Endothelial and smooth muscle cells derived from human cardiac explants demonstrate angiogenic potential and suitable for design of cell-containing vascular grafts.

BACKGROUND: Endothelial and smooth muscle cells are considered promising resources for regenerative medicine and cell replacement therapy. It has been shown that both types of cells are heterogeneous depending on the type of vessels and organs in which they are located. Therefore, isolation of endothelial and smooth muscle cells from tissues relevant to the area of research is necessary for the adequate study of specific pathologies. However, sources of specialized human endothelial and smooth muscle cells are limited, and the search for new sources is still relevant. The main goal of our study is to demonstrate that functional endothelial and smooth muscle cells can be obtained from an available source-post-surgically discarded cardiac tissue from the right atrial appendage and right ventricular myocardium. METHODS: Heterogeneous primary cell cultures were enzymatically isolated from cardiac explants and then grown in specific endothelial and smooth muscle growth media on collagen IV-coated surfaces. The population of endothelial cells was further enriched by immunomagnetic sorting for CD31, and the culture thus obtained was characterized by immunocytochemistry, ultrastructural analysis and in vitro functional tests. The angiogenic potency of the cells was examined by injecting them, along with Matrigel, into immunodeficient mice. Cells were also seeded on characterized polycaprolactone/chitosan membranes with subsequent analysis of cell proliferation and function. RESULTS: Endothelial cells isolated from cardiac explants expressed CD31, VE-cadherin and VEGFR2 and showed typical properties, namely, cytoplasmic Weibel-Palade bodies, metabolism of acetylated low-density lipoproteins, formation of capillary-like structures in Matrigel, and production of extracellular matrix and angiogenic cytokines. Isolated smooth muscle cells expressed extracellular matrix components as well as α-actin and myosin heavy chain. Vascular cells derived from cardiac explants demonstrated the ability to stimulate angiogenesis in vivo. Endothelial cells proliferated most effectively on membranes made of polycaprolactone and chitosan blended in a 25:75 ratio, neutralized by a mixture of alkaline and ethanol. Endothelial and smooth muscle cells retained their functional properties when seeded on the blended membranes. CONCLUSIONS: We established endothelial and smooth muscle cell cultures from human right atrial appendage and right ventricle post-operative explants. The isolated cells revealed angiogenic potential and may be a promising source of patient-specific cells for regenerative medicine.

Induced Pluripotent Stem Cells of Microtus levis x Microtus arvalis Vole Hybrids: Conditions Necessary for Their Generation and Self-Renewal.

Every year, the list of mammalian species for which cultures of pluripotent stem cells (PSCs) are generated increases. PSCs are a unique tool for extending the limits of experimental studies and modeling different biological processes. In this work, induced pluripotent stem cells (iPSCs) from the hybrids of common voles Microtus levis and Microtus arvalis, which are used as model objects to study genome organization on the molecular-genetic level and the mechanisms of X-chromosome inactivation, have been generated. Vole iPSCs were isolated and cultured in a medium containing cytokine LIF, basic fibroblast growth factor (bFGF), ascorbic acid, and fetal bovine serum. Undifferentiated state of vole iPSCs is maintained by activation of their endogenous pluripotency genes - Nanog, Oct4, Sox2, Sall4, and Esrrb. The cells were able to maintain undifferentiated state for at least 28 passages without change in their morphology and give rise to three germ layers (ectoderm, mesoderm and endoderm) upon differentiation.

[Effect of high dose tamoxifen therapy in conservative treatment of patients with III-IV stages of non-small cell lung cancer].

Lung cancer is currently the leading cause of cancer-related death. The effectiveness of treatment of locally advanced and metastatic non-small cell lung cancer (NSCLC) remains low. One the promising new directions in NSCLC treatment is the inclusion of antiestrogens (tamoxifen) to standard chemotherapy regimens. Expression of estrogen (Eralpha) and progesterone (PR) receptors in the tumor tissue, objective effect and NSCLC patients' survival at chemohormonal treatment with tamoxifen use were analysed. ERalpha and PR-receptor status study in patients with NSCLC revealed presence of expression only in 1 (2%) patient. One-year and median survival (55% and 13.1 months respectively) at high-dose tamoxifen therapy use in combination with chemotherapy were established to be significantly higher (P = 0,00055) than in patients, which received only chemotherapy (32% and 9.5 months respectively).

