DNMAML allows differentiation of CD34⁺ thymocytes into CD4⁺CD8β⁺ DP thymocytes in FTOC. (A-C) Human CD34⁺ thymocytes, sorted for EGFP after infection with MigR1 and DNMAML1 retroviral constructs, were subjected to FTOC. Cultures were analyzed after 11 and 19 days as indicated, and at each time point, 3 lobes of each viral construct were pooled. Results shown are derived from 3 independent experiments. (A) Reduction in the frequency of DNMAML1 transduced EGFP⁺ cells in FTOC compared with the frequency of control MigR1 transduced EGFP⁺ thymic progenitors. Numbers in histograms indicate the percentage of EGFP⁺ cells. (B) Reduction in the absolute number of DNMAML1 (□) transduced EGFP⁺ cells that are generated in FTOC after 11 and 19 days of culture compared with the MigR1 (■) control. Numbers indicate the average number of EGFP⁺ cells (± SD) that were generated per lob, initiated with 10 000 EGFP⁺ cells. (C) Flow cytometric analysis of FTOCs after 11 days of culture, gated on EGFP⁺ cells as shown in histograms on the left in panel A. Numbers in dot plots indicate the percentage of cells in the indicated areas. (D) Gene expression analysis of genes involved in Notch signaling (top) and T-cell development (bottom) in CD34⁺ thymocytes, transduced with MigR1 (■) control and DNMAML1 (□) and sorted for human CD45 and EGFP after 24 hours of OP9-DL1 coculture as shown in Figure S2B. Expression levels for all genes are presented in units relative to those from EGFP⁻ cells from the same cultures and normalized to β-actin, calculated by the ΔΔC_T method. Data shown are the average of 2 independent experiments that consisted of triplicate wells each, and error bars indicate data range.