Figure 1: Phenotypic changes during T-cell development.

(A) HM1 ESCs transduced with either GFP or with a GFP-HoxB4 construct, respectively, were analyzed for Nanog1 and Oct4 expression by flow cytometry to confirm pluripotency. For differentiation into HPCs the cells were cultured on monolayers of OP9 stromal cells. By day 5, the cells were expressing Flk-1. CD45 and CD41 emerged by day 8. (B) After transferring the cells onto OP9-DL1 cells, the cells transitioned into the DN1, DN2, DN3 and DN4 stages becoming CD4⁺CD8⁺ DP T cells by day 22. There were no characteristic differences between the HoxB4 and non-HoxB4 cultures. (C) As controls, thymocytes were phenotyped for CD4 and CD8 expression. (D) To confirm HoxB4 expression, cell lysates were analyzed by Western blotting. Only cell lysates of transduced cells were positive for HoxB4. Thymocytes were used as controls.