Elena Tolkunova

Oct4 is required for primordial germ cell survival

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4‐deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline.

The Caudal-related protein Cdx2 promotes trophoblast differentiation of mouse embryonic stem cells

Besides holding great promise in clinics, embryonic stem (ES) cells represent a valuable tool for studying regulation of early developmental processes, such as cell differentiation in preimplantation embryos. The caudal‐related homeobox protein Cdx2 is a transcriptional regulator essential for trophoblast lineage, functioning as early as implantation. Using an inducible system, we show that gain of Cdx2 function in ES cells triggers trophoblast‐like morphological differentiation, accompanied by ploidy increase, onset of expression of trophoblast‐specific markers, and loss of pluripotency‐associated gene expression. These data provide an insight into the genetic network that controls lineage specification and functioning in early mammalian development.

Derivation, Characterization, and Stable Transfection of Induced Pluripotent Stem Cells from Fischer344 Rats

The rat represents an important animal model that, in many respects, is superior to the mouse for dissecting behavioral, cardiovascular and other physiological pathologies relevant to humans. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here, we report an improved lentivirus-based hit-and-run riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We demonstrate that the excision of proviruses does not affect either the karyotype or the differentiation ability of these cells. We show that the established riPS cells are readily amenable to genetic manipulations such as stable electroporation. Finally, we propose a genetic tool for an improvement of riPS cell quality in culture. These data may prompt iPS cell-based gene targeting in rat as well as the development of iPS cell-based therapies using disease models established in this species.

Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA and beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer.

Protecting a transgene expression from the HAC-based vector by different chromatin insulators

Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoidtetO-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoidtetO-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors.

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

In the present publication we describe for the first time the derivation 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.

Chromosomal instability of mouse pluripotent cells cultured in vitro

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

Generation of rat-induced pluripotent stem cells: Reprogramming and culture medium

The rat represents an animal model highly attractive for studying pharmacology, physiology, aging, cardiovascular diseases, etc., that in many aspects is more adequate than the mouse model. Derivation of induced pluripotent stem cells from rats (riPS) opens the opportunity for gene targeting in specific rat strains, as well as for the development of new protocols for the treatment of different degenerative diseases. Here we report an improved protocol for riPS cell generation, which is based on lentivirus delivery of reprogramming factors with their subsequent excision from the genome, application of serum-free media and chemical inhibitors MEK and GSK. We compared various conditions for riPS cell derivation, analyzed the cell karyotype, and assessed the pluripotency of the established cells. These data may prompt further iPS cell-based gene targeting in rat, as well as the development of iPS-based cell therapy, using this animal model.

Primary cultures of human colon cancer as a model to study cancer stem cells

The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient’s tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.

The Role of the Carcinoembryonic Antigen Receptor in Colorectal Cancer Progression

Clinical and experimental data suggest that carcinoembryonic antigen (CEA, CD66e, CEACAM-5) plays a key role in the formation of hepatic metastasis from colorectal and other types of epithelial cancers. The molecular events involved in CEA-induced metastasis have yet to be defined. Our group first cloned the gene (CEAR) for CEA-binding protein from the surface of fixed liver macrophages, (Kupffer cells). In this study to further elucidate the role of CEAR in colorectal cancer progression, its expression in colorectal cancer cells was suppressed by short hairpin RNAs (shRNAs) in CEA-overexpressing and CEA - negative MIP-101 colorectal cancer cell lines. The data show that targeted suppression of endogenous CEAR in tumor cells resulted in changes in cell invasiveness. RT-PCR data indicated reduced levels of E-cadherin, Snail, MMP-2, and Oct-4 in the clones with suppressed CEAR suggesting a role in the epithelial mesenchymal transition. The comparative analysis of tumorigenic activity to the liver of the cell lines with suppressed CEAR has also been conducted using an intrasplenic injection model in immuno-deficient mice. This data shows a decrease in tumor progression associated with CEAR suppression. In summary the results of this study revealed a novel role for CEAR gene in the regulation of colorectal cancer cell invasiveness and progression.