ABSTRACT
OBJECTIVES: Angiogenesis, the process of formation of blood vessels, is essential for many physiological as well as pathological processes. It has been shown that human adipose tissue contains a population of non-characterized cells, called stromal-vascular fraction (SVF) cells, which are able to differentiate into several lineages. The aim of this study was to determine conditions for promoting differentiation of human adipose tissue progenitors towards endothelial cells, as well as to show that SVF cells cooperate with differentiated endothelium in capillary network formation. MATERIALS AND METHODS: Stromal vascular fraction cells were isolated according to modified Hauner's method and after adaptation they were cultured in pro-angiogenic or pro-adipogenic medium. Cells were characterized by presence of surface antigens by flow cytometry, and by expression of genes characteristic for endothelial cells or for adipocytes, quantitative real-time polymerase chain reaction. A number of tests were performed to verify their differentiation. RESULTS: Differentiation of human SVF cells towards endothelium was stimulated by the presence of serum and absence of adipogenic factors, documented by the pattern of gene expression as well as different functional in vitro assays. SVF cells were found to work together with human umbilical vein endothelial cells to form capillary networks. CONCLUSIONS: Here, we show that differentiation of SVF cells to endothelial cells or adipocyte-like cells depended on the medium used. Our work provides a clear model for analysing the differentiation capacity of SVF cells.
Subject(s)
Adipose Tissue/cytology , Blood Vessels/cytology , Cell Differentiation , Culture Media/metabolism , Stromal Cells/cytology , Adipocytes/cytology , Adult , Capillaries/cytology , Cell Movement , Cell Proliferation , Cell Separation , Cells, Cultured , Collagen/metabolism , Culture Media, Serum-Free , Drug Combinations , Female , Gene Expression Regulation , Humans , Laminin/metabolism , Middle Aged , Neovascularization, Physiologic , Proteoglycans/metabolism , Time FactorsABSTRACT
Gene transfer into neurons both in vivo and in vitro may aid in understanding of gene regulation and function in nerve cells. Especially desirable is ability to control the gene expression. In this study we developed conditions for transfection of hippocampal dentate gyrus neurons in dissociated cultures in vitro by calcium-phosphate method. Furthermore, we describe an effective use of tetracycline responsive gene promoter (Tet-On) system for the controlled and very efficient expression of transfected genes. Under optimal conditions as established in this study, efficiency of transfection of neurons with green fluorescent protein (GFP) driven by constitutive cytomegalovirus (CMV) early promoter reached 2.7%. With tetracycline responsive promoter percentage of GFP-positive neurons raised in the presence of tetracycline analog, doxycycline up to 20%. Application of the Tet-On system resulted in almost 10-fold induction of GFP expression.