Browsing by Subject "fluorescence activated cell sorting"

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    Expression profiling of stem cell signaling alters with spheroid formation in CD133high/CD44high prostate cancer stem cells
    (Spandidos Publications, 2014) Oktem G.; Bilir A.; Uslu R.; Inan S.V.; Demiray S.B.; Atmaca H.; Ayla S.; Sercan O.; Uysal A.
    Cancer stem cells (CSC) isolated from multiple tumor types differentiate in vivo and in vitro when cultured in serum; however, the factors responsible for their differentiation have not yet been identified. The first aim of the present study was to identify CD133high/CD44high DU145 prostate CSCs and compare their profiles with non-CSCs as bulk counterparts of the population. Subsequently, the two populations continued to be three-dimensional multicellular spheroids. Differentiation was then investigated with stem cell-related genomic characteristics. Polymerase chain reaction array analyses of cell cycle regulation, embryonic and mesenchymal cell lineage-related markers, and telomerase reverse transcriptase (TERT) and Notch signaling were performed. Immunohistochemistry of CD117, Notch1, Jagged1, Delta1, Sox2, c-Myc, Oct4, KLF4, CD90 and SSEA1 were determined in CSC and non-CSC monolayer and spheroid subcultures. Significant gene alterations were observed in the CD133high/CD44high population when cultured as a monolayer and continued as spheroid. In this group, marked gene upregulation was determined in collagen type 9 α1, Islet1 and cyclin D2. Jagged1, Delta-like 3 and Notch1 were respectively upregulated genes in the Notch signaling pathway. According to immunoreactivity, the staining density of Jagged1, Sox2, Oct4 and Klf-4 increased significantly in CSC spheroids. Isolated CSCs alter their cellular characterization over the course of time and exhibit a differentiation profile while maintaining their former surface antigens at a level of transcription or translation. The current study suggested that this differentiation process may be a mechanism responsible for the malignant process and tumor growth.
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    The investigation of immunomodulatory effects of adipose tissue mesenchymal stem cell educated macrophages on the CD4 T cells
    (Elsevier GmbH, 2019) Özdemir R.B.Ö.; Özdemir A.T.; Sarıboyacı A.E.; Uysal O.; Tuğlu M.İ.; Kırmaz C.
    Mesenchymal stem cells (MSCs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. Macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that MSCs can differentiate between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype of macrophages. However, it has not yet been fully clarified whether these differentiated macrophages are functional. In this study, we compared the immunomodulatory effects on the CD4 T cells of M1, M2a and M2c macrophages with the macrophages that directly and indirectly cultured with MSCs. We analyzed the changes in CD14, CD64, CD80, CD163 and CD200R expression to evaluate macrophage phenotypes, and the changes in CD4, IFN-g, IL-4, IL-17a and FoxP3 expression to evaluate T helper subsets using the FACS method. The changes in IL-1b, IL-4, IL-10, IL-12p70, IL-17a and IFN-g in the media supernatants were analyzed using the Luminex method. We also performed WST-1 and Caspase-3 ELISA analyses to observe the proliferation and apoptosis status of the T cells. MSCs were found to differentiate macrophages into a distinctive phenotype, which was close to the M2c phenotype, but was not considered as an M2c cell due to the low expression of CD163, a characteristic marker for M2c. While MEM-D, MEM-ID and MSCs showed similar inhibitory effects on the Th2 and Th17 cells, the most significant increase in Treg cell frequencies was seen in MEM-D cells. Macrophages can alter their phenotypes and functions according to the stimuli from the environment. The fact that macrophages educated with MSCs suppressed the production of all the cytokines we evaluated even after the removal of MSCs suggests that these cells may be differentiated by MSCs into a suppressive macrophage subgroup. However, the Treg cell activation caused by direct interactions between MSCs and macrophage cells may be the most prominent observation of this study compared to previous work. As a result, according to our data, the interactions between MSCs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the T lymphocyte subgroups at least as effectively as MSCs. However, our data obtained from in vitro experiments should be supported by future in vivo studies. © 2019 Elsevier GmbH