A promoter-based bioassay that predicts the effect of the osteoarthritic microenvironment on cartilage formation

Abstract

Purpose: Recent developments have demonstrated that intrinsic cartilage repair is possible in certain osteoarthritis (OA) patients. A growing body of literature suggests an influence of joint microenvironment on intrinsic cartilage repair via affecting chondrogenic capacity of stem cells; i.e. this environment can be permissive or non-permissive for repair. A method able to discriminate between patients with permissive or non-permissive articular joint microenvironments would be of great use to predict the outcome of cartilage repair therapies. We postulate this can be best done with a bioassay, as in a bioassay different cellular signals are integrated and multiple reporters can be measured simultaneously. Therefore, our goal is to develop a rapid bioassay that will be able to discriminate between permissive and non-permissive joint conditions. Methods: To construct our bioassay, promoter sequences of four different genes were isolated from human gDNA using PCR; i.e. IL6 (-600 till +78), IL8 (-572 till +100 bp), ADAMTS5 (-1432 till +37) and WISP1 (-833 till +50) and cloned in the pNL1.2 vector (Promega) which expressed the unstable Nano luciferase. Promoter reporter constructs were efficiently introduced by transient transfection with lipofectamine in H11 chondrocytes. Transfected cells were stimulated for 24 hours with OA-synovium conditioned medium (OAs-cm), as a substitute for OA synovial fluid, from sixteen individual patients and luciferase activity was measured on a luminometer. OAs-cm was obtained by culturing synovium from end-stage OA patients for 24 hours. Fold change was calculated relative to control stimulation. These results hereof were correlated (Pearson) with the ability of these OAs-cm to inhibit sGAG formation during chondrogenesis. This was tested by chondrogenic differentiation of human fetal bone-marrow derived MSCs in an established three-dimensional pellet culture model. Pellets were cultured for 14 days in serum-free chondrogenic medium (without dexamethasone). After 10 days of differentiation, MSCs were exposed to the OAs-cm from the same sixteen individual donors. Cartilage formation was determined at day 14 by measuring sulfated glycosaminoglycan (sGAG) content. Results: Stimulation of the hIL6, hIL8 and hADAMTS5 promoter reporters with OAs-cm form sixteen different patients resulted in an up-regulation of promoter activity compared to control stimulation, whereas stimulation of the hWISP1 promoter reporter resulted in a down-regulation of the promoter activity compared to control. Interestingly, large heterogeneity was observed in the response of the promoter reporters between the OAs-cm from distinct patients with hIL6 (FC 33.9 ± 32.3), hIL8 (FC 32.2 ± 27.7), hADAMTS5 (FC 3.7 ± 2.4) and hWISP1 (FC 0.4 ± 0.24). Furthermore, individual OAs-cm reduced sGAG content of differentiating MSCs by a range between 3% and 65%. Significant correlations were found between the fold change of the promoter activity and the percentage of inhibition on chondrogenesis. We observed a positive correlation with hIL6 (p=0.01 and r=0.67), hIL8 (p=0.01 and r=0.66) and with hADAMTS5 (p=0.01 and r=0.72), whereas a negative correlation was seen with hWISP1 (p=0.002 and r=-0.78). To validate if this correlation will predict the effect of other OAs-cm, sixteen newly collected OAs-cm were tested and eleven out of sixteen OAs-cm were within the 95% prediction interval. Conclusions: The current study demonstrates that our reporter assays are able to predict the effect of the joint microenvironment (OAs-cm) on cartilage formation as measured by sGAG production. Significant correlations were observed between the fold change of these promoter reporters and the inhibition of chondrogenic capacity of hMSCs, with positive correlations for hIL6, hIL8 and hADAMTS5 and a negative correlation with hWISP1. Our promoter constructs appear to be able to predict repair capacity of stem cells and could be of use for a personalized treatment strategy.