Figure 7

Transgene regulation of Lent-On-Plus (CEETnl2 and CEETnl2Is2) in mesenchymal and hematopoietic cells derived from hESCs.

(a) AND-1 hESC were transduced with concentrated Lent-On-Plus (CEETnl2 and CEETnl2Is2) LVs. eGFP and SSA3 (a pluripotency marker) expressions were analyzed in the presence (left plots) or absence (right plots) of Dox (0.1 μg/ml). The gate of the eGFP+ populations were set to have 0.5–1-% of SSE3+eGFP+ cells in untransduced population (Mock; top plot). (b) Lent-On-Plus regulatable hESCs were differentiated to mesenchymal stromal cells (see M&M for details). At day 10 of differentiation Dox was added and eGFP expression levels analyzed 5 days later. The plots show eGFP expression levels in the CD105 population of untransduced (Mock; top plot), CEETnl2Is2-transduced (middle plots) and CEETnl2 (bottom plots) in the presence or absence of Dox as indicated at the top of the plots. (c) Lent-On-Plus regulatable hESCs were differentiated toward the hematopoietic lineage using the OP9 differentiation protocol (see M&M for details). The cells were maintained in the presence or absence of Dox from day 1 of differentiation and the eGFP expression analyzed in the different hematopoietic populations (CD45 − CD34+, CD45+CD34+ and CD45+CD34−) as indicated at the different days (Day 8; Top-Left plot and Day 15; Top-right plot). Background levels in untransduced hESCs were set between 4.6–8.3% for each population (arrows) at each day of differentiation (Mock, second top plots). The percentage of cells expressing eGFP in the presence or absence of Dox of the CEETnl2- and CEETnl2Is- transduced hESCs are shown inside each plot for each hematopoietic population.