Supplementary Figure 4: Enhanced T cell positive selection and maturation in ATOs compared with OP9-DL1 monolayer co-cultures.

Full flow cytometry data for Fig. 2a is provided, showing T cell differentiation from three different cord blood donors (#1, #2 and #3) at (a) 4 weeks and (b) 6 weeks. ATO and OP9-DL1 cultures were started in parallel with CD34+CD3- HSPCs from the same cord blood units. Cells are gated on total CD14-CD56- cells or CD3+TCRαβ+ T cells as indicated. (c) Absolute cell numbers of T cell subsets at week 4 and 6 in OP9-DL1 co-cultures versus ATOs using the gating strategy shown in (a) and (b). Each OP9-DL1 culture was initiated with 1.5x104 CD34+CD3- CB HSPCs cells, and each ATO was initiated with 7.5x103 HSPCs from the same cord blood unit, with technical duplicate ATOs harvested and pooled at the indicated times for comparison of cell counts. Bars represent the mean and SD of three independent experiments.