Formation of cartilage matrix proteins by BMP-transfected murine mesenchymal stem cells encapsulated in a novel class of alginates.
Proliferation and differentiation of wild-type, BMP-2 and BMP-4 transfected cells of C3H10T1/2, a mouse mesenchymal stem cell line that can differentiate into chondrocytes, were studied under monolayer (2D-) and encapsulation (3D-) conditions. Cells were encapsulated in a novel class of alginate. The alginate was of clinical grade (CG) because of complete removal of mitogenic and cytotoxic contaminants by chemical means. Compared to commercial alginates used so far for encapsulation it was characterized by ultra-high viscosity (UHV; viscosity of a 0.1% w/v solution of about 20 cP). In contrast to monolayer cultures, proliferation of cells was prevented when the cells were encapsulated in UHV/CG alginate at the same suspension density. As revealed by immunohistochemistry and quantitative RT-PCR, transfected and wild-type monolayer cells showed synthesis of type I collagen after transfer into differentiation medium, while culture in an alginate scaffold resulted in an upregulation of type II collagen and other hyaline cartilage proteins. BMP-4 transfected cells produced considerably more type II collagen than BMP-2 transfected and wild-type cells. BMP-4 transfected cells were also characterized by type I collagen production up to Day 10 and exhibited transient alkaline phosphatase activity levels that were much higher than the peak values observed for the other two cell lines. The coincidence of the ALP peak values with downregulation of type I collagen in BMP-4 transfected cells suggested that C3H10T1/2 cells differentiate into chondrocytes via a chondroprogenitor-like cell.
Weber
M
M
Department of Biotechnology, University of Würzburg, Am Hubland, Biozentrum, Germany.
Steinert
A
A
Jork
A
A
Dimmler
A
A
Thürmer
F
F
Schütze
N
N
Hendrich
C
C
Zimmerman
U
U
D000464:Alginates / Q000737:chemistry*; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002356:Cartilage / Q000378:metabolism*; D002455:Cell Division; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D006801:Humans; D007150:Immunohistochemistry; D008648:Mesoderm / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D008934:Mitogens; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012624:Seaweed / Q000378:metabolism; D013234:Stem Cells / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta
D000464:Alginates; C525718:BMP2 protein, human; C525809:BMP4 protein, human; C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D018076:DNA, Complementary; D008934:Mitogens; D016212:Transforming Growth Factor beta; D002118:Calcium