Document Detail


Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2.
MedLine Citation:
PMID:  11683496     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
A L Carlberg; B Pucci; R Rallapalli; R S Tuan; D J Hall
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Differentiation; research in biological diversity     Volume:  67     ISSN:  0301-4681     ISO Abbreviation:  Differentiation     Publication Date:  2001 Jun 
Date Detail:
Created Date:  2001-10-30     Completed Date:  2002-01-17     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0401650     Medline TA:  Differentiation     Country:  Germany    
Other Details:
Languages:  eng     Pagination:  128-38     Citation Subset:  IM    
Affiliation:
Dept. of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.
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MeSH Terms
Descriptor/Qualifier:
Aggrecans
Animals
Bone Morphogenetic Protein 2
Bone Morphogenetic Proteins / genetics,  metabolism*
Cell Culture Techniques / methods*
Cell Cycle
Cell Differentiation / genetics
Cell Line
Cells, Cultured
Chondrocytes / cytology,  metabolism
Chondrogenesis / genetics*
Cloning, Molecular
Collagen / genetics,  metabolism
Cyclin-Dependent Kinase Inhibitor p21
Cyclins / genetics,  metabolism
Extracellular Matrix Proteins*
Fibroblasts / cytology
Genetic Vectors / genetics
Humans
Immunohistochemistry
Lectins, C-Type
Mesoderm / cytology*,  metabolism*
Mice
Proteoglycans / genetics
RNA, Messenger / genetics,  metabolism
Retroviridae / genetics*
Reverse Transcriptase Polymerase Chain Reaction
Transduction, Genetic
Transforming Growth Factor beta*
Transgenes / genetics*
Grant Support
ID/Acronym/Agency:
AR39740/AR/NIAMS NIH HHS; CA67032/CA/NCI NIH HHS; DE12864/DE/NIDCR NIH HHS; ES07005/ES/NIEHS NIH HHS
Chemical
Reg. No./Substance:
0/Agc1 protein, mouse; 0/Aggrecans; 0/BMP2 protein, human; 0/Bmp2 protein, mouse; 0/Bone Morphogenetic Protein 2; 0/Bone Morphogenetic Proteins; 0/CDKN1A protein, human; 0/Cdkn1a protein, mouse; 0/Cyclin-Dependent Kinase Inhibitor p21; 0/Cyclins; 0/Extracellular Matrix Proteins; 0/Lectins, C-Type; 0/Proteoglycans; 0/RNA, Messenger; 0/Transforming Growth Factor beta; 9007-34-5/Collagen

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