Figure 3

In vitro study of the immune modulation of CB-SCs on monocytes. (A) Phase contrast microscopy shows the co-culture of CB-SC with monocytes (bottom left panel) for 18 hrs. CB-SCs co-culture with lymphocytes (top right panel) served as the control. The impaired CB-SCs after co-culture with monocytes were restored to expansion and became 90 to approximately 100% confluence after 7 to 10 days (bottom right). Original magnification, × 100. (B) Apoptotic analysis of floating cells from the co-culture of CB-SCs with monocytes for 18 hrs. (C) Western blotting shows the expression of the cellular inhibitor of apoptosis protein (cIAP) 1, not cIAP2, in four preparations of CB-SCs. (D) Western blotting shows the expression of tumor necrosis factor receptor II (TNF-RII), not TNF-RI, in four preparations of CB-SCs. (E) TNFα suppresses the proliferation of CB-SCs in a dose–response manner. Cell proliferation was evaluated using CyQUANTR Cell Proliferation Assay Kit [25]. (F) The blocking experiment with iNOS inhibitor 1400W demonstrates that CB-SC-derived nitric oxide (NO) contributes to the immune modulation of CB-SCs on monocytes. Monocytes were initially stimulated with lipopolysaccharide (LPS, 10 μg/ml) for 8 hrs, and then co-cultured with CB-SCs at ratio 1:5 of CB-SCs:monocytes for 48 hrs in the presence or absence of 1400W (100 nM), followed by real time PCR analysis by using Human Th17 for Autoimmunity and Inflammation PCR Array kit (SABiosciences, Valencia, CA, USA).