Direct Reprogramming of Somatic Skin Cells from a Patient with Huntington's Disease into Striatal Neurons to Create Models of Pathology.

A new in vitro model of Huntington's disease (HD) was developed via a direct reprogramming of dermal fibroblasts from HD patients into striatal neurons. A reprogramming into induced pluripotent stem (iPS) cells is obviated in the case of direct reprogramming, which thus yields neurons that preserve the epigenetic information inherent in cells of a particular donor and, consequently, the age-associated disease phenotype. A main histopathological feature of HD was reproduced in the new model; i.e., aggregates of mutant huntingtin accumulated in striatal neurons derived from a patient's fibroblasts. Experiments with cultured neurons obtained via direct reprogramming make it possible to individually assess the progression of neuropathology and to implement a personalized approach to choosing the treatment strategy and drugs for therapy. The in vitro model of HD can be used in preclinical drug studies.

Human Artificial Chromosomes and Their Transfer to Target Cells.

Human artificial chromosomes (HACs) have been developed as genetic vectors with the capacity to carry large transgenic constructs or entire gene loci. HACs represent either truncated native chromosomes or de novo synthesized genetic constructs. The important features of HACs are their ultra-high capacity and ability to self-maintain as independent genetic elements, without integrating into host chromosomes. In this review, we discuss the development and construction methods, structural and functional features, as well as the areas of application of the main HAC types. Also, we address one of the most technically challenging and time-consuming steps in this technology - the transfer of HACs from donor to recipient cells.

The Profile of Post-translational Modifications of Histone H1 in Chromatin of Mouse Embryonic Stem Cells.

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.

KH-Domain Poly(C)-Binding Proteins as Versatile Regulators of Multiple Biological Processes.

Five known members of the family of KH-domain poly(C)-binding proteins (Pcbp1-4, hnRNP-K) have an unusually broad spectrum of cellular functions that include regulation of gene transcription, regulation of pre-mRNA processing, splicing, mRNA stability, translational silencing and enhancement, the control of iron turnover, and many others. Mechanistically, these proteins act via nucleic acid binding and protein-protein interactions. Through performing these multiple tasks, the KH-domain poly(C)-binding family members are involved in a wide variety of biological processes such as embryonic development, cell differentiation, and cancer. Deregulation of KH-domain protein expression is frequently associated with severe developmental defects and neoplasia. This review summarizes progress in studies of the KH-domain proteins made over past two decades. The review also reports our recent finding implying an involvement of the KH-factor Pcbp1 into control of transition from naïve to primed pluripotency cell state.

Proteasomes in Protein Homeostasis of Pluripotent Stem Cells.

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are subjects of high interest not only in basic research, but also in various applied fields, particularly, in regenerative medicine. Despite the tremendous interest to these cells, the molecular mechanisms that control protein homeostasis in these cells remain largely unknown. The ubiquitin-proteasome system (UPS) acts via post-translational protein modifications and protein degradation and, therefore, is involved in the control of virtually all cellular processes: cell cycle, self-renewal, signal transduction, transcription, translation, oxidative stress, immune response, apoptosis, etc. Therefore, studying the biological role and action mechanisms of the UPS in pluripotent cells will help to better understand the biology of cells, as well as to develop novel approaches for regenerative medicine.

Post-translational modifications of linker histone H1 variants in mammals.

The covalent modifications of the linker histone H1 and the core histones are thought to play an important role in the control of chromatin functioning. Histone H1 variants from K562 cell line (hH1), mouse (mH1) and calf (cH1) thymi were studied by matrix-activated laser desorption/ionization fourier transform ion cyclotron resonance mass-spectroscopy (MALDI-FT-ICR-MS). The proteomics analysis revealed novel post-translational modifications of the histone H1, such as meK34-mH1.4, meK35-cH1.1, meK35-mH1.1, meK75-hH1.2, meK75-hH1.3, acK26-hH1.4, acK26-hH1.3 and acK17-hH1.1. The comparison of the hH1, mH1 and cH1 proteins has demonstrated that the types and positions of the post-translational modifications of the globular domains of the H1.2-H1.4 variants are very conservative. However, the post-translational modifications of the N- and C-terminal tails of H1.2, H1.3 and H1.4 are different. The differences of post-translational modifications in the N- and C-terminal tails of H1.2, H1.3 and H1.4 likely lead to the differences in DNA-H1 and H1-protein interactions.

[SUPPRESSION OF TUMOR GROWTH AFTER XENOGRAFTING OF HUMAN COLORECTAL CARCINOMA CELLS].

Previously we've described the obtainment of a subpopulation of cancer stem cells from a human colorec- tal carcinoma cell line MIP101. These cells possess elevated clonogenic and tumorigenic capacities. According to our data, depletion of stem compartment in a cancer cell population blocks its tumorigenicity. The current work is dedicated to the comparison of tumorigenic potential between cell populations with enriched or depleted stem compartment. We show that tumor growth following xenografting of enriched stem cell population can be suppressed by intramuscular injections of ganciclovir. Thus, we report a method to obtain a cell population with high Oct4 promoter expression within the MIP101 colorectal carcinoma cell line and to eliminate these cells from the population in vitro as well as in vivo.

