Culture and Phenotyping of Chondrocytes in Primary Culture

Thirion, Sylvie; Bérenbaum, Francis

doi:10.1385/1-59259-810-2:001

Cited by 67 publications

(69 citation statements)

References 34 publications

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“…Cells were treated with fibroblast growth factor (FGF1) (10 ng/ml) (a kind gift from M. Mohamadi, New York University [NYU]) and heparin (5 g/ml) as indicated in the figure legends. Chondrocytes from the ribs of newborn rats were isolated in accordance with the standard procedure (25) and treated with FGF after 48 h for the periods of time indicated in the figures legends. 32 To induce oxidative stress, H 2 O 2 (final concentration of 800 M in PBS solution) was administered to the RCS cells for the periods of time indicated in the figure legends.…”

Section: Methodsmentioning

confidence: 99%

The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

Kolupaeva

Daempfling

Basilico

2013

Molecular and Cellular Biology

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Fibroblast growth factor (FGF)-induced growth arrest of chondrocytes is a unique cell type-specific response which contrasts with the proliferative response of most cell types and underlies several genetic skeletal disorders caused by activating FGF receptor (FGFR) mutations. We have shown that one of the earliest key events in FGF-induced growth arrest is dephosphorylation of the retinoblastoma protein (Rb) family member p107 by protein phosphatase 2A (PP2A), a ubiquitously expressed multisubunit phosphatase. In this report, we show that the PP2A-B55␣ holoenzyme (PP2A containing the B55␣ subunit) is responsible for this phenomenon. Only the B55␣ (55-kDa regulatory subunit, alpha isoform) regulatory subunit of PP2A was able to bind p107, and this interaction was induced by FGF in chondrocytes but not in other cell types. Small interfering RNA (siRNA)-mediated knockdown of B55␣ prevented p107 dephosphorylation and FGF-induced growth arrest of RCS (rat chondrosarcoma) chondrocytes. Importantly, the B55␣ subunit bound with higher affinity to dephosphorylated p107. Since the p107 region interacting with B55␣ is also the site of cyclin-dependent kinase (CDK) binding, B55␣ association may also prevent p107 phosphorylation by CDKs. FGF treatment induces dephosphorylation of the B55␣ subunit itself on several serine residues that drastically increases the affinity of B55␣ for the PP2A A/C dimer and p107. Together these observations suggest a novel mechanism of p107 dephosphorylation mediated by activation of PP2A through B55␣ dephosphorylation. This mechanism might be a general signal transduction pathway used by PP2A to initiate cell cycle arrest when required by external signals.T he response of cells to growth factor signaling is often cell type specific, so that different cells exposed to the same growth factor will show a totally different biological response ranging from stimulation of proliferation, differentiation, or growth inhibition. While in some cases this could be due to the utilization of distinct, although cognate receptors, in many other cases it can be shown that different biological outcomes result from activation of the same receptor in a different biological context.An example of such behavior is the response of chondrocytes to fibroblast growth factor (FGF) signaling. Chondrocyte proliferation and differentiation are required for the process of endochondral ossification that mediates the formation and growth of long bones and vertebrae. One of the major regulators of this process is FGF signaling. Excessive or unregulated FGF signaling caused by activating FGF receptor (FGFR) mutations strongly inhibits chondrocyte proliferation and affects their differentiation, resulting in several bone morphogenetic disorders (1), and it is now quite clear that the major biological response of chondrocytes to FGF is inhibition of cell proliferation.This response is cell type specific and contrasts with the proliferative FGF response in most other cells. We have sought to identify the determinants of the growth i...

