Reexpression of Cartilage-Specific Genes by Dedifferentiated Human Articular Chondrocytes Cultured in Alginate Beads

We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibri...

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Veröffentlicht in:Experimental cell research 1994-05, Vol.212 (1), p.97-104
Hauptverfasser: Bonaventure, J., Kadhom, N., Cohen-Solal, L., Ng, K.H., Bourguignon, J., Lasselin, C., Freisinger, P.
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container_end_page 104
container_issue 1
container_start_page 97
container_title Experimental cell research
container_volume 212
creator Bonaventure, J.
Kadhom, N.
Cohen-Solal, L.
Ng, K.H.
Bourguignon, J.
Lasselin, C.
Freisinger, P.
description We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoreticanalysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (proα 1II, p 0α 1II, and p nα 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the α 1(II) and α 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly α 1(II) mRNA, whereas α 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no α 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells.
doi_str_mv 10.1006/excr.1994.1123
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After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoreticanalysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (proα 1II, p 0α 1II, and p nα 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the α 1(II) and α 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly α 1(II) mRNA, whereas α 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no α 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. 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Psychology</topic><topic>Gene Expression</topic><topic>Humans</topic><topic>Hydroxyproline - metabolism</topic><topic>Molecular Sequence Data</topic><topic>Polymerase Chain Reaction</topic><topic>Procollagen - secretion</topic><topic>Proline - metabolism</topic><topic>RNA, Messenger - biosynthesis</topic><topic>Skeleton and joints</topic><topic>Vertebrates: osteoarticular system, musculoskeletal system</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bonaventure, J.</creatorcontrib><creatorcontrib>Kadhom, N.</creatorcontrib><creatorcontrib>Cohen-Solal, L.</creatorcontrib><creatorcontrib>Ng, K.H.</creatorcontrib><creatorcontrib>Bourguignon, J.</creatorcontrib><creatorcontrib>Lasselin, C.</creatorcontrib><creatorcontrib>Freisinger, P.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Experimental cell research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bonaventure, J.</au><au>Kadhom, N.</au><au>Cohen-Solal, L.</au><au>Ng, K.H.</au><au>Bourguignon, J.</au><au>Lasselin, C.</au><au>Freisinger, P.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Reexpression of Cartilage-Specific Genes by Dedifferentiated Human Articular Chondrocytes Cultured in Alginate Beads</atitle><jtitle>Experimental cell research</jtitle><addtitle>Exp Cell Res</addtitle><date>1994-05-01</date><risdate>1994</risdate><volume>212</volume><issue>1</issue><spage>97</spage><epage>104</epage><pages>97-104</pages><issn>0014-4827</issn><eissn>1090-2422</eissn><coden>ECREAL</coden><abstract>We have used the three-dimensional culture system in alginate beads to redifferentiate human articular chondrocytes which were first expanded on a plastic support. After 15 days in alginate beads, electron microscopy showed that cells had synthesized an extracellular matrix containing collagen fibrils. Electrophoreticanalysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (proα 1II, p 0α 1II, and p nα 1II). After pepsin digestion a small amount of collagen type XI was also detected. These results were confirmed by Northern blot analysis of total RNAs. Hybridization with collagen cDNA probes coding for the α 1(II) and α 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly α 1(II) mRNA, whereas α 1(I) mRNA transcripts were almost undetectable. Such a result was observed even after several passages on plastic flasks, suggesting that dedifferentiated cells were able to revert to a chondrocytic phenotype in this three-dimensional system. However, SV40-transformed chondrocytes were not able to redifferentiate in alginate as no α 1(II) mRNAs were detected. Total RNA was converted into cDNA by reverse transcription and amplified by polymerase chain reaction. This technique was employed to amplify mRNAs specific for collagen type II and type X and the large aggregating proteoglycan aggrecan. Two transcripts resulting from an alternative splicing of the complement regulatory protein (CRP)-like domain of aggrecan were originally identified in chondrocytes in monolayers. Like intact cartilage, chondrocytes in alginate expressed only the larger transcript with the CRP domain, whereas the two transcripts were equally expressed in SV40-transformed chondrocytes. Thus, the alginate system appears to represent a relevant model for the redifferentiation of human chondrocytes, especially when only a small cartilage biopsy is available, and could prove useful for pulse-chase studies of patients with skeletal chondrodysplasias. However it was unable to restore the chondrocytic phenotype in virally transformed cells.</abstract><cop>Orlando, FL</cop><pub>Elsevier Inc</pub><pmid>8174647</pmid><doi>10.1006/excr.1994.1123</doi><tpages>8</tpages></addata></record>
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subjects Alginates
Base Sequence
Biological and medical sciences
Blotting, Northern
Capsules
Cartilage, Articular - cytology
Cartilage, Articular - embryology
Cartilage, Articular - metabolism
Cell Differentiation - physiology
Collagen - biosynthesis
Collagen - genetics
Culture Techniques - methods
Fundamental and applied biological sciences. Psychology
Gene Expression
Humans
Hydroxyproline - metabolism
Molecular Sequence Data
Polymerase Chain Reaction
Procollagen - secretion
Proline - metabolism
RNA, Messenger - biosynthesis
Skeleton and joints
Vertebrates: osteoarticular system, musculoskeletal system
title Reexpression of Cartilage-Specific Genes by Dedifferentiated Human Articular Chondrocytes Cultured in Alginate Beads
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