Objective. To investigate whether adult human articular chondrocytes (AHACs), dedifferentiated by monolayer expansion, can differentiate toward diverse mesenchymal lineages and, if so, whether this ability is regulated by growth factors during monolayer expansion.Methods. AHACs were expanded as multiclonal or clonal populations in medium without (control) or with factors enhancing cell dedifferentiation (transforming growth factor ␤1, fibroblast growth factor 2, and platelet-derived growth factor type BB [TFP]). Cells were then cultured under conditions promoting chondrogenic, osteogenic, or adipogenic differentiation, and the acquired phenotypes were assessed histologically, biochemically, and by real-time reverse transcriptase-polymerase chain reaction.Results Conclusion. Dedifferentiated AHACs exhibit differentiation plasticity, which is modulated by growth factors used during monolayer expansion and is highly heterogeneous across different clones. Clonal culture of AHACs in the presence of regulatory molecules could lead to the identification of AHAC subpopulations with enhanced cartilage repair capacity.

Growth in three-dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10-30,000 cells were generated upon overnight culture in poly-Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with >/=threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, >/=threefold, upregulation of the expression of >100 transcripts while 73 were >/=threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-alpha, -beta, and gamma), IL-8, CCL20 (MIP-3alpha), and Angiopoietin-like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process.