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Fig. 2 | Stem Cell Research & Therapy

Fig. 2

From: Cyclosporine A and IFNγ licencing enhances human mesenchymal stromal cell potency in a humanised mouse model of acute graft versus host disease

Fig. 2

CsA enhances MSC immunomodulatory gene expression induced by IFNγ but not TNFα. MSCs were seeded at 2 × 105 per well in a 6 well plate and left alone or stimulated with IFNγ (50 ng/ml) or TNFα (20 ng/ml) and/or CsA (1000 ng/ml) at different timepoints. In some groups, MSCs were first stimulated with one cytokine for 24 h followed by CsA for a further 24 h. After a total of 48 h, supernatant was collected for detection of CXCL9 by ELISA and cells were harvested for Quantitative PCR (qPCR) analysis. In other groups, MSCs were stimulated with a cytokine and CsA and harvested after 24 h. Controls for exposure of cytokines at both 24 h and 48 h timepoints were performed. RNA from each sample was extracted, cDNA was synthesised and used as a template for qPCR. mRNA expression of CCL2 (a), COX2 (b) and CXCL9 (c) was calculated relative to the house keeping gene GAPDH. The concentration of CXCL9 was examined by ELISA (d). These experiments were repeated using four MSC donors (n = 4) (passage 5–7), and the results shown are representative of this. Statistical analysis was carried out using unpaired Student’s t test where * < 0.05, ** < 0.01 and *** < 0.001. Stars with no bar are in comparison to the MSC alone group

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