Gene expression profiling of primary human articular chondrocytes in high-density micromasses reveals patterns of recovery, maintenance, re- and dedifferentiation

Dehne, Tilo; Schenk, R; Perka, Carsten; Morawietz, Lars; Pruß, Axel; Sittinger, Michael; Kaps, Christian; Ringe, Jochen

doi:10.1016/j.gene.2010.04.006

Cited by 55 publications

(48 citation statements)

References 55 publications

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“…Elevated levels of expression are observed for both genes in response to different cell-proliferative assays. In hyalocytes, ascorbic acid was observed to increase their proliferation in combination with increasing COL11A1 expression, whereas in a different study, chondrocytes exhibited high-density micromass growth in conjunction with COL11A1 [82,83]. Similarly, COL12A1 was upregulated with the growth of glioblastoma multiforme compared with normal brain, and when the proliferative potential of SKOV3 cells was reduced by knocking down the expression of RUNX1 , COL12A1 expression also decreased [84,85].…”

Section: Discussionmentioning

confidence: 99%

Gene markers of cellular aging in human multipotent stromal cells in culture

et al. 2014

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IntroductionHuman multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined.MethodsCommercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology.ResultsThe phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7.ConclusionsOur results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development of assays to test the quality of MSCs before clinical use.

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Section: Discussionmentioning

confidence: 99%

Gene markers of cellular aging in human multipotent stromal cells in culture

et al. 2014

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“…Especially, the chondrocyte micromass culture model built of primary cells is an easy to manage 3D model, which has already been shown to mimic essential aspects of human chondrocyte biology, pathophysiology, and differentiation. 24 The downside of the model is the extensive need of primary chondrocytes impeding highthroughput approaches including human cells. The use of cells from animal tissue sources such as porcine byproducts like cartilage used in this study can overcome these limitations, but the suitability of such porcine cell-based in vitro cultures to model human cartilage and its pathophysiology in OA was not investigated comprehensively so far.…”

Section: ■ Discussionmentioning

confidence: 99%

Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

et al. 2014

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In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.

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“…In addition, several other downregulated genes such as DPT , FGFR1 and TGFB1 are likely to have pro-chondrogenic effects [15], [47]. One positive effect of BMP2 was the downregulation of COL3A1 , a collagen frequently coexpressed with type I collagen in connective tissues [48].…”

Section: Resultsmentioning

confidence: 99%

Analysis of the Effects of Five Factors Relevant to In Vitro Chondrogenesis of Human Mesenchymal Stem Cells Using Factorial Design and High Throughput mRNA-Profiling

et al. 2014

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The in vitro process of chondrogenic differentiation of mesenchymal stem cells for tissue engineering has been shown to require three-dimensional culture along with the addition of differentiation factors to the culture medium. In general, this leads to a phenotype lacking some of the cardinal features of native articular chondrocytes and their extracellular matrix. The factors used vary, but regularly include members of the transforming growth factor β superfamily and dexamethasone, sometimes in conjunction with fibroblast growth factor 2 and insulin-like growth factor 1, however the use of soluble factors to induce chondrogenesis has largely been studied on a single factor basis. In the present study we combined a factorial quality-by-design experiment with high-throughput mRNA profiling of a customized chondrogenesis related gene set as a tool to study in vitro chondrogenesis of human bone marrow derived mesenchymal stem cells in alginate. 48 different conditions of transforming growth factor β 1, 2 and 3, bone morphogenetic protein 2, 4 and 6, dexamethasone, insulin-like growth factor 1, fibroblast growth factor 2 and cell seeding density were included in the experiment. The analysis revealed that the best of the tested differentiation cocktails included transforming growth factor β 1 and dexamethasone. Dexamethasone acted in synergy with transforming growth factor β 1 by increasing many chondrogenic markers while directly downregulating expression of the pro-osteogenic gene osteocalcin. However, all factors beneficial to the expression of desirable hyaline cartilage markers also induced undesirable molecules, indicating that perfect chondrogenic differentiation is not achievable with the current differentiation protocols.

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Gene expression profiling of primary human articular chondrocytes in high-density micromasses reveals patterns of recovery, maintenance, re- and dedifferentiation

Cited by 55 publications

References 55 publications

Gene markers of cellular aging in human multipotent stromal cells in culture

Gene markers of cellular aging in human multipotent stromal cells in culture

Suitability of Porcine Chondrocyte Micromass Culture To Model Osteoarthritis in Vitro

Analysis of the Effects of Five Factors Relevant to In Vitro Chondrogenesis of Human Mesenchymal Stem Cells Using Factorial Design and High Throughput mRNA-Profiling

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