[Effect of the intercellular adhesion molecule E-cadherin on the prognosis of non-small cell lung cancer].

The effect of E-cadherin on the prognosis of Stage I-II non-small cell lung cancer (NSCLC) was investigated. Tumor E-cadherin expression and its correlation with the clinical and morphological characteristics of a patient, as well as the prognostic role of this marker were studied. It was ascertained that the E-cadherin expression did not depend on patient gender and age, tumor histological pattern, location, grade, and pT category, but it was significantly related to the pN1 category (p < 0.0001). Univariate analysis revealed that the histological pattern of a tumor, nodal status, resection type (lobectomy/pulmonectomy), and E-cadherin expression level were significantly correlated with patient survival. Multivariate analysis showed that the important predictors were tumor histological pattern and resection type (p = 0.01 and p = 0.00004, respectively). A more complete study of the prognostic and predictive role of E-cadherin expression in patients with Stage I-II NSCLC will help identify a prognostically unfavorable group of patients who may be given additional treatment in the postoperative period.

The evolutionary pathway of x chromosome inactivation in mammals.

X chromosome inactivation is a complex process that occurs in marsupial and eutherian mammals. The process is thought to have arisen during the differentiation of mammalian sex chromosomes to achieve an equal dosage of X chromosome genes in males and females. The differences in the X chromosome inactivation processes in marsupial and eutherian mammals are considered, and the hypotheses on its origin and evolution are discussed in this review.

Characteristics of induced human pluripotent stem cells using DNA microarray technology.

We performed transcriptome analysis of some human induced pluripotent stem cells, embryonic stem cells, and human somatic cells using DNA microarrays. PluriTest bioinformatic system was used for evaluation of cell pluripotency. Changes in the genome structure and status of X-chromosome gene expression was analyzed using microarray technology.

[About the impact of the dendritic cell autovaccine on the results of treatment of non-small-cell lung cancer patients].

In thoracic department of the National Cancer Institute studied the effectiveness of dendritic cell autovaccine in the postoperative period in non-small-cell lung cancer patients. The results, showing good tolerance dendritic cell autovaccine. Shows the formation of the expressed antigen immune response after repeated injections dendritic cell autovaccine, as manifested after 4 revaccination. Results of survival patients non-small-cell lung cancer who received postoperative dendritic cell autovaccines demonstrate the high efficiency of the method and its applicability with a minimum of side effects. Further study of survival of patients non-small-cell lung cancer who received immunotherapy treatment, monitoring of compliance with the best mode of repeated injections.

[The influence of microvessel density in the tumor on the prognosis in patients with stage I-II non-small cell lung cancer after surgical treatment].

A study of the prognostic role of microvessel density in tumors of patients with stage I-II non-small cell lung cancer (NSCLC) was performed from June 2008 to June 2011. Determination of microvessel density using the marker CD31 was conducted in 114 patients. Microvessel density was significantly correlated with tumor size: so at greater than 3 cm in diameter, the content of microvessels was 68.7 +/- 3.1, with a smaller tumor size--48.6 +/- 4.5. Independent prognostic markers in patients with stage I-II NSCLC after surgery are histological type (HR = 2.97), tumor size (HR = 4.5), and the number of microvessels in tumor (HR = 3.2).

Stem cells giving rise to extraembryonic tissues.

The review is devoted to characterization of stem cells involved in the formation of extraembryonic tissues during the early development of mammalian embryos. Here we present our results of characterization of stem cells from the trophoblast and extraembryonic endoderm of voles and comparative analysis of these cells and the corresponding mouse cells and discuss possible signal pathways maintaining these cells in undifferentiated state.

[Meiotic inactivation of sex chromosomes in mammals].

During meiosis, heteromorphic mammalian X and Y chromosomes in males undergo transcription silencing and form a compact structure, the XY body, containing specific modifications of the chromatin. In this review, we consider the dynamics of sex chromosome inactivation and discuss the suggestion that the paternally inherited X chromosome preserve inactivated state in zygote. This state results from meiotic silencing and is prone to imprinted inactivation, which his observed in mammalian females at early embryogenetic stages.

[Expression of early developmental genes in vole Microtus rossiaemeridionalis].

