ABSTRACT
Mice were generated in which a Col2-GFP transgene serves as a reporter for the chondrocyte lineage and for chondrogenesis in live embryos and newborn pups. Cells actively engaged in chondrogenesis were identified by confocal optical sectioning within their native environments in embryos and in thick tissue slices. Chondrocytes exhibiting GFP fluorescence were purified from rib cages by high-speed cell sorting of crude cell suspensions. Intensity of fluorescence correlated with biosynthesis of procollagen II in these cells. The use of these mice and their cells provides a novel approach for studying chondrocyte differentiation and chondrogenesis during skeletal development.
Subject(s)
Bone and Bones/embryology , Chondrocytes/cytology , Chondrogenesis/physiology , Collagen , Genes, Reporter , Luminescent Proteins , Animals , Cell Lineage , Cell Separation , Chondrocytes/metabolism , Collagen/genetics , Female , Fluorescence , Green Fluorescent Proteins , Luminescent Proteins/genetics , Mice , Mice, Transgenic , Microscopy, ConfocalABSTRACT
Fibroblast growth factor (FGF) 9 was compared with FGF2 in its ability to influence proliferation, differentiation, terminal differentiation and apoptosis in a rat calvaria-derived cell line (RCJ 3.1C5.18) that spontaneously undergoes chondrocyte differentiation in vitro. Like FGF2, FGF9 promoted proliferation, but to a lesser extent. In contrast to FGF2, which blocked chondrocytic differentiation, FGF9 had no effect on differentiation but inhibited terminal differentiation. FGF9 also stimulated expression of the mitotic inhibitor p21 to a greater extent than FGF2. Neither ligand influenced apoptosis. The results indicate that FGF9 could account for many of the physiological responses attributed to FGF-receptor activation in the growth plate.
Subject(s)
Chondrocytes/metabolism , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factors , Growth Substances/pharmacology , Animals , Cell Differentiation/drug effects , Cell Division/drug effects , Cells, Cultured , Chondrocytes/drug effects , Fibroblast Growth Factor 9 , Humans , In Vitro Techniques , Rats , Receptors, Fibroblast Growth Factor/metabolismABSTRACT
We used a combination of morphologic and histochemical methods to demonstrate that rat calvaria-derived mesenchymal cells, RCJ 3.1C5. 18, in culture progress through the differentiation pathway exhibited by chondrocytes in the endochondral growth plate. The cells were grown either as monolayer or suspension cultures. Subconfluent monolayer cultures did not express markers typical of chondrocyte phenotypes. However, after reaching confluency the cells formed nodules of chondrocytic cells separated by cartilage-appearing matrix and encapsulated by fibroblast-like cells. Suspension culture produced cell aggregates with similar characteristics. Matrix in both the nodules and aggregates stained for collagen Types II and XI and aggrecan, and some cells displayed a distinctive pericellular matrix that stained for Type X collagen. Mineralization was evident in older cultures. By electron microscopy, most cells in the aggregates appeared as typical chondrocytes. However, some larger cells were surrounded by a "mat" of matrix comprised of hexagonal arrays of dense nodules interconnected by a filamentous network. Immunogold localization confirmed the presence of collagen Type X in this matrix. Analysis of markers of chondrocyte differentiation and terminal differentiation over time showed that these markers were acquired sequentially over 2 weeks of culture. This model system will be useful to study the regulation of various steps in the chondrocyte differentiation pathway.