[THE EFFECT OF PLAKOPHILIN-2 GENE MUTATIONS ON ACTIVITY OF THE CANONICAL Wnt SIGNALING PATHWAY].

Plakophilin-2 is a desmosomal protein encoded by PKP2 gene. Desmosomal proteins are usually considered as structural proteins with the main function of maintaining intercellular interactions. Genetic studies revealed that mutations in desmosomal genes could lead to arrhythmogenic right ventricular cardiomyopathy, heart disease characterized by substitution of cardiomyocytes by adipose and fibrotic tissue predominantly in right ventricle. Wnt signaling pathway is one of the signal transduction pathways which could be involved in the formation of the pathology. The purpose of this study was to investigate Wnt activity changes caused by PKP2 mutations during adipogenic and cardiomyogenic differentiation. We used multipotent mesenchymal stromal cells and iPS cells generated from patient carrying PKP2 gene mutation. We show that Wnt activity is lower in the cells with mutant PKP2. This data indicate a possible signaling role of plakophilin-2 by regulating Wnt activity.

[Design of cell line stable expressing proteasomal subunit PSMD14 fused to the fluorescent protein EGFP and HTBH tag based on HEK293 cells].

A stable cell line based on HEK293 cells that expresses proteasome subunit PSMD14 fused to the fluorescent protein EGFP and HTBH tag has been selected. This chimera was shown to be incorporated completely into proteolitic active proteasomes. The created cell line can be used for further fluorescent studies of proteasomes localization in the cell.

[Reprogramming of somatic cells. Problems and solutions].

An adult mammal is composed of more than 200 different types of specialized somatic cells whose differentiated state remains stable over the life of the organism. For a long time it was believed that the differentiation process is irreversible, and the transition between the two types of specialized cells is impossible. The possibility of direct conversion of one differentiated cell type to another was first shown in the 80s of the last century in experiments on the conversion of fibroblasts into myoblasts by ectopic expression of the transcription factor MyoD. Surprisingly, this technology has remained unclaimed in cell biology for a long time. Interest in it revived after 200 thanks to the research of Novel Prize winner Shinya Yamanaka who has shown that a small set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is capable of restoring pluripotency in somatic cells which they lost in the process of differentiation. In 2010, using a similar strategy and the tissue-specific transcription factors Vierbuchen and coauthors showed the possibility of direct conversion of fibroblasts into neurons, i. e. the possibility of transdifferentiation of one type of somatic cells in the other. The works of these authoras were a breakthrough in the field of cell biology and gave a powerful impulse to the development of cell technologies for the needs of regenerative medicine. The present review discusses the main historical discoveries that preceded this work, evaluates the status of the problem and the progress in the development of methods for reprogramming at the moment, describes the main approaches to solving the problems of reprogramming of somatic cells into neuronal, and briefly discusses the prospect of application of reprogramming and transdifferentiation of cells for such important application areas as regenerative medicine, cell replacement therapy and drug screening.

[A comparative analysis of colorectal carcinoma cell lines that differ in metastatic potential].

In the current work we make an attempt to compare cancer cells of one origin, but differing in the expression of CEA protein, a clinical marker of metastatic carcinomas, presumably one of the key factors in metastatic activity. We have explored the morphology of cell colonies in vitro, expression patterns of epithelial markers, the ability of these cells to form tumors and metastases in vivo, and evaluated their stem compartment with the aid of a suicidal genetic construct sensitive to the embryonic stem cell marker, Oct4.

[Investigation of transcriptional regulation of Oct4 (POU5F1) gene with distal enhancer].

Investigations of transcriptional regulation of Oct4 gene in mouse embryonic stem cells have revealed an important cis-element--the distal enhancer (DE). DE consists of two functionally significant elements--DEa and DEb. Both elements are necessary to complete the DE-mediated expression of Oct4 gene in pluripotent cells. The most likely candidates for the binding site DEb are Oct4 itself in complex with Sox2 protein. It remains unclear which transcriptional proteins bind to the DEa site and what is the mechanism of the co-operation between the DEa and the DEb. Through the use of using the EMSA and chromatographic fractionation of proteins from extracts of mouse embryonic stem cells and mouse tissues, were isolated proteins specifically interacting with the sequence DEa Oct4 gene.

[In vitro derivation and characterization of a colorectal cancer stem cell subpopulation].

In present publication we describe for the first time the obtainment of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells' primary role in the development of malignant neoplasms.

[The interaction between CDX2 transcription factor and DDX5 RNA helicase].