The expression of genes Sox2, Klf4, Myc, Sall4, Gata6, Foxa2, Hnf4a, Cdx2, Esrrb, Hand1 in cultivated cells, embryos and organs of adult voles Microtus rossiaemeridionalis was studied. High resemblance of the expression patterns of these genes in the organs of adult voles, mice and humans was demonstrated. It was established that genes Gata6, Foxa2 and Hnf4a were specifically expressed in vole extraembryonic endoderm cells, while Cdx2 and Handl genes, in trophoblast stem cells. This shows that these genes can be used markers for corresponding vole cell lines. Indirect confirmation pointing to the fact that Oct4 gene is a marker gene for epiblast cells both in the vole and mouse was obtained.

[Comparative analysis of the DXPas34 regulatory region in rodents].

Mouse X chromosome inactivation center contains the DXPas34 minisatellite locus which plays an important role in expression regulation of the Tsix and Xist genes, involved into female dosage compensation. Comparative analysis of the DXPas34 locus from mouse, rat, and four common vole species revealed similar organization of this region in the form of tandem repeat blocks. A search for functionally important elements in this locus showed that all the species examined carried the conservative motif monomers, which could be involved in regulation of X inactivation.

Induced Pluripotent Stem Cells: Problems and Advantages when Applying them in Regenerative Medicine.

Induced pluripotent stem cells (iPSCs) are a new type of pluripotent cells that can be obtained by reprogramming animal and human differentiated cells. In this review, issues related to the nature of iPSCs are discussed and different methods of iPSC production are described. We particularly focused on methods of iPSC production without the genetic modification of the cell genome and with means for increasing the iPSC production efficiency. The possibility and issues related to the safety of iPSC use in cell replacement therapy of human diseases and a study of new medicines are considered.

Derivation of induced pluripotent stem cells from fetal human skin fibroblasts.

The isolation and study of autologous human stem cells remain among the most urgent problems in cell biology and biomedicine to date. Induced pluripotent stem cells can be derived from human somatic cells by the overexpression of a number of genes. In this study we reprogrammed fetal human skin fibroblasts by transduction with retroviral vectors carrying murine Oct4 , Sox2 , Klf4 , and c-Myc cDNAs. As a result, cells with the protein expression and gene transcription pattern characteristic of human embryonic stem cells were derived. These induced pluripotent cells are capable of differentiation in vitro into the ectoderm, mesoderm, and endoderm derivatives.

Molecular basis of Mammalian embryonic stem cell pluripotency and self-renewal.

Mammalian embryonic stem cells (ESC) have a number of specific properties that make them a unique object of fundamental and applied studies. In culture, ESC can remain in an infinitely undifferentiated state and differentiate into descendants of all three germ layers - ectoderm, endoderm, and mesoderm - that is, they can potentially produce more than 200 cell types comprising the body of an adult mammal. These properties of ESC are refered to as self-renewal and pluripotency. In this review, the basic signal pathways implicated in the maintenance of ESC pluripotency are considered. The major genes comprising a subsystem of "internal regulators of pluripotency," their protein products and regulators, are characterized, and interaction with other factors is described as well. The role of epigenetic mechanisms and microRNAs in the system of ESC self-renewal and pluripotency, as well as the relationship between pluripotency and X-chromosome inactivation in female mammals, is discussed.

[Induced pluripotent stem cells].

Induced pluripotent stem cells (iPS) result from a reprogramming of somatic cells via transduction with viral vectors expressing the Oct4, Sox2, c-Myc, Klf4, Nanog, and Lin28 genes, which are essential for the establishment and maintenance of the pluripotent state. In properties, iPS are almost fully similar to embryonic stem cells (ESC). To date, iPS have been obtained from various differentiated cells of mice and humans. Along with ESC, iPS are highly promising for research and medicine.

[OCT4 and NANOG are the key genes in the system of pluripotency maintenance in mammalian cells].

Embryonic stem cells are able to give rise after differentiation to derivatives of three germinal layers (ectoderm, endoderm, and mesoderm) and to functional gametes. This property of cells is referred to as pluripotency. The pluripotent status of preimplantation embryo cells and embryonic stem cells is maintained by a complicated system of molecular signaling pathways and transcription factors. The key regulators in this system are the transcription factors OCT4 and NANOG. The role and place of these factors in the pluripotency-sustaining system and their interaction with other factors are considered in the review. Data are presented on the structure, chromosomal location, expression, and regulation of the Oct4 and Nanog genes in mammals.