MSC educated tolerogenic DC induce regulatory T cells. C57BL/6 DC were cultured in the absence (immature DC) or presence (mature DC) of LPS without or with MSC (tolerogenic DC), and harvested from adherent MSC via gentle aspiration after 48 hours. These DC cells were washed and subsequently cultured with sorted CD4+CD25−FoxP3− T cells at a ratio of 1:4 DC to T cell, for 72 hours. T cells were gated on CD3 to discriminate them from the DC. Expression of CD25 and FoxP3-GFP was examined by flow cytometry following co-culture with immature DC (A), mature DC (B) or tolerogenic DC (C). MHCIIlow CD86low tolerogenic DC significantly increased the induction of Tregs compared to immature DC or mature DC (**, P <0.01) (D). Data in figures A, B and C are representative flow cytometry plots while figure D shows statistics from three studies. DC, dendritic cells; LPS, lipopolysaccharide; MHC, major histocompatibility complex; MSC, mesenchymal stromal cells.