Chondrogenic differentiation of bovine bone marrow mesenchymal stem cells in pellet cultural system.
OBJECTIVE
Pluripotent mesenchymal stem cells (MSC) have been isolated and well characterized from several tissue sources, including bone marrow stroma. MSC from different animals showed slight differences in morphology and in the potential to differentiate. In the present study, we isolated MSC from bovine bone marrow and induced chondrogenesis in order to establish a new experimental model of stem cell research.


METHODS
Bone marrow was harvested from 8 calves. For inducing chondrogenesis, MSC were cultured in pellet culture system in a chemically defined medium supplemented with 0 and 10 ng/mL of transforming growth factor beta1 (TGF-beta1). Chondrogenic differentiation was evaluated by histological, immunohistochemical, and in situ hybridization techniques. The degrees of genes expression were measured by quantitative RT-PCR.


RESULTS
Metachromatic alcian blue staining and immunoreactivity for type II collagen were detected in both pellet groups (0 and 10 ng/mL TGF-beta1) after 7 days of culturing. In situ hybridization demonstrated strong expression of type II collagen and aggrecan mRNAs in the round cells located at the center region of pellets and at densely organized areas. On the other hand, type I collagen mRNA was strongly expressed in the superficial layer of the pellets. After 20 days of pellet culture, expression of type II collagen mRNA in the cells which were not treated by TGF-beta1 was 1.7-fold higher compared with that treated by TGF-beta1.


CONCLUSIONS
Independent, spontaneous chondrogenesis of bovine MSC in pellet culture occurred without addition of any external bioactive stimulators, namely factors from TGF-beta family, which were previously considered necessary.
Bosnakovski
Darko
D
Laboratory of Veterinary Surgery, Department of Clinical Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. darko@vetmed.hokudai.ac.jp
Mizuno
Morimichi
M
Kim
Gonhyung
G
Ishiguro
Taketo
T
Okumura
Masahiro
M
Iwanaga
Toshihiko
T
Kadosawa
Tsuyoshi
T
Fujinaga
Toru
T
D053674:Aggrecans; D000818:Animals; D001854:Bone Marrow Cells; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects; D020219:Chondrogenesis / Q000187:drug effects; D024043:Collagen Type II / Q000032:analysis; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D011509:Proteoglycans / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D017154:Stromal Cells; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D053674:Aggrecans; D024043:Collagen Type II; D016326:Extracellular Matrix Proteins; D037181:Lectins, C-Type; D011509:Proteoglycans; D012333:RNA, Messenger; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1