Purpose: To gain insight in etio-pathophysiological effects of dysfunctional collagen type VI (COL6) fibrils, we studied the effect of an identified high impact osteoarthritis (OA) mutation in COL6A3. Since COL6 is abundant in the pericellular matrix surrounding chondrocytes and known to regulate responses to loading, we studied effect of the COL6A3 mutation in interaction with injurious mechanical strain. Henceforth, we employed genetically COL6A3 edited human induced pluripotent stem cell (iPSC) to an established in-vitro cartilage organoid model and studied transcriptome wide changes in chondrocytes with COL6A3 mutation in interaction with hyper-physiological mechanical strain (MS).
Methods: CRISPR-Cas9 technology was applied to introduce an identified OA risk mutation (rs144223596) in human iPSCs. COL6A3-edited and unedited isogenic control hiPSCs were subjected to our previously established 3D chondrogenesis protocol. To apply mechanical loading, chondrocytes were either released from their cartilage extracellular matrix using collagenase and embedded into a cylindrical shaped 2% w/v agarose and cultured for additional 14 days in presence of 10ng/mL TGFB3, or directly exposed to MS. The spherical and cylindrical cartilage pellets were dynamically loaded with a strain of 20% and 10%, respectively, at a frequency of 5hz for 10 minutes. Transcriptomics of both models where jointly analyzed using DEseq2 with SVA correction in R. Pathway enrichment and protein-protein interaction network analyses were performed in R statistical language with the clusterProfiler package and with online available tool STRING, respectively.
Results: Differential expression analysis between isogenic controls and COL6A3 mutants revealed 3,700 differentially expressed genes (DEGs). Amongst others, notable DEGs were; MMP9 (FC= 3.67, FDR = 7*10-16), FGFR3 (FC=2.63, FDR=6*10-4), COL27A1 (FC=0.70, P=4*10-4), COLGATL2 (FC=0.70, FDR=2*10-5) (Fig. 1a). Pathway analysis on the DEGs were enriched for biological processes such as skeletal system development and an inflammatory response (Fig 1b). Differential expression analysis between unloaded and loaded cartilage organoids resulted in 179 DEGs, amongst others, notable DEGs were; PLAUR (FC=1.50, FDR=0.025), CD44 (FC=1.45, FDR =0.019), CAV1 (FC=1.52, FDR=4*10-4), ITGA5 (FC=1.50, FDR=0.021) (Fig 2a). Pathway analysis on the DEGs were enriched for biological processes such as wound healing, response to heat and ‘de novo’ protein folding (Fig 2b). Finally, the effect of the COL6A3 mutation on mechano-transduction was assessed. 135 genes show an interacting effect between the COL6A3 mutation and hyper-physiological mechanical stress, as shown in the resulting protein-protein interaction network (Fig 3a). Of note, ADAMTS5 and IGFBP5, are genes that previously have been shown to be responsive to MS, have a significant interaction effect between mechanical loading and the COL6A3 mutation (Fig 3b).
Conclusions: The identified COL6A3 mutation affects the chondrocytes transcriptomic landscape, enriched for matrix related pathways, suggesting a link of the mutation to a cartilage phenotype. Furthermore, in this model of hyper-physiological mechanical stress, gene expression of mechano-senor genes is upregulated. Moreover, the COL6A3 mutation interferes with normal wild-type response to mechanical stress, providing a potential causal mechanism for the observed OA risk with this mutation.
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