Clonal chondroprogenitors maintain telomerase activity and Sox9 expression during extended monolayer culture and retain chondrogenic potential.
OBJECTIVE
Articular cartilage contains mesenchymally derived chondroprogenitor cells that have the potential to be used for stem cell therapy. The aim of this study was to characterise the growth kinetics and properties of in vitro expanded cloned chondroprogenitors and determine if critical determinants of the progenitor phenotype were maintained or lost in culture.


METHODS
Chondroprogenitors were isolated from immature bovine metacarpalphalangeal joints by differential adhesion to fibronectin. Cloned colonies were expanded in vitro up to 50 population doublings (PD). Growth characteristics were assessed by cell counts, analysis of telomere length, telomerase activity, expression of senescence-associated beta-galactosidase activity and real-time quantitative polymerase chain reaction to analyse the gene expression patterns of sox9 and Notch-1 in chondroprogenitors.


RESULTS
Cloned chondroprogenitors exhibited exponential growth for the first 20 PD, then slower linear growth with evidence of replicative senescence at later passages. Mean telomere lengths of exponentially growing chondroprogenitors were significantly longer than dedifferentiated chondrocytes that had undergone a similar number of PD (P<0.05). Chondroprogenitors also had 2.6-fold greater telomerase activity. Chondroprogenitors maintained similar sox9 and lower Notch-1 mRNA levels compared to non-clonal dedifferentiated chondrocytes. Chondroprogenitors were induced to differentiate into cartilage in 3D pellet cultures, immunological investigation of sox9, Notch-1, aggrecan and proliferating cell nuclear antigen (PCNA) expression showed evidence of co-ordinated growth and differentiation within the cartilage pellet.


CONCLUSIONS
Clonal chondroprogenitors from immature articular cartilage provide a useful tool to understand progenitor cell biology from the perspective of cartilage repair. Comparisons with more mature progenitor populations may lead to greater understanding in optimising repair strategies.
Khan
I M
IM
Connective Tissue Laboratories, Cardiff University, Museum Avenue, Cardiff CF10 3US, Wales, UK.
Bishop
J C
JC
Gilbert
S
S
Archer
C W
CW
D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology; D019902:Chondrocytes / Q000378:metabolism* / Q000502:physiology; D020219:Chondrogenesis / Q000502:physiology*; D008297:Male; D012333:RNA, Messenger / Q000235:genetics; D051881:Receptor, Notch1 / Q000235:genetics / Q000378:metabolism; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism* / Q000502:physiology; D019098:Telomerase / Q000378:metabolism*
D012333:RNA, Messenger; D051881:Receptor, Notch1; D055758:SOX9 Transcription Factor; D019098:Telomerase