MSC promote the expansion of regulatory T cells in a Notch dependent manner, specifically through Jagged 1 signalling. T cells from eGFP-FoxP3 transgenic mice were isolated by magnetic bead isolation (CD4) or cell sorting (CD4+ CD25− FoxP3−), and subsequently cultured with or without MSC for 72 hours and examined for CD25 and FoxP3-GFP expression by flow cytometry. Culture of CD4+ CD25− FoxP3− cells cultured with MSC did not result in an increase in regulatory T cells whereas co-culture of CD4+ T cells with MSC resulted in expansion of the existing regulatory population (A,B). MSC significantly increased the expansion of Treg cells, compared to CD4 cells cultured alone (***, P <0.001), bar chart displaying n = 7 (B). To examine the role of Notch, CD4+ T cells were cultured with MSC in the presence or absence of GSI (1 μM) or vehicle control DMSO and examined for CD25 and GFP expression by flow cytometry (C). CD4+ T cells were cultured with wild type MSC or Jagged-1 knockdown MSC for 72 hours and then examined for CD25 and FoxP3-GFP expression by flow cytometry (D). Counting beads were used to determine the number of cells expressing both CD25 and FoxP3 (E). Data are representative of three studies. DMSO, dimethyl sulfoxide; GSI, gamma secretase inhibitor; MSC, mesenchymal stromal cells; Treg, regulatory T cells.