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 fibrils. Electrophoretic analysis of proline-labeled cells demonstrated that redifferentiated chondrocytes synthesized mainly type II collagen and its precursors (pro alpha 1II, pc alpha 1II, and pn alpha 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 alpha 1(II) and alpha 1(I) chains of collagen types II and I showed that chondrocytes cultured in alginate expressed mainly alpha 1(II) mRNA, whereas alpha 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 alpha 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.
Bonaventure
J
J
CNRS URA 584, Hôpital Necker, Paris, France.
Kadhom
N
N
Cohen-Solal
L
L
Ng
K H
KH
Bourguignon
J
J
Lasselin
C
C
Freisinger
P
P
D000464:Alginates; D001483:Base Sequence; D015152:Blotting, Northern; D002214:Capsules; D002358:Cartilage, Articular / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics; D046508:Culture Techniques / Q000379:methods*; D015870:Gene Expression; D006801:Humans; D006909:Hydroxyproline / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011347:Procollagen / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis*
D000464:Alginates; D002214:Capsules; D011347:Procollagen; D012333:RNA, Messenger; D003094:Collagen; D011392:Proline; D006909:Hydroxyproline