Unravelling the role of osteoprotegerin in osteoarthritis by applying a human isogenic induced pluripotent stem cell model

Abstract

Purpose: Founded by the hypothesis that the OA disease pathway of the previously identified high impact gain-of-function mutation in TNFRSF11B could be extrapolated to common, age-related, OA disease phenotypes, we set out to functionally study its effect. To this end, we established in vitro hiPS derived spherical chondrogenic and osteogenic pellets and compared TNFRSF11B mutated cells generated from affected family members to CRISPR/Cas9 repaired isogenic control cells. Given that the mutant OPG decoy receptor more efficiently antagonizes osteoclastogenesis, we expect that the mutation affects matrix mineralization hence inflicting the observed chondrocalcinosis phenotype. Methods: Using skin fibroblasts, hiPSCs of an affected family member were created (FOA) and differentiated to induced mesenchymal stromal stem cells (iMSCs). These were differentiated for 6 weeks to deposit neo-cartilaginous and neo-osseous pellets or neo-bone in parallel with a CRISPR-Cas9 repaired hiPSC line (FOA-R) serving as an isogenic control. Presence of glycosaminoglycans was determined by Alcian blue while mineralization was determined by Alizarin red staining. Gene expression analyses were performed to study anabolic markers and hypertrophy (ie, COL2A1, COL1A1, COL10A1) and matrix mineralization (ie, ALPL, MGP). Results: As demonstrated by the intensity of Alcian blue and Alizarin red staining of spherical neo-cartilaginous and neo-osseous pellets, respectively (Figure 1) the bi-directional differentiation via iPSC derived iMSCs appeared successful. Additionally, while only subtle differences in intensity were observed for the Alcian blue staining, the Alizarin red staining intensity in FOA mutated neo-osseous pellets was clearly higher as that of the FOA-repaired pellets, indicating increased matrix mineralization (Figure 1). Gene expression analyses of neo-cartilaginous pellets, showed, however, that in the presence of the FOA TNFRSF11B mutation, MGP levels were considerably decreased (FD=0.14 P=1.6x10-13), indicating cell signaling towards matrix mineralization also in the mutated neo-cartilaginous tissues. In line with this, neo-cartilaginous tissues from FOA mutated cells compared to FOA repaired cells expressed lower levels of COL2A1 (FD=-6.8, P-value=3.9x10-3) concomitant with higher levels of COL1A1 (FD=1.7, P-value=7.4x10-3). Additionally, in the FOA mutated neo-osseous pellets gene expression analysis showed a higher expression of RUNX2 (FD=2.6, P-value= 8.29x10-3) and a trend towards a higher expression of COL1A1 (FD=1.54, P-value= 0.079) and ALPL (FD=1.57, P-value=0.055) relative to FOA repaired neo-osseous pellets.supplements. Conclusions: Employing hiPS-cells from early-onset OA family members (FOA) and CRISPR/Cas9 repaired isogenic controls (FOA-R) in an established spherical 3D in vitro chondrogenic and osteogenic pellet model confirmed that the previously identified gain of function mutation in TNFRSF11B encoding OPG acts via increased matrix mineralization of cartilage and bone. In cartilage, likely, this is modulated via MGP expression as suggested by the significant lower levels in the mutant cells. This may raise the possibility to slow down OA progression in early-onset family members with the use of vitamin K.