ABSTRACT
During continuous cultivation cell lines can lose a number of innate characteristics or acquire new ones. In this work we compared growth and phenotypic characteristics of human glioblastoma À172 and Ò98G lines from cell culture collection of Research Institute of Influenza of Ministry of Health of Russian Federation (St. Petersburg). The activity of genes encoding intracellular proteins that determine cell lines belonging to mesenchymal type, as well as several growth factor genes and extracellular matrix genes were estimated. Cell lines A172 and T98G varied in morphology and surface markers expression. A172 cells were characterized by higher expression of mesenchymal markers CD90, CD105, fibroblast activation protein, and tenascin C. Both cell lines showed high level of a2 smooth muscle actin expression. The obtained data indicating high activity of genes encoding major inductors of angiogenesis (VEGF, FGF2 (b), TGFb1) and thrombospondin-1 in foregoing cell lines are in agreement with published data. Reduction of fetal serum in culture medium from 10 to 5 % in both cell lines resulted in the increase of proportion of cells with surface antigens CD73 and CD105. Both A172 and T98G cell lines sustain the main features of glioblastomas and therefore can serve as research objects in investigation of this kind of neoplasms.
Subject(s)
Antigens, Differentiation/biosynthesis , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Glioblastoma , Neoplasm Proteins/biosynthesis , Cell Line, Tumor/metabolism , Cell Line, Tumor/pathology , Glioblastoma/metabolism , Glioblastoma/pathology , HumansABSTRACT
Endoglin (CD105) is the marker of endothelial and mesenchymal stem cells and the component of TGF-ß, BMP-9 and BMP-10-binding receptor complexes. Its expression is significantly increased on blood vessels endothelium of ischemic tissues and growing tumors. Measurement of concentration of the soluble endoglin in the serum or urine is used as a method for diagnosing cancer and pregnancy disorders. The aim of this work was to create a novel family of monoclonal antibodies recognizing endoglin on the cell surface and in biological fluids. Murine myeloma cells' derived recombinant protein representing the whole extracellular part of endoglin was used as an antigen. F1(SJL/JxBALB/c) mice were the donors of immune splenocytes. Hybridoma screening procedures were performed using E. coli-produced copies of the antigen, endoglin-expressing immortalized human cell lines, and primary cultures of human mesenchymal stromal cells. Ten novel monoclonal antibodies recognizing at least eight distinct epitopes were produced. Eight antibodies bind membrane form of endoglin on the surface of normal and transformed human cells derived from different tissue sources. Two antibodies recognize linear antigenic determinants of the molecule and can be used to detect endoglin by western blot. Sandwich ELISA system was designed in order to measure soluble endoglin in cell culture medium.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/immunology , Antigens, CD/immunology , Receptors, Cell Surface/immunology , Animals , Antibodies, Monoclonal, Murine-Derived/biosynthesis , Antibodies, Monoclonal, Murine-Derived/chemistry , Antigens, CD/metabolism , Endoglin , Female , Humans , Mice , Neoplasms/diagnosis , Neoplasms/immunology , Neoplasms/metabolism , Pregnancy , Pregnancy Complications/diagnosis , Pregnancy Complications/immunology , Pregnancy Complications/metabolism , Receptors, Cell Surface/metabolismABSTRACT
There are contradictory data concerning the influence of mesenchymal stromal cells (MSC) on immunoglobulin (Ig) production. Most of them were obtained using MSC from bone marrow. Properties of MSC from other tissues are elusive. In the present work MSC cultures were derived from umbilical cord, adipose tissue, and bone marrow of healthy donors, as well as from bone marrow of patients with autoimmune diseases. MSC from all these sources had similar surface markers phenotype. The influence of co-cultivation with MSC at exponential or stationary phase on IgM and IgE content in Namalva and U266 cells was evaluated. MSC from bone marrow of healthy donors had no effect on IgM and IgE production. Proliferating MSC obtained from patients with Crohn's disease and multiple sclerosis stimulated Ig production. Exponentially growing MSC derived from umbilical cord and adipose tissue also stimulated Ig synthesis. MSC at stationary cultures amplified IgM production in Namalva cells and suppressed IgE synthesis in U266. Thus, MSC with similar phenotype but derived from different sources differ in their capacity to modulate Ig production in B-lymphoid cells. The effect of MSC depends on their growth stage and may differ for lymphoblastoid and myeloma cells.
Subject(s)
Immunoglobulin E/biosynthesis , Immunoglobulin M/biosynthesis , Mesenchymal Stem Cells/pathology , Adipose Tissue/cytology , Adipose Tissue/immunology , B-Lymphocytes/chemistry , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Bone Marrow Cells/immunology , Bone Marrow Cells/pathology , Case-Control Studies , Cell Cycle , Cell Differentiation , Cell Line, Tumor , Cell Proliferation , Coculture Techniques , Crohn Disease/immunology , Crohn Disease/pathology , Female , Fetal Blood/cytology , Fetal Blood/immunology , Humans , Immunoglobulin E/immunology , Immunoglobulin M/immunology , Male , Mesenchymal Stem Cells/immunology , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Organ SpecificityABSTRACT
Mesenchymal stromal cells were isolated from the adipose tissue obtained during surgery for breast cancer and cultured under conditions of normal or low oxygen concentrations. In patients that had received a course of radiation and polychemotherapy prior to surgery, the proliferative potential of mesenchymal stromal cells was irreversibly disturbed. In patients receiving no therapy prior to surgery, the morphological, growth, phenotypic, and differentiation characteristics of mesenchymal stromal cells did not differ from the corresponding parameters of mesenchymal stromal cells from healthy donors. Culturing under hypoxic conditions increased adipogenic differentiation potencies of mesenchymal stromal cells from donors and patients.
Subject(s)
Breast Neoplasms/pathology , Mesenchymal Stem Cells/pathology , Breast Neoplasms/immunology , Female , Humans , ImmunophenotypingABSTRACT
A number of publications contain contradictory data about influence of mesenchymal stromal cells (MSC) on B-lymphocyte growth, differentiation and production of immunoglobulins (Ig). The aim of the study was to investigate the influence of MSC derived from adipose tissue of healthy donors and cancer patients on the proliferation and Ig synthesis of lymphoblastoid cell line Namalva and myeloma cell line U266. Co-cultivation of Namalva cells with MSC stimulated their proliferation, decreased the doubling time and the minimal effective seeding dose and therefore made cloning of these lymphoblastoid cells possible. The presence of MSC supported the survival and proliferation of Namalva cells cultivated in growth factor deficient medium. MSC also stimulated proliferation of U266 myeloma cells. Both MSC derived from adipose tissue from the healthy donors and from patients with breast cancer effectively stimulated B-cell lines proliferation. Presence of MSC in mixed cultures had no influence on the production of IgM or IgE by Namalva or U266 cells respectively. Co-cultivation of Namalva or U266 with MSC resulted in the formation of close intercellular contacts between cells of both types.
