Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.

2.50
Hdl Handle:
http://hdl.handle.net/10541/84455
Title:
Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.
Authors:
Li, Ying; Tew, Simon R; Russell, Amanda M; Gonzalez, Karin R; Hardingham, Timothy E; Hawkins, Robert E
Abstract:
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.
Affiliation:
UK Centre for Tissue Engineering, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
Citation:
Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9., 10 (3-4):575-84 Tissue Eng.
Journal:
Tissue Engineering
Issue Date:
19-Oct-2009
URI:
http://hdl.handle.net/10541/84455
DOI:
10.1089/107632704323061933
PubMed ID:
15165474
Language:
en
ISSN:
1076-3279
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorLi, Ying-
dc.contributor.authorTew, Simon R-
dc.contributor.authorRussell, Amanda M-
dc.contributor.authorGonzalez, Karin R-
dc.contributor.authorHardingham, Timothy E-
dc.contributor.authorHawkins, Robert E-
dc.date.accessioned2009-10-19T15:57:25Z-
dc.date.available2009-10-19T15:57:25Z-
dc.date.issued2009-10-19T15:57:25Z-
dc.identifier.citationTransduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9., 10 (3-4):575-84 Tissue Eng.en
dc.identifier.issn1076-3279-
dc.identifier.pmid15165474-
dc.identifier.doi10.1089/107632704323061933-
dc.identifier.urihttp://hdl.handle.net/10541/84455-
dc.description.abstractChondrocytes 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.en
dc.language.isoenen
dc.subject.meshAdenoviridae-
dc.subject.meshAlginates-
dc.subject.meshCell Division-
dc.subject.meshChondrocytes-
dc.subject.meshCollagen Type II-
dc.subject.meshGenes, Reporter-
dc.subject.meshGenetic Vectors-
dc.subject.meshGlucuronic Acid-
dc.subject.meshHexuronic Acids-
dc.subject.meshHigh Mobility Group Proteins-
dc.subject.meshHumans-
dc.subject.meshLentivirus-
dc.subject.meshMicrospheres-
dc.subject.meshSOX9 Transcription Factor-
dc.subject.meshTranscription Factors-
dc.subject.meshTransduction, Genetic-
dc.titleTransduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.en
dc.contributor.departmentUK Centre for Tissue Engineering, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.en
dc.identifier.journalTissue Engineeringen

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