Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2

Autor: Ravikumar Rallapalli, Bruna Pucci, Rocky S. Tuan, David J. Hall, Alyssa L. Carlberg
Rok vydání: 2001
Předmět:
Cyclin-Dependent Kinase Inhibitor p21
Cancer Research
animal structures
Genetic Vectors
Cell Culture Techniques
Bone Morphogenetic Protein 2
Biology
Bone morphogenetic protein
Bone morphogenetic protein 2
Cell Line
Mesoderm
Mice
Chondrocytes
Transduction
Genetic
Transforming Growth Factor beta
Cyclins
Animals
Humans
Lectins
C-Type
Aggrecans
RNA
Messenger
Transgenes
Cloning
Molecular
Molecular Biology
Cells
Cultured
Aggrecan
Extracellular Matrix Proteins
Reverse Transcriptase Polymerase Chain Reaction
Cell Cycle
Mesenchymal stem cell
Genetic transfer
Cell Differentiation
Cell Biology
Fibroblasts
Chondrogenesis
Immunohistochemistry
Embryonic stem cell
Cell biology
Retroviridae
Cell culture
Bone Morphogenetic Proteins
embryonic structures
Immunology
Proteoglycans
Collagen
Developmental Biology
Zdroj: Differentiation. 67:128-138
ISSN: 0301-4681
DOI: 10.1046/j.1432-0436.2001.670405.x
Popis: The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.
Databáze: OpenAIRE
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