A high-risk osteoarthritis mutation in COL6A3 alters the mechanobiologic response of chondrocytes derived from human induced pluripotent stem cells
Osteoarthritis (OA) is a complex disease arising from a combination of genetic, biological, and mechanical risk factors that act, in part, to alter chondrocyte homeostasis. Our recent exome sequencing studies identified a causal OA variant in COL6A3, a monomeric unit of collagen type VI that serves as a distinguishing component of the pericellular matrix (PCM), a key transducer of mechanical and biochemical signals for the chondrocyte. To study the effect of this mutation, human induced pluripotent stem cell (hiPSC)-derived chondrocytes, genetically edited to harbor the COL6A3 mutation, were used as an in vitro model to investigate chondrocyte mechanobiology and pathobiology. COL6A3 mutant chondrocytes exhibited reduced expression of key matrix proteins, suggesting altered PCM structure and composition.  Functional analyses revealed altered mechanotransduction, characterized by heightened osmotically induced calcium signaling and reduced anabolic response to TRPV4 activation, at the transcriptional level and in matrix biosynthesis. RNA sequencing identified dysregulated pathways and aberrant TRPV4 signaling in mutant chondrocytes following mechanical loading. The presence of the COL6A3 mutation resulted in disrupted circadian rhythms, with increased BMAL1 expression and a significant phase shift, suggesting a link between the PCM and the circadian clock. Finally, COL6A3 mutant chondrocytes exhibited exacerbated catabolic response to interleukin-1, a key inflammatory cytokine in OA. Our study demonstrates the utility of a human iPSC-based system for studying the pathophysiology of specific OA risk alleles. These findings highlight the impact of the COL6A3 variant on chondrocyte physiology and support targeting mechanotransduction signaling pathways as a potential strategy for OA intervention in this case. Overall design: To investigate the impact of a high-risk osteoarthritis mutation in COL6A3 on chondrocyte mechanotransduction, human induced pluripotent stem cells (hiPSCs) were gene-edited to harbor the mutation and differentiated into chondrocytes alongside unedited, wildtype hiPSCs. Wildtype and COL6A3 mutant hiPSC-derived chondrocytes were encapsulated in an agarose hydrogel and cultured for 14 days to form tissue-engineered cartilage, and subjected to physiologic levels of dynamic, compressive mechanical loads (10% stain, 1Hz) over 3 hours, or left uncompressed to serve as a free-swelling control. Loaded samples were collected at three time-points immediatly after mechanical loading (0hrs, 3hrs, 24hrs). Tissue-engineereed cartilage was also cultured with the TRPV4 specific antagonist GSK205 (10 uM) under these specific free-swelling and loaded conditions.