigenetic and molecular mechanisms of PGC specification and formation, as well as related infertility disorders, such as azoospermia and teratozoospermia.

Mitochondrial Translation Initiation Factor 3: Structure, Functions, Interactions, and Implication in Human Health and Disease.

Mitochondria are essential organelles of eukaryotic cell that provide its respiratory function by means of the electron transfer chain. Expression of mitochondrial genes is organized in a bacterial-like manner; however multiple evolutionary differences are observed between the two systems, including translation initiation machinery. This review is dedicated to the mitochondrial translation initiation factor 3 (IF3mt), which plays a key role in the protein synthesis in mitochondria. Involvement of IF3mt in human health and disease is discussed.

Detection of small numbers of iPSCs in different heterogeneous cell mixtures with highly sensitive droplet digital PCR.

Induced pluripotent stem cells (iPS cells) are a prospective resource for regenerative biomedicine. iPS cells can differentiate into any type of stem, progenitor and somatic cells to help replace structures within damaged organs or tissues. However, iPS cells themselves, can produce malignant tumors if they are injected into the body of an immunocompatible or immunodeficient recipient. Thus, it is necessary to detect any residual iPS cells content in biomedical cell products obtained from iPS cells and destined for transplantation. In this article we describe searches for iPS cells in heterogeneous cell mixtures, using two different methods-quantitative RT-PCR and droplet digital PCR (ddPCR). In experiments with various heterogeneous mixtures, including mixtures with neural stem cells, we found that the OCT4, TDGF1 and LIN28 genes are the best markers for such a search, and droplet digital PCR provides the greatest measurement accuracy, which is 0.002%. Thus, we have confirmed the advantage of using droplet digital PCR in the search for pluripotent stem cells in heterogeneous cell mixtures. We hope that this data can be useful for biosafety control in regenerative biomedicine.

Clonal Composition of Human Multipotent Mesenchymal Stromal Cells: Application of Genetic Barcodes in Research.

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

[The role of laboratory biomarkers in monitoring of rituximab biosimilar therapy (Acellbia, "BIOCAD") in patients with rheumatoid arthritis].

AIM: to evaluate the role of laboratory biomarkers in monitoring effectiveness of rituximab (RTM) biosimilar therapy in a total dose of 1200 mg. MATERIALS AND METHODS: 20 patients (pts) with rheumatoid arthritis (RA) (18 woman, mean age 61.5(54-66.5) years, mean disease duration 39.5(20-84) months, mean DAS28 5.6(4.9-6.8)) received two intravenous RTM biosimilar infusions (600 mg №2) in combination with DMARDs and glucocorticoids. Laboratory biomarkers were assessed at baseline and weeks 12 and 24 after the first infusion of RTX. RESULTS: RTM biosimilar induced decreases in DAS28, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) at week 12 and 24, p.

New genes for accurate normalization of qRT-PCR results in study of iPS and iPS-derived cells.

iPSC-derived cells (from induced pluripotent stem cells) are a useful source that provide a powerful and widely accepted tool for the study of various types of human cells in vitro. Indeed, iPSC-derived cells from patients with hereditary diseases have been shown to reproduce the hallmarks of these diseases in vitro, phenotypes that can then also be manipulated in vitro. Quantitative reverse transcription PCR (qRT-PCR) is often used to characterize the progress of iPSC differentiation, validate mature cell types and to determine levels of pathological markers. Quantitative reverse transcription PCR (qRT-PCR) is used to quantify mRNA levels. This method requires some way of normalizing the data, typically by relating the obtained levels of gene expression to the levels of expression of a "house keeping gene", a gene whose expression is presumed not to change during manipulation of the cells. In the literature, typically only one such reference gene is used and its stability of expression during cell manipulation is not demonstrated. We are not aware of any study systematically looking at the expression of such genes in human iPSC or during their differentiation into neurons. Here we compare the expression of 16 reference genes in iPSC, neural stem cells (NSC) and neurons derived from iPSC. The applications GeNorm and NormFinder were used to identify the most suitable reference genes. We showed that ACTb, C1orf43, PSMB4, GAPDH and HMBS have the most stable expression. The use of these reference genes allows an accurate normalization of qRT-PCR results in all the cell types discussed above. We hope that this report will help to enable the performance of proper qRT-PCR results normalization in studies with iPSC-derived cells and in disease-modeling reports.