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Section: Methodsmentioning

confidence: 99%

The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

Kolupaeva

Daempfling

Basilico

2013

Molecular and Cellular Biology

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“…22,24 Moreover, in vitro expansion of chondrocytes is associated with a decreased capacity for chondrogenesis due to dedifferentiation or terminal differentiation of chondrocytes with passaging. [25][26][27][28] Some limitations to the ACI procedure may be related to the lack of a scaffold that provides the physical and biological signals required for proper growth and differentiation. Hence, there has been growing interest in determining appropriate combinations of cells, biologically active molecules, and biomaterial scaffolds to promote a strong differentiated phenotype.…”

mentioning

confidence: 99%

Chondrogenic Differentiation of Adipose-Derived Adult Stem Cells by a Porous Scaffold Derived from Native Articular Cartilage Extracellular Matrix

Cheng

Estes

Awad

et al. 2009

Tissue Engineering Part A

256

237

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Adipose-derived adult stem cells (ASCs) have the ability to differentiate into a chondrogenic phenotype in response to specific environmental signals such as growth factors or artificial biomaterial scaffolds. In this study, we examined the hypothesis that a porous scaffold derived exclusively from articular cartilage can induce chondrogenesis of ASCs. Human ASCs were seeded on porous scaffolds derived from adult porcine articular cartilage and cultured in standard medium without exogenous growth factors. Chondrogenesis of ASCs seeded within the scaffold was evident by quantitative RT-PCR analysis for cartilage-specific extracellular matrix (ECM) genes. Histological and immunohistochemical examination showed abundant production of cartilage-specific ECM components-particularly, type II collagen-after 4 or 6 weeks of culture. After 6 weeks of culture, the cellular morphology in the ASC-seeded constructs resembled those in native articular cartilage tissue, with rounded cells residing in the glycosaminoglycan-rich regions of the scaffolds. Biphasic mechanical testing showed that the aggregate modulus of the ASC-seeded constructs increased over time, reaching 150 kPa by day 42, more than threefold higher than that of the unseeded controls. These results suggest that a porous scaffold derived from articular cartilage has the ability to induce chondrogenic differentiation of ASCs without exogenous growth factors, with significant synthesis and accumulation of ECM macromolecules, and the development of mechanical properties approaching those of native cartilage. These findings support the potential for a processed cartilage ECM as a biomaterial scaffold for cartilage tissue engineering. Additional in vivo evaluation is necessary to fully recognize the clinical implication of these observations.

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“…Primary valvular cells from AV were prepared according to a standardized protocol described in literature elsewhere [15,16]. Description of experiments and methodology of both 2D and 3D cultures are given below.…”

Section: Tissue Processingmentioning

confidence: 99%

Histochemical and Molecular Characterization of Spongiosal Cells in Native Tissue, Two- and Three-Dimensional Cultures of Rat Aortic Valve

Nanduri

Moein

Thatipalli

et al. 2016

Journal of Histology

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The histocytochemical and molecular analysis of cells that constitute the aortic valve (AV) of the rat heart was done in this study. We have focussed on the identity of cells in the spongiosal layer of the valve by immunofluorescence studies using lineage specific markers and cytochemical staining. We have established two-dimensional (2D) cultures of cells from isolated rat AV leaflets and maintained endothelial and interstitial valvular cells (IVC) over a period of six to eight weeks. Using "passage 0" cells from 2D valvular cultures, we could reconstruct the three-dimensional (3D) valvular tissue in collagen gels that showed very similar cellular organization and marker expression profile, as that of the native tissue. Lineage specific markers in the native tissue and cell cultures were studied by Reverse Transcriptase-PCR and immunofluorescence for VCAM-I, -SMA, collagen I, CD71, collagen II, and Ecadherin markers. This is the first report on the identification of cell lineages in the spongiosal layer of AV and the successful reconstruction of 3D valvular tissue from primary cell cultures of AV.

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Culture and Phenotyping of Chondrocytes in Primary Culture

Cited by 67 publications

References 34 publications

The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

The B55α Regulatory Subunit of Protein Phosphatase 2A Mediates Fibroblast Growth Factor-Induced p107 Dephosphorylation and Growth Arrest in Chondrocytes

Chondrogenic Differentiation of Adipose-Derived Adult Stem Cells by a Porous Scaffold Derived from Native Articular Cartilage Extracellular Matrix

Histochemical and Molecular Characterization of Spongiosal Cells in Native Tissue, Two- and Three-Dimensional Cultures of Rat Aortic Valve

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