Effect of TNF-α Dose and Exposure on TSG-6 Activation by Neonatal and Adult Mesenchymal Stromal Cells

Hamidian Jahromi, Shiva 1, 2 ;  Li, Yunqing 1, 2 ;  Davies, John E.  1, 2

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2. Faculty of Dentistry, University of Toronto

Background: Tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) is a major anti-inflammatory mediator released by activated Mesenchymal Stromal Cells (MSCs).[1] Neonatal MSCs are considered more metabolically active than cells derived from adult tissues, and potentially less heterogeneous.[2] We hypothesized that a TNF-a activated neonatal MSC population (human umbilical cord perivascular cells – HUCPVCs) would express an enhanced level of TSG-6 activation compared to adult bone marrow MSCs (bmMSCs). In preparation for an in-vivo xenotransplantation study in mice, we compared the TNF-a stimulation response of HUCPVCs to both human and mouse bmMSCs. 

Materials and Methods: HUCPVCs, hbmMSCs and mbmMSCs were seeded at passage 3 and cell density of 5000 cells/cm2 (n=3). After 24 hours, cells were stimulated with 1, 10, 50, and 100ng/mL of human recombinant TNF-a for HUCPVCs and hbmMSCs, and mouse recombinant TNF-a for mbmMSCs. Supernatant was collected for measurement of secreted TSG-6 protein and total RNA of the same cells was isolated for TSG-6 expression analysis. 

Results and Discussion: 1ng/mL TNF-a stimulation of HUCPVCs caused a significant upregulation of TSG-6 within the first 30 minutes of exposure (~11.5 fold). By contrast, hbmMSCs showed 2-fold increase by 1 hour that increased to 9.5-fold with 50ng/mL of TNF-a for the same exposure time. However, mbmMSC showed a 2-fold increase after 24 hours that was independent of TNF-a concentration. Hence, both human MSC populations exhibited enhanced TSG-6 upregulation upon TNF-a stimulation compared to mbmMSCs. 

Conclusions: Regarding TSG-6 target enhancement, both human MSCs showed high sensitivity to stimulation with TNF-a compared with mbmMSCs. HUCPVCs showed a higher sensitivity, and more prompt response to low dose (1ng/mL) TNF-a stimulation compared to both adult hbmMSCs and mbmMSCs. 

Significance: Neonatal MSCs may be a stronger candidate when treating inflammatory diseases. 

References: [1] Milner C.M. and Day, A.J., J. Cell Sci., 2003; 116:1863-1873 [2]  Sarugaser R. et al., PLoS ONE, 2009; 4(8):e6498