Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.
Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.
Li
Ying
Y
UK Centre for Tissue Engineering, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
Tew
Simon R
SR
Russell
Amanda M
AM
Gonzalez
Karin R
KR
Hardingham
Timothy E
TE
Hawkins
Robert E
RE
D000256:Adenoviridae; D000464:Alginates; D002455:Cell Division / Q000502:physiology; D019902:Chondrocytes / Q000166:cytology / Q000502:physiology*; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006609:High Mobility Group Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus; D008863:Microspheres; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D000464:Alginates; D024043:Collagen Type II; D006603:Hexuronic Acids; D006609:High Mobility Group Proteins; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D014157:Transcription Factors; D020723:Glucuronic Acid