MON-262 Aggrecan Is Required for Chondrocyte Differentiation in ATDC5 Chondroprogenitor Cells

Background Aggrecan is an integral part of the extracellular matrix in cartilaginous tissue, including that of the growth plate. Heterozygous defects in the aggrecan gene have been identified as a cause of autosomal dominant short stature, bone age acceleration, and premature growth cessation, all of which are consistent with a role for aggrecan in the cartilaginous growth plate. Previous animal studies of aggrecanopathies have shown disruption of growth plate architecture and acceleration of chondrocyte hypertrophy, although the mechanisms accounting for this phenotype remains unknown. We undertook studies to elucidate the mechanism by which aggrecan loss-of-function mutations lead to impaired long bone growth in concert with bone age acceleration. Methods We used ATDC5 chondroprogenitor cells, an established model of chondrogenesis, to evaluate the effects of aggrecan deficiency. ATDC5 aggrecan knock down cell lines (AggKD) were generated using shRNA lentiviral transduction particles. Cells were stimulated with insulin to induce chondrogenesis for 21 days and studied. Aggrecan qPCR and Alcian blue staining were used to assess levels of aggrecan knock down. Control and AggKD cells were cultured in media containing 10 μg/ml insulin to stimulate chondrogenesis. Cells were studied at multiple time points over 21 days using qPCR, hemocytometer counting, and flow cytometry with propidium iodide staining for cell cycle analysis. Extracellular matrix (ECM) pre-conditioning experiments and co-culture experiments of AggKD cells with wild-type (WT) ATDC5 cells were also used to evaluate effects on chondrogenesis. Results Control ATDC5 cells showed induction of col2a1 starting at day 8 and induction of col10a1 starting at day 12. AggKD cells had significantly reduced expression of col2a1 and col10a1 (p<0.0001) with only minimal increases in expression over time, indicating that chondrogenesis was markedly impaired. This reduction of col2a1 and col10a1 was not rescued by culturing AggKD cells in wells pre-conditioned with WT ATDC5 ECM, or even by co-culturing with WT ATDC5 cells. Sox9 and ihh levels were similar between control and AggKD cell lines. Hemocytometer counts showed increased cells in the control line compared to AggKD lines at Day 8 (p<0.0005), but there was no difference in cell cycle analysis using flow cytometry. Conclusion Our studies indicate that AggKD cells do not undergo normal chondrogenesis, suggesting for the first time that aggrecan has an integral role in this cell development process. Experiments that reintroduced aggrecan via extracelluar and pericellular matrices from pre-conditioning wells and co-culturing cells were unsuccessful at rescuing chondrogenesis in AggKD cells. This suggests that the role of aggrecan in chondrogenesis appears to be mediated through intracellular mechanisms.