Cartilage degeneration in the course of osteoarthritis (OA) is associated with an alteration in chondrocyte metabolism. In order to identify molecules representing putative key regulators for diagnosis and therapeutic intervention, we analyzed gene expression and microRNA (miR) levels in OA and normal knee cartilage using a customized cartilage cDNA array and quantitative RT-PCR. Among newly identified candidate molecules, H19, IGF2, and ITM2A were significantly elevated in OA compared to normal cartilage. H19 is an imprinted maternally expressed gene influencing IGF2 expression, whose transcript is a long noncoding (lnc) RNA of unknown biological function harboring the miR-675. H19 and IGF2 mRNA levels did not correlate significantly within cartilage samples suggesting that deregulation by imprinting effects are unlikely. A significant correlation was, however, observed for H19, COL2A1, and miR-675 expression levels in OA tissue, and functional regulation of these candidate molecules was assessed under anabolic and catabolic conditions. Culture of chondrocytes under hypoxic signaling showed co-upregulation of H19, COL2A1, and miRNA-675 levels in close correlation. Proinflammatory cytokines IL-1β and TNF-α downregulated COL2A1, H19, and miR-675 significantly without close statistical correlation. In conclusion, this is the first report demonstrating deregulation of an lncRNA and its encoded miR in the context of OA-affected cartilage. Stress-induced regulation of H19 expression by hypoxic signaling and inflammation suggests that lncRNA H19 acts as a metabolic correlate in cartilage and cultured chondrocytes, while the miR-675 may indirectly influence COL2A1 levels. H19 may not only be an attractive marker for cell anabolism but also a potential target to stimulate cartilage recovery.

Multipotent mesenchymal stromal cells (MSCs) are an attractive cell source for cell therapy in cartilage. Although their therapeutic potential is clear, the requirements and conditions for effective induction of chondrogenesis in MSCs and for the production of a stable cartilaginous tissue by these cells are far from being understood. Different sources of MSCs have been considered for cartilage tissue engineering, mainly based on criteria of availability, as for adipose tissue, or of proximity to cartilage and the joint environment in vivo, as for bone marrow and synovial tissues. Focussing on human MSCs, this review will provide an overview of studies featuring comparative analysis of the chondrogenic differentiation of MSCs from different sources. In particular, it will examine the influence of the cells' origin on the requirements for the induction of chondrogenesis and on the phenotype achieved by the cells after differentiation.

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White (WAT) and brown (BAT) adipose tissue are tissues of energy storage and energy dissipation, respectively. Experimental evidence suggests that brown and white preadipocytes are differentially determined, but so far not much is known about the genetic control of this determination process. The aim of this study was to identify differentially expressed genes involved in brown and white preadipocyte development. Using representational difference analysis (cDNA RDA) and DNA microarray screening, we identified four genes with higher expression in white preadipocytes (three different complement factors and ␦-6 fatty acid desaturase) and seven genes with higher expression levels in brown preadipocytes, of which three are structural genes implicated in cell adhesion and cytoskeleton organization (fibronectin, ␣-actinin-4, metargidin) and four that might function in gene transcription and protein synthesis (vigilin, necdin, snRNP polypeptide A, and a homolog to human hepatocellular carcinomaassociated protein). The expression profile of these genes was analyzed during preadipocyte differentiation, upon ␤-adrenergic stimulation, and in WAT and BAT tissue in vivo compared with references genes such as peroxisome proliferatoractivated receptor-␥ (PPAR␥), uncoupling protein 1 (UCP1), cytochrome c oxidase. adipocyte differentiation; thermogenesis; preadipocyte marker genes; uncoupling protein; cDNA representational-difference analysis; DNA microarray analysis WHITE AND BROWN ADIPOSE TISSUES represent counter actors in energy partitioning, channeling lipid energy either to accumulation in white fat (WAT) or to oxidation, i.e., dissipation in brown fat (BAT) a highly thermogenic tissue (23). Throughout the last years considerable progress has been made in elucidating the molecular mechanisms of adipocyte differentiation which involves sequential activation of numerous transcription factors from several families like different members of the CCAAT/enhancer binding proteins (C/ EBP) and peroxisome proliferator-activated receptors (PPAR) (1,15,30,44,52). However, most of these studies focused on differentiation of white preadipocytes using established white preadipocyte cell lines such as 3T3-L1 and 3T3-F442A cells (37). One of the remaining questions is how and at which stage of development the differentiation of BAT vs. WAT is regulated, of which very little is currently known. Brown and white adipocytes show distinct morphological and biochemical phenotypes in vivo (9). When differentiated in vitro, brown adipocytes show a higher respiratory capacity than white adipocytes and express the BAT specific uncoupling protein 1 (UCP1), which is considered to be a marker for brown adipocytes (21). It is still not clear whether BAT and WAT derive from the same adipose precursor cells or arise independently from distinct mesenchymal stem cells (44), although recently PGC-1, a coactivator of PPAR␥, has been identified, which induces genes important in the development of brown adipocyte phenotype (41).We have performed parallel culture...

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Background: Mesenchymal stromal cells isolated from bone marrow (MSC) represent an attractive source of adult stem cells for regenerative medicine. However, thorough research is required into their clinical application safety issues concerning a risk of potential neoplastic degeneration in a process of MSC propagation in cell culture for therapeutic applications. Expansion protocols could preselect MSC with elevated levels of growth-promoting transcription factors with oncogenic potential, such as c-MYC. We addressed the question whether c-MYC expression affects the growth and differentiation potential of human MSC upon extensive passaging in cell culture and assessed a risk of tumorigenic transformation caused by MSC overexpressing c-MYC in vivo. Methods: MSC were subjected to retroviral transduction to induce expression of c-MYC, or GFP, as a control. Cells were expanded, and effects of c-MYC overexpression on osteogenesis, adipogenesis, and chondrogenesis were monitored. Ectopic bone formation properties were tested in SCID mice. A potential risk of tumorigenesis imposed by MSC with c-MYC overexpression was evaluated. Results: C-MYC levels accumulated during ex vivo passaging, and overexpression enabled the transformed MSC to significantly overgrow competing control cells in culture. C-MYC-MSC acquired enhanced biological functions of c-MYC: its increased DNA-binding activity, elevated expression of the c-MYC-binding partner MAX, and induction of antagonists P19ARF/P16INK4A. Overexpression of c-MYC stimulated MSC proliferation and reduced osteogenic, adipogenic, and chondrogenic differentiation. Surprisingly, c-MYC overexpression also caused an increased COL10A1/COL2A1 expression ratio upon chondrogenesis, suggesting a role in hypertrophic degeneration. However, the in vivo ectopic bone formation ability of c-MYC-transduced MSC remained comparable to control GFP-MSC. There was no indication of tumor growth in any tissue after transplantation of c-MYC-MSC in mice. Conclusions: C-MYC expression promoted high proliferation rates of MSC, attenuated but not abrogated their differentiation capacity, and did not immediately lead to tumor formation in the tested in vivo mouse model. However, upregulation of MYC antagonists P19ARF/P16INK4A promoting apoptosis and senescence, as well as an observed shift towards a hypertrophic collagen phenotype and cartilage degeneration, point to lack of safety for clinical application of MSC that were manipulated to overexpress c-MYC for their better expansion.

cDNA-array analysis identified SERPINA1 and A3 as new differentiation-relevant genes for cartilage. Since SERPINA1 secretion correlated with both chondrogenesis of MSCs and dedifferentiation during chondrocyte expansion, it represents an attractive marker for refinement of chondrocyte differentiation.

Phenotypic and molecular parallels between the development of chondrosarcoma and the differentiation of chondrocytes in normal growth plate suggest that chondrosarcoma may arise from mesenchymal precursor cells driven towards chondrogenesis. We hypothesized that a comparison between cartilaginous tumours and their possible physiological cells of origin, mesenchymal stem cells (MSCs), might have biological and clinical relevance. MSCs from eight donors were submitted to chondrogenic differentiation in spheroid cultures. Expression profiles of MSCs at days 0, 7, 14, 28 and 42 of chondrogenesis and of 18 chondrosarcomas with different histological grades were studied using a customized cDNA array. Hierarchical clustering of MSC gene expression during chondrogenesis allowed the classification of samples in a pre-chondrogenic and a chondrogenic cluster corresponding to the phenotypes of early and late differentiation stages. The 74 genes differentially expressed between the two clusters were defined as chondrogenesis-relevant genes. Gene expression profiles of chondrosarcoma were submitted to hierarchical clustering on the basis of these chondrogenesis-relevant genes. This analysis allowed clear distinction between grade I and grade III chondrosarcoma and separated grade II chondrosarcoma into two groups. All grade II chondrosarcomas with occurrence of metastasis were found together with the grade III chondrosarcomas in the pre-chondrogenic cluster. This analysis shows that a molecular approach based on the comparison of tumour samples to an in vitro model for chondrogenic differentiation allows a new classification of chondrosarcoma in two clusters. These data suggest that the identification of a pre-chondrogenic and a chondrogenic phenotype for chondrosarcoma by gene expression profiling could develop into a useful tool to predict the clinical behaviour of chondrosarcoma.