Analysis of the Correlation between CD133 Expression on Human Colorectal Adenocarcinoma Cells HT-29 and Their Resistance to Chemotherapeutic Drugs.

A correlation was found between chemoresistance of HT-29CD133+ and HT-29CD133- sublines obtained after cell sorting and high expression of CD133. On the other hand, knockout of the PROM1 gene and, as a consequence, the absence of CD133 expression did not increase the sensitivity of tumor cells to chemotherapy, which indicates the absence of a direct effect of CD133 on the formation of chemoresistance in colorectal cancer cells. Variants of the HT-29 line with complete or partial knockout of the PROM1 gene were equally sensitive to protein kinase inhibitors sorafenib and sunitinib. Notably, the highest resistance to mTOR inhibitors, temsirolimus and everolimus, was shown by cells with complete knockout of the PROM1 gene (KO-HT-29 (P1)). These findings suggest that CD133 is associated with the chemoresistance of colorectal cancer cells, but is not involved in its formation.

[Expression of ganglioside GD2 on colorectal adenocarcinoma cells]. / Ékspressiia gangliozida GD2 na kletkakh kolorektal'noi adenokartsinomy.

Using flow cytometry GD2 ganglioside expression was evaluated both on colorectal adenocarcinoma cell lines and on tumor tissue samples from colorectal cancer patients. The marker was found on EpCAM-positive tumor cells in 6 of 12 patients' samples but not on the HT29 and CaCo-2 cell lines. GD2 expression was not an exceptional feature of cancer stem cells, since its expression level was similar on CD133-positive and CD133-negative tumor cells. Thus, the presence of GD2 ganglioside was revealed on colorectal adenocarcinoma cells for the first time. This finding makes it possible to use targeted therapy to treat this disease.

Involvement of Actin Filaments in the Cytotoxic Effect of GD2-Specific Antibodies.

Induction of direct cell death is one of the mechanisms of the antitumor effect of GD2-specific antibodies used for the therapy of high-risk neuroblastoma. The mechanisms of the cytotoxic signal triggered by antibody binding to GD2 ganglioside on the surface of the tumor cell remain insufficiently studied. Using inhibitor analysis we demonstrated that actin microfilaments are involved in the cell death induced by GD2-specific antibodies. Specifically, a strong antagonistic influence of cytochalasin D on the cytotoxic effect induced by GD2-specific antibodies was demonstrated in GD2+ tumor cell lines, which was expressed in at least 20% increase in cell survival and a significant decrease of the fraction of cells with fragmented DNA.

Cell Therapy as a Tool for Induction of Immunological Tolerance after Liver Transplantation.

Transplantation of solid organs, including liver, induces a number of serious complications related to immune incompatibility and requiring long-term use of immunosuppressive drugs. Finding the ways to inducing recipient immunological tolerance to the grafts is a top priority in organ transplantation and immunology. Along with the search for immunosupressive therapy, the development of alternative approaches to induction of immunological tolerance based on cell technologies is now in progress. In this regard, studies of the so-called spontaneous operational tolerance observed in ~20% patients after orthotopic liver transplantation is a promising trend. Understanding of this phenomenon can shed light on the mechanisms of immunological tolerance to allografts and will help to identify specific tolerance biomarkers and cell types with the aptitude for the induction of tolerance to liver allografts.

Isolation of Induced Pluripotent Cells from Stromal Liver Cells of Patients with Alcoholic Cirrhosis.

Stromal liver cells obtained from liver biopsy specimens of a patient with alcoholic cirrhosis can proliferate for a long time in culture passing more than 30 passages. In the course of culturing from early to late passages, acceleration of cell proliferation, decrease of the expression of some markers, and loss of hepatogenic differentiation potential were observed. On passage 30, induced pluripotent stem cells were obtained from these cells and comparative analysis of adipogenic and hepatic differentiation potencies of these cells and original liver stromal cells was performed. Induced pluripotent stem cells differentiated into both directions more efficiently and more rapidly than initial cells. Under conditions of hepatic differentiation, liver stromal cells started to express markers of definitive endoderm, but not markers of immature/mature hepatocytes, whereas induced pluripotent stem cells consistently expressed markers of definitive endoderm, immature/mature hepatocytes.

[The hepatic differentiation of adult and fetal liver stromal cells in vitro]. / Gepatogennaia differentsirovka stromal'nykh kletok vzrosloi i fetal'noi pecheni in vitro.

The liver has a marked capacity for regeneration. In most cases the liver regeneration is determined by hepatocytes. The regenerative capacity of hepatocytes is significantly reduced in acute or chronic damage. In particular, repair mechanisms are not activated in patients with alcoholic cirrhosis. Organ transplantation or advanced methods of regenerative medicine can help such patients. The promising results were obtained in clinical trials involving patients with various forms of liver disease who received transplantation of autologous bone marrow stem cells. However, to improve the effectiveness of such treatment it is necessary to search for more optimal sources of progenitor cells, as well as to evaluate the possibility of using descendants of these cells differentiated in vitro. In this study we isolated stromal cells from the liver biopsies of three patients with alcoholic cirrhosis, conducted their morphological and phenotypic analysis, and evaluated the hepatic potential of these cells in vitro. The stromal cells isolated from fetal liver were used for comparison. The results of this can serve as a basis for the development of a new method for the treatment of end-stage liver disease. The stromal cells isolated from the liver biopsies for a long time proliferate in a culture and this which makes it possible to expand them to large amounts for subsequent differentiation into hepatocyte-like cells and autologous transplantation.

Mesenchymal-Epithelial Transition in Culture of Stromal Progenitor Cells Isolated from the Liver of a Patient with Alcoholic Cirrhosis.

The cells isolated from biopsy specimen of a patient with alcoholic liver cirrhosis and cultured under standard conditions for obtaining stromal cell culture clearly diverged during early passages into two morphologically and phenotypically different subtypes: epithelial and mesenchymal. Mesenchymal cells expressed CD90 and CD44 and epithelial cells expressed CD166, CD227, and hepatocyte growth factor receptor Met. Starting from passage 6, the culture underwent spontaneous morphological changes and by passages 8-10 contained only epithelium-like cells. CD90 and CD44 expression disappeared, CD166 and CD227 expression remained unchanged, and Met expression increased. A small fraction of cells expressed GATA-4, HNF3ß, HNF1α, and HNF4α. After addition of inducers of hepatogeneic differentiation, the cells started producing albumin.

[Experimental analysis of human specific protein coding open reading frame c11orf72].

Gene c11orf72 (also known as FLJ90834) included in human gene reference list was previously predicted on the basis oftranscriptome analysis. We show that c11orf72 predicted protein coding open reading frame is specific for human genome and that it is absent from DNAs of other investigated primate species (chimpanzee, macaque). For the first time, we systematically analyzed c11orf72 expression in five normal and two cancerous human tissues (testicles, heart, brain, lung, bladder, bladder tumor and testicular tumor) and found no transcriptional activity there. Promoter of c11orf72, located close to promoter of a housekeeping gene NDUFV1, has shown high methylation level, whereas NDUFV1 promoter was almost free from methylation. The protein product for cllorf72 was analyzed using heterologous expression in human cell lines NT2/D1 (Tera2) and HepG2, in N- and C-terminal fusion constructs with the fluorescent protein TurboGFP. C11orf72 protein showed no cytotoxic or promitotic activity and was distributed diffusely through the cell. Our data confirm the possibility of gain of new protein-coding genes during human evolution due to simple accumulation of point mutations. However, we found no evidence for the functional significance of gene c11orf72.

Preparation of Fab-fragments of GD2-specific antibodies and analysis of their antitumor activity in vitro.

Monoclonal antibodies ME361 specific to ganglioside GD2 were isolated from the conditioned medium of hybridoma HB9326 and mouse ascitic fluid by the method of affinity chromatography; their Fab-fragments were obtained by proteolytic cleavage with papain. Evaluation of Fab-fragment specificity by flow cytometry and dot-blot analysis showed that binding effectiveness of fragments with antigens was close to that for the full-length molecule of antigen. It was shown that Fab-fragments and whole antibodies ME361 dose-dependently inhibit the proliferation of cells of mice T-lymphoma EL-4, and induce apoptosis of these cells 24 h after incubation.

Intravenous xenotransplantation of human placental mesenchymal stem cells to rats: comparative analysis of homing in rat brain in two models of experimental ischemic stroke.

Mesenchymal stem cells from human placenta obtained after term natural delivery were cultured and labeled with vital dye Dil of magnetic fluorescing microparticles. The labeled cells were transplanted intravenously to rats with occlusion of the median cerebral artery. Penetration of cells through the brain-blood barrier and their distribution in the brain of experimental animals were studied on serial cryostat sections. Two models of cerebral artery occlusion associated with different traumatic consequences were used. The efficiency of crossing the blood-brain barrier by transplanted cells, the number of mesenchymal cells attaining the ischemic focus and neurogenic zones, and the time of death of transplanted cells largely depended on the degree and nature of injury to the central nervous system, which should be taken into account when planning the experiments for evaluation of the effects of cell therapy on the models of neurological diseases and in clinical studies in the field of regenerative neurology.

Functional properties of mesenchymal stem cells labeled with magnetic microparticles in vitro and analysis of their distribution after transplantation.

Mesenchymal stem cells enzymatically isolated from human placenta were labeled with magnetic fluorescent microparticles (d=0.96 µ). We showed that microparticles in high doses (>10 µl stock suspension per 1 ml culture medium) significantly inhibited cell proliferation in culture. In our work we determined the optimal concentration of particles not affecting physiological properties of mesenchymal stem cells: it does not change cell proliferation, does not induce apoptosis, and does not modulate their transdifferentiation into neuronal cells. In vivo experiments showed that the chosen particles allow easy visualization of transplanted cells ex vivo on sections of different tissues.

Mouse retinal progenitor cell (RPC) cocultivation with retinal pigment epithelial cell culture affects features of RPC differentiation.

We provide evidence that coculturing of retinal progenitor cells (RPC) with retinal pigment epithelial cells significantly biases the standard in vitro RPC differentiation patterns. In particular, in cocultivation experiments RPCs lost the ability to differentiate spontaneously and displayed approximately 2.1-2.4-fold increase in immunoreactivity to the neural stem cell marker nestin and approximately 1.6-1.7-fold increase in rod photoreceptor cell rhodopsin marker immunoreactivity. The data suggest the influence of the intercellular interaction networks on RPC differentiation.

Gangliosides induce cell apoptosis in the cytotoxic line CTLL-2, but not in the promyelocyte leukemia cell line HL-60.

Gangliosides induce apoptosis in the cells of the IL-2-dependent cytotoxic mouse line CTLL-2. Upon incubation with gangliosides for 24 h, their effect resulting in appearance of apoptotic cells, falls in a series GM2 > GM3 > GM1 > GD1a > GD1b > GT1b. In the presence of rIL-2, apoptosis induced by GM1 is suppressed, whereas that induced by GM2 is enhanced (the effect of intracellular agent C2-Cer is independent of this cytokine). The GM1-induced apoptosis is cancelled by the caspase I inhibitor. The gangliosides under study are not able to induce apoptosis in the promyelocyte leukemia cell line HL-60. Physiological aspects of the phenomenon found are discussed.