Mutations in the Parkinson's Disease-Associated PARK2 Gene Are Accompanied by Imbalance in Programmed Cell Death Systems.

Parkinson's disease is caused by the degeneration of midbrain dopaminergic neurons. A rare recessive form of the disease may be caused by a mutation in the PARK2 gene, whose product, Parkin, controls mitophagy and programmed cell death. The level of pro- and anti-apoptotic factors of the Bcl-2 family was determined in dopaminergic neurons derived from the induced pluripotent stem cells of a healthy donor and a Parkinson's disease patient bearing PARK2 mutations. Western blotting was used to study the ratios of Bax, Bak, Bcl-2, Bcl-XL, and Bcl-W proteins. The pro-apoptotic Bak protein level in PARK2-neurons was shown to be two times lower than that in healthy cells. In contrast, the expression of the anti-apoptotic factors Bcl-XL, Bcl-W, and Bcl-2 was statistically significantly higher in the mutant cells compared to healthy dopaminergic neurons. These results indicate that PARK2 mutations are accompanied by an imbalance in programmed cell death systems in which non-apoptotic molecular mechanisms play the leading role.

Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells.

Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.

Molecular mechanisms of induced pluripotency.

In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered.

[Cell technologies in complex treatment of venous trophic ulcers].

Live skin equivalent and fibroblasts in gel were used in complex treatment of venous trophic ulcers to evaluate efficacy of cell transplants. Their efficacy depended on extent of trophic ulcer and time of their existence. Cell culture method is minimally traumatic, can be used in elder patients and seniors and gives positive results in 85% of cases.

[Enhanced control of proliferation in telomerized cells].

Clones of telomerized fibroblasts of adult human skin have earlier been obtained. It was shown that despite their fast growth in mass cultures, these cells poorly form colonies. Conditioned medium, antioxidants, and reduced partial oxygen pressure enhanced their colony formation, but not to the level characteristic of the initial cells. The conditioned medium of telomerized cells enhanced colony formation to a much greater extent than that of the initial cells. A study of proteome of the telomerized fibroblasts has revealed changes in the activities of tens of genes. A general trend consists in weakening and increased lability of the cytoskeleton and in activation of the mechanisms controlling protein degradation. However, these changes are not very pronounced. During the formation of immortal telomerized cells, selection takes place, which appears to determine changes in the expression of some genes. It was proposed that a decrease in the capacity of telomerized cells for colony formation is due to increased requirements of these cells to cell-cell contacts. The rate of cell growth reached that characteristic of mass cultures only in the largest colonies. In this respect, the telomerized fibroblasts resembled stem cells: they are capable of self-maintenance, but "escape" to differentiation in the absence of the corresponding microenvironment (niche), which is represented by other fibroblasts. Non-dividing cells in the test of colony formation should be regarded as differentiated cells, since they have no features of degradation, preserve their viability, actively move, grow, phagocytized debris, etc. It was also shown that telomerization did not prevent differentiation of myoblasts and human neural stem cells. Thus, the results obtained suggest the existence of normal mechanisms underlying the regulation of proliferation in the telomerized cells, which opens possibilities of their use in cell therapy, especially in the case of autotransplantation to senior people, when the cell proliferative potential is markedly reduced and accessibility of stem cells is significantly restricted.

[Role of telomerase in reactivation of macrophage nuclei in heterokaryons].

It was shown that the duration of stay of macrophages in the peritoneal cavity of mice and method of their isolation did not affect markedly their capacity for resumption of DNA synthesis in heterokaryons. This means that mouse macrophage undergo such changes during differentiation that reactivation of DNA synthesis in their nuclei is only possible after interaction of telomeres with telomerase, since it was already shown that telomerase was involved in reactivation of DNA synthesis in the macrophage nuclei. The results of experiments did not reveal differences in the length of telomeres in mouse macrophages and other somatic cells. This could depend on the significant length of mouse telomeres and, as a result, their shortening, sufficient for the inhibition of proliferation, is beyond the limits of sensitivity of the current methods. It is also possible that changes in DNA properties in the macrophages occurring during their differentiation depend on changes in the conformation of the telomere complex in these cells. Testing of this suggestion is relevant with respect to recent data that cell hybridization, specifically in the form of heterokaryons, may be essential in realization of the therapeutic effect caused by the introduction of cells during cell therapy.