Previously, our results of a two-hybrid screening essay allowed us to recognize p68 (DDX5) as a possible partner of CDX2. The recent part of research was carried out to confirm this interaction. We show the co-localization of these proteins in the nuclei of colon carcinoma cell line and of epithelium of villi. By means of GST-pulldown we reveal DDX5 as a part of a complex with CDX2. During the investigation of the effect of DDX5-CDX2 interaction upon beta-catenin-mediated transcription regulation we note that in each of three investigated cell lines Cdx2 acts as an activator of luciferase expression. In T98G and U20S cell lines we observe a partial decline of beta-catenin transcription enhancing effect while interacting with CDX2. In the cell systems studied, DDX5 acts as a weak repressor both solely and together with CDX2 and beta-catenin. Concerning the influence upon D1 cyclin promoter, we find that, depending on environment, CDX2 may either decline its transcription (U20S line) or raise it (T98G). Besides, PDGF reduces CDX2 activity both in activation and repression. When DDX5 and CDX2 are transfected in T98G cells together, the repressing activity of DDX5 is leveled with activation by Cdx2. In both cell lines the native DDX5 acts as a weak repressor of D1 cycline; PDGF treatment does no significant effect on its activity.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

[Genetic manipulation and studying of differentiation properties of rat induced pluripotent stem cells].

Induced pluripotent stem (iPS) cells are derived from somatic cells reprogrammed to the pluripotent state by the induced expression of defined transcription factors, achieved for the first time by the seminal work of Takahashi and Yamanaka. This new type of pluripotent cells has offered new exciting options in regenerative medicine allowing the replacement of cells and organs with the patient's own cells thereby avoiding immunological complications. In order to develop such technologies in approved animal models, iPS cells were also generated from rodents. Of course, the most important model for studying of different diseases is rat. In this study, we present a method suitable for rat iPS cells genetic modification by stable transfection and show necessary conditions for the first stages of direct differentiation.

[Chromosomal instability of in vitro cultured mouse embryonic stem cells and induced pluripotent stem cells].

A perspective of using embryonic stem (ES) and induced pluripotent stem (iPS) cells in clinical medicine makes karyological analysis of these cells an important issue. Using methods of classical and molecular cytogenetics chromosomal analysis, we have carried out karyological study of two mouse ES and two iPS cell lines derived de novo. We have found monosomy of X chromosome in all studied ES and iPS cell lines, thus making a modal number of chromosomes in these cell lines 39. A chromosomal instability (aneuploidy) was revealed in both studied iPS cell lines. Moreover, we have detected chromosomal rearrangements and chromosomal fragments in one of iPS cell line. Our findings underline the importance of careful cytogenetic evaluation of pluripotent cell lines, especially iPS cell lines, which should be carried out prior to any clinical use of these cells.

[E-cadherin as a novel surface marker of spermatogonial stem cells].

Spermatogenesis is a fundamental biological process that ensures gee transmission from one generation to another trough gametes. This process relies on a rare population of testicular cells, called spermatogonial stem cells (SSCs), that self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding research of SSCs, their biological properties remain largely unknown which is partly due to very limited availability of these cells. Here we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs, which can be successfully used for their enrichment.

[Spontaneous transformation and immortalization of mesenchymal stem cells in vitro].

Mesenchymal stem cells (MXC) possess plasticity and unlimited proliferative activity in vitro that makes them an attractive subject of the studies focused on searching new resources for regenerative medicine. The usage of MSC is effective for treatment of patients with degenerative and traumatic diseases of different tissues, however, biological basis of therapeutic efficacy of MSC are still obscure. We found that long term culture of MSC expressing transgenic green fluorescence protein (GFP) led to increase in their proliferative activity, decrease in adhesion, and loss of differentiation potential and production of the GFP. MSC at the first passages showed karyotipic features of transformation, that at the later passages were complicated with developing of tumorigenic abilities detected after transplantation into normal syngenic recipients. When explanted into cell culture conditions the cells of the tumor tissue originated from the MSC did not express GFP and were not inducible to differentiation, but in contrast to the parent cells showed decreased clonogenic and proliferative activities. We suggest that growth of MSC in vitro results in their spontaneous transformation at early passages. Immortalization making physiological basis for the unlimited proliferation of MSC in vitro may be a feature of MSC transformation but not an initial characteristic of the stem cells.

[The application of lentiviral vectors for tissue-specific gene manipulations].

The trophectoderm (TE) ofblastocysts, the first epithelium established in mammalian development, 1) plays signaling, supportive, and patterning functions during pre-implantation development, 2) ensures embryo implantation into the uterine wall, and 3) gives rise to extra-embryonic tissues essential for embryo patterning and growth after implantation. We show that mouse TE, itself permissive to lentiviral (LV) infection, represents a robust non-permeable physical barrier to the virus particles, thereby shielding the cells of the inner cell mass (ICM) from viral infection. This LV feature will allow modulations of gene expression in a lineage-specific manner, thus having significant applications in mouse functional genetics.