Isolation of a Population of Cells Co-Expressing Markers of Embryonic Stem Cells and Mesenchymal Stem Cells from the Rudimentary Uterine Horn of a Patient with Uterine Aplasia.

More than 50% cells isolated from the endometrial cavity scraping and the myometrium of the rudimentary horn of an underdeveloped uterus removed from a patient with uterine aplasia and maintained under culturing conditions normal for mesenchymal stem cells (MSC) expressed embryonic transcription factors Oct4 and Nanog, embryonic cell membrane sialyl glycolipid SSEA4, and MSC markers. After 2-3 passages, the cells lost the expression of the early embryogenesis markers, but retained MSC markers. The presence of dormant stem cells in the underdeveloped endometrium and in the uterus indicates that this tissue has a regenerative potential that can be activated and used for completion of organ morphogenesis. This task requires the development of methods of early diagnosis of morphogenesis impairment and tools for safe reactivation of the ontogenesis.

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 Epithelial Cell Adhesion Molecule (EpCAM) in Tumor Spheroids of Human Colorectal Adenocarcinoma Cells.

We studied the formation of spheroids by Caco-2, SW480, and HCT116 human colorectal adenocarcinoma cell lines under low-adhesion culturing conditions. Of these three cell lines, only HCT116 formed stable tumor spheroids. Flow cytometry analysis of 19 surface markers in monolayer HCT116 culture and spheroids formed by these cells revealed considerable similarity of the expression profiles in these two culturing modes. The only exception was EpCAM molecule: its expression in spheroids was 3-fold higher than in the monolayer culture. Scanning confocal laser microscopy showed equal EpCAM distribution in the inner mass of the spheroids.

Proliferative Activity of Colorectal Cancer Cells with Different Levels of CD133 Expression.

We studied proliferative activity of colorectal cancer cells with different expression level of CD133 molecule associated with cancer stem cells phenotype. Analysis of BrdU incorporation into Caco-2 and HT-29 cell lines showed that the percentage of cells in the DNA synthesis phase in the CD133+/high population is higher than in CD133-/low population. The expression of proliferation marker Ki-67 and the percentage of Ki-67+ cells were also higher in the CD133+/high population. Colorimetric analysis with crystal violet dye showed that the number of cells after 10-days culturing was higher in the CD133+/high population in both cell lines. These findings suggest that cells with high level of CD133 expression are characterized by higher proliferative activity, which can contribute to the tumor progression.

Surface Molecular Markers of Cancer Stem Cells: Computation Analysis of Full-Text Scientific Articles.

The data on cancer stem cell surface molecular markers of 27 most common cancer diseases were analyzed using natural language processing and data mining techniques. As a source, 8933 full-text open-access English-language scientific articles available on the Internet were used. Text mining was based on searching for three entities within one sentence, namely a tumor name, a phrase "cancer stem cells" or its synonym, and a name of differentiation cluster molecule. As a result, a list of surface molecular markers was formed that included markers most frequently mentioned in the context of certain tumor diseases and used in studies of human and animal tumor cells. Based on similarity of the associated markers, the tumors were divided into five groups.

CD133+ human colorectal adenocarcinoma cells are resistant to staining with fluorescent dyes used for analysis of ABC transporter activities.

Flow cytometry measurement of the expression of surface marker CD133 simultaneously with the analysis of fluorescent dye exclusion was performed in order to develop new methods for detection of cancer stem cell populations in tumor tissue samples from patients with colorectal adenocarcinoma. No correlation was found between the count of CD133(+) cancer cells and the volume of the "population" formed from cells actively pumping off the fluorescent dye. On the other hand, the fluorescence distribution plot showed predominant location of CD133(+) cancer cells among cells stained with neither DyeCycle Violet DNA-binding dye, nor rhodamine 123 mitochondrial dye. These cells did not show the properties of the classical "side population", because they did not shift to the area of stained cell after treatment with ionic channel blocker verapamil.

Subpopulation of colorectal adenocarcinoma cells co-expressing CD133 and cancer stem cells markers of other tumors.

Co-expression of colorectal adenocarcinoma cancer stem cells marker CD133 and a set of surface molecules described in published reports as possible cancer stem cell markers of other solid tumors was analyzed by flow cytometry. Minor cell populations expressing CD29, CD34, CD90, and CD117 against the background of CD133 expression were detected in cancer cells suspensions from the patients with colorectal adenocarcinoma. Our findings suggest that these markers can be used as additional markers of cancer stem cells of human colorectal adenocarcinoma.

Detection of minor subpopulations of colorectal adenocarcinoma cells expressing cancer stem cell markers.

The expression of puitative surface molecular markers of cancer stem cells on human colorectal adenocarcinoma cells was analyzed by flow cytofluorometry. Cell subpopulations expressing markers of epithelial and malignant cells and stem cell markers were identified. Four minor subpopulations with CD24(+)/CD133(+), CD44(+)/CD133(+), CD90(+)/CD71(+), or CD90(+)/CD24(+) phenotypes meeting this requirement were detected; presumably, those were cancer stem cell subpopulations. These results extend our knowledge on heterogeneity of human colorectal adenocarcinoma cell population and outline new trends of research of cancer stem cell phenotype in these tumors.

[Identification of the side population associated with ATP-binding cassette transporters activity using imaging flow cytometry]. / Vyiavlenie "bokovoi populiatsii", assotsiirovannoi s aktivnost'iu ATP-zavisimykh transporterov, pri pomoshchi protochnoi tsitometrii s vizualizatsiei.

DyeCycle Violet efflux, caused by ATP-binding cassette transporters activity, was analyzed in human colorectal adenocarcinoma cell lines SW480, HT-29, Caco-2 by neans of FACSAria III flow cytometer and ImageStreamX Mk II imaging flow cytometer. Along with similarity of cytometry data obtained on the two instruments, the use of imaging flow cytometry made it possible to characterize the morphology of side population cells, as well as morphology of other cell populations differing in the degree of dye accumulation. The population of cells, which are smaller than the side population cells and practically do not take the dye, is of the special interest. Probably, this population may contribute to the tumor resistance to chemotherapy.

Standardization of biochemical profile of mesenchymal cell materials by probing the level of dehydrogenase activity.

It is demonstrated that the output optical signal of MTT test is directly proportional to the number of viable cells in the primary culture of mesenchymal cells (skin fibroblasts and mesenchymal stem cells from the bone marrow, placenta, and umbilical cord). The slope of the best curve in coordinates "cell number - optical signal" reflecting specific productivity of MTT-formazan characterizes mean dehydrogenase activity of cells and their physiological activity. It was found that in vitro dehydrogenase activity of primary cultures of mesenchymal cells increased during the first 3-5 passages and then tended to decrease. The variant of MTT method presented here can be used for standardization of cell materials.

[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.

[Identification of "side population" associated with cancer stem cells by flow cytometry with violet laser].

The possibility of identification of cancer stem cells "side population" in solid tumors by using the flow cytometer equipped with 405 nm violet laser was investigated. Ovarian cancer (Skov-3) and colon cancer (Colo 320) cell lines formed the "side population" after vital staining with Hoechst 33342. Analysis of cells isolated from tumor tissue of malignant melanoma and colorectal cancer, also revealed "side population" that was a result of the fluorescent dye exclusion. The percentage of melanoma cells included in the "side population" was the same as that of cells co-expressing cancer stem cells markers--CD34 and CD44. In contrast, the colon cancer "side population" was significantly smaller than the minor populations of colon cancer cells identified by either CD133 expression or exclusion of Rhodamine 123.