The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of permanent cartilage.
OBJECTIVE
To regenerate permanent cartilage, it is crucial to know not only the necessary conditions for chondrogenesis, but also the sufficient conditions. The objective of this study was to determine the signal sufficient for chondrogenesis.


METHODS
Embryonic stem cells that had been engineered to fluoresce upon chondrocyte differentiation were treated with combinations of factors necessary for chondrogenesis, and chondrocyte differentiation was detected as fluorescence. We screened for the combination that could induce fluorescence within 3 days. Then, primary mesenchymal stem cells, nonchondrogenic immortalized cell lines, and primary dermal fibroblasts were treated with the combination, and the induction of chondrocyte differentiation was assessed by detecting the expression of the cartilage marker genes and the accumulation of proteoglycan-rich matrix. The effects of monolayer, spheroid, and 3-dimensional culture systems on induction by combinations of transcription factors were compared. The effects of the combination on hypertrophic and osteoblastic differentiation were evaluated by detecting the expression of the characteristic marker genes.


RESULTS
No single factor induced fluorescence. Among various combinations examined, only the SOX5, SOX6, and SOX9 combination (the SOX trio) induced fluorescence within 3 days. The SOX trio successfully induced chondrocyte differentiation in all cell types tested, including nonchondrogenic types, and the induction occurred regardless of the culture system used. Contrary to the conventional chondrogenic techniques, the SOX trio suppressed hypertrophic and osteogenic differentiation at the same time.


CONCLUSIONS
These data strongly suggest that the SOX trio provides signals sufficient for the induction of permanent cartilage.
Ikeda
Toshiyuki
T
Laboratory for Bone & Joint Diseases, SNP Research Center, RIKEN (The Institute of Physical and Chemical Research), University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
Kamekura
Satoru
S
Mabuchi
Akihiko
A
Kou
Ikuyo
I
Seki
Shoji
S
Takato
Tsuyoshi
T
Nakamura
Kozo
K
Kawaguchi
Hiroshi
H
Ikegawa
Shiro
S
Chung
Ung-il
UI
D000328:Adult; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002356:Cartilage / Q000196:embryology* / Q000254:growth & development; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D047108:Embryonic Development / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D006609:High Mobility Group Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology; D010641:Phenotype; D055758:SOX9 Transcription Factor; D055755:SOXD Transcription Factors; D015398:Signal Transduction / Q000502:physiology*; D012867:Skin / Q000166:cytology; D013234:Stem Cells / Q000378:metabolism*; D040521:Subcutaneous Tissue / Q000187:drug effects / Q000254:growth & development; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism* / Q000494:pharmacology / Q000502:physiology
D015415:Biomarkers; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D055755:SOXD Transcription Factors; C089186:Sox5 protein, mouse; C095939:Sox6 protein, mouse; C527933:Sox9 protein, mouse; D014157:Transcription Factors