Gene expression profiles of human chondrocytes during passaged monolayer cultivation

Abstract

Chondrocyte phenotype has been shown to dedifferentiate during passaged monolayer cultivation. Hence, we have investigated the expression profile of 27 chondrocyte-associated genes from both osteoarthritic cartilage tissue and healthy passaged human articular chondrocytes by quantitative real-time PCR. Our results indicate that the gene expression levels of matrix proteins and proteases in chondrocytes from monolayer culture decrease compared with those from cartilage tissue, while monolayer cultured chondrocytes from normal and osteoarthritic cartilage exhibit similar gene expression patterns. However, chondrocytic gene expression profiles were differentially altered at various stages of passage. The expression of the matrix proteins aggrecan, type II collagen, and fibromodulin inversely correlated with increasing passage number, while fibronectin and link protein exhibited a marked increase with passage. The expression of matrix proteinases MMP-3/9/13 and ADAMTS-4/5 decreased with passage, whereas proteinase inhibitors TIMP-2/3 were elevated. The cytokine IL-1 also showed increased expression with monolayer chondrocyte culture, while IGF-1 expression levels were diminished. No significant changes in TGF-b, or the chondrogenic transcription factors Sox-9, c-fos, or c-jun were observed. Our data indicates that cultured chondrocytes undergo dedifferentiation during monolayer culture, although the gene expression level of transcription factors necessary for chondrogenesis remains unchanged. This data may prove important for the future development of more specific and efficacious cultivation techniques for human articular chondrocyte-based therapies. ß

Discrimination of meniscal cell phenotypes using gene expression profiles

“…As a preliminary proof of concept, we additionally evaluated the expression profi les of passaged chondrocytes, a possible source for meniscal tissue engineering. Although this preliminary application involved cells derived from a single animal, our results were in agreement with the widely reported dedifferentiation of chondrocytes with passage; Col-2 rapidly decreased while Col-1 either increased continuously or stabilized after a certain number of passages (von der Mark et al, 1977;Darling and Athanasiou, 2005;Lin et al, 2008). However, the set of best predictors for passage number did not match the best set of tissue origin predictors.…”

“…While the two ratios showed signifi cant difference between osteoarthritic and control cartilage tissues, only Col-1/ Col-2 seemed indicate dedifferentiation of chondrocytes with passage. Another approach to capture gene expression trends in dedifferentiation has involved statistical methods like clustering and principal component analysis (Lin et al, 2008). Although such approaches may be helpful in identifying groups of genes that behave similarly under certain conditions, they do not provide a clear quantitative marker that indicates cell phenotype.…”

Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications

“…The ability to expand cells without standard passaging abolishes cell enzymatic trypsin exposure, which is directly associated with cellular dedifferentiation. NP cells are often compared to chondrocytes and, as such, several studies have shown the negative effects of passaging on chondrogenic gene expression (Darling et al, 2005;Lin et al, 2008). The rationale is that non-passaged cells do not need to waste time and energy on perhaps a poor re-differentiation process and, therefore, will be far more efficient in cell based therapy for tissue repair.…”

“…where dedifferentiation decreases expression of collagen type II and aggrecan and increases expression of collagen type I (Darling et al, 2005;Lin et al, 2008). However, it is unclear if chondrocyte-like NP cells change in the same way during expansion (Kluba et al, 2005).…”

“…Preserving maximal expression of ECM proteins in NP cells during culture is important to maximise the effectiveness of autologous cell implantation therapy in the NP of degenerate IVDs. Standard monolayer culture of bovine and human chondrocytes is known to cause dedifferentiation though the interaction with rigid culture surfaces, enzymatic passaging of cells, increased proliferation and increased cell density (Darling et al, 2005;Homicz et al, 2002;Lefebvre et al, 1998;Lin et al, 2008). To circumvent this fundamental problem, we developed a culture method that reduces the need for passaging by growing the cells on a soft, continuously expanding culture surface (Majd et al, 2009).…”