Primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 BMPs and Sox9 maintain appropriate phenotype.
OBJECTIVE
To confirm that primary intervertebral disc cells cultured in monolayer transduced with adenovirus maintained their phenotype, hence is an appropriate system to test gene therapy agents.


METHODS
Adult bovine nucleus pulposus and anulus fibrosus cells cultured in monolayer were transduced with adenoviruses expressing human bone morphogenetic proteins (AdBMPs) or Sox9 (AdSox9), or green fluorescence protein (AdGFP, as control). Chondrocyte phenotypic markers (e.g., type II collagen and aggrecan) and the chondrocyte hypertrophy marker (type X collagen) were measured 6 days after viral transduction by reverse-transcription polymerase chain reaction.


RESULTS
Primary nucleus pulposus and anulus fibrosus cells transduced with AdBMPs, AdSox9, or adenovirus-expressing green fluorescence protein only (AdGFP, as control) continue to express healthy chondrocyte phenotypic markers and showed no evidence of the expression of the chondrocyte hypertrophy marker (type X collagen gene). Thus, we have shown that bovine nucleus pulposus and anulus fibrosus cells transduced with adenovirus overexpressing 12 different bone morphogenetic proteins or Sox9 maintain their phenotype in short-term culture.


CONCLUSIONS
In this study, primary bovine intervertebral disc cells transduced with adenovirus overexpressing 12 bone morphogenetic proteins or Sox9 preserved their phenotype in short-term culture. These cells did not express the type X collagen gene, an undesirable chondrocyte hypertrophic gene that could lead to ossification. Therefore, low-passage intervertebral disc cells cultured in monolayer is an appropriate culture system to test therapeutic genes. We further suggest that these cells may also be appropriate for engineering tissues or for cell therapy for degenerative disc diseases.
Zhang
Yejia
Y
Department of Rehabilitation Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.
Markova
Dessislava
D
Im
Hee-Jeong
HJ
Hu
Wenyang
W
Thonar
Eugene J-M A
EJ
He
Tong-Chuan
TC
An
Howard S
HS
Phillips
Frank M
FM
Anderson
D Greg
DG
D000256:Adenoviridae / Q000235:genetics; D053674:Aggrecans / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism* / Q000821:virology; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism*; D024121:Collagen Type X / Q000235:genetics / Q000378:metabolism; D007403:Intervertebral Disc / Q000166:cytology*; D010641:Phenotype; D012333:RNA, Messenger / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000378:metabolism; D014161:Transduction, Genetic
D053674:Aggrecans; D019485:Bone Morphogenetic Proteins; D024043:Collagen Type II; D024121:Collagen Type X; D012333:RNA, Messenger; D055758:SOX9 Transcription